IMMUNOPHARMACOLOGY (IP)
Α,Β-AMYRONE ISOLATED FROM PROTIUM PANICULATUM INHIBITS LPS-STIMULATED J774 MACROPHAGES
EMERSON SILVA LIMA; PATRICIA D.O. ALMEIDA; ANA P.A. BOLETI; ANDRE L. RUDIGER; VALDIR F. VEIGA- JUNIOR.
UNIVERSIDADE FEDERAL DO AMAZONAS, MANAUS - AM - BRASIL.
Introduction: Protium is the main genus belonging to the family Burseraceae and one of the most common in South America. In folk medicine, natural gums and resins of Protium are used for various diseases, as tonic and stimulating, for the treatment of ulceration and inflammation. Objectives: The present study aimed to investigate the anti- inflammatory activity of triterpene α,β-amyrone isolated oleoresin of Protium paniculatum Engler (Burseraceae). Methods and Results: Firstly, we evaluated the cytotoxic potential of α,β-amyrone in the murine macrophages J774 lines by Alamar Blue method. Also, cells were treated with α,β-amyrone, stimulated with LPS and after 24 hours the cell supernatant was collected for evaluation of nitric oxide (NO•) and cytokines TNF-α, IL-6, IL-10, and IFN-γ. The anti-inflammatory activity of α,β-amyrone was also evidenced by the expression of COX-2 by western blotting and inhibition of paw edema induced by carrageenan in rats. In the evaluation of cytotoxicity was observed that α,β- amyrone did not alter the viability of J774 murine macrophages (IC50> 50 µg/mL). Analyzing the effects on the production of inflammatory mediators was observed that the α,β-amyrone in a concentration of 10 µg/ml inhibited more than 80% production of NO• and inhibited the production of IL-6 and induced the production of IL-10. The COX-2 expression was inhibited in a concentration dependent manner as paw edema formation in rats induced by carrageenan, producing a fast and immediate response. Conclusion: This study may provide a basis for future investigations on the therapeutic role of triterpene α, β-amyrone in the treatment of inflammation.
A HYDROGEN SULFIDE-RELEASING CYCLOOXYGENASE INHIBITOR MARKEDLY ACCELERATES RECOVERY FROM EXPERIMENTAL SPINAL CORD INJURY
SALVATORE CUZZOCREA1; MICHELA CAMPOLO2; ESPOSITO EMANUELA3; AKBAR AHMAD4; ROSANNA DI PAOLA5; JOHN L WALLACE6.
1,2,3,4,5.UNIVERSITY OF MESSINA, MESSINA - ITÁLIA; 6.ANTIBE THERAPEUTICS INC., TORONTO - CANADÁ.
Spinal cord trauma results in loss of motor function that is in part due to the ensuing inflammatory response. Hydrogen sulfide (H2S) is a potent, endogenous anti-inflammatory and neuroprotective substance that has been exploited in the design of a novel nonsteroidal anti-inflammatory drugs. In the present study, we have evaluated the potential beneficial effects ATB-346, an H2S-releasing derivative of naproxen, in a murine model of spinal cord injury (SCI). SCI was induced in male CD1 mice by spinal cord compression, produced through the application of vascular clips to the dura via a T5 to T8 laminectomy. ATB-346, naproxen (both at 30 µmol•kg−1) or vehicle was orally administered to the mice 1 and 6 hours after SCI and once daily thereafter for 10 days. ATB-346 significantly reduced levels of inflammation (pro-inflammatory cytokines, apoptosis) that characterize the secondary events of SCI, as well as markedly improving motor function. The effects of ATB-346 were superior to those of naproxen. This included a significant reduction of pro-inflammatory cytokines and apoptosis of neural tissue, as well as a marked reduction of tissue nitrosative stress. Motor function (Basso mouse index of locomotion) improved gradually in mice treated with naproxen. However, mice treated with ATB-346 exhibited a significantly more rapid and sustained recovery of motor function, achieving greater than double the increase in locomotion score of the naproxen group by the 10th day of treatment. These results demonstrate marked beneficial effects of an H2S-releasing derivative of naproxen in an animal model of SCI, significantly reducing secondary inflammation and tissue injury, and markedly enhancing recovery of motor function. The combination of inhibition of cyclooxygenase and delivery of H2S may offer a promising alternative to existing therapies for traumatic injury.
A HYDROGEN SULPHIDE DONOR REDUCES ACUTE PANCREATITIS INDUCED BY DUCT LIGATION OR L- ARGININE
DANIELLE GOMES SANTANA1; MARCELA SANTOS SANTANA2; ALAN SANTOS OLIVEIRA3; MARCELO NICOLAS MUSCARÁ4; SORAIA KÁTIA PEREIRA COSTA5; ENILTON APARECIDO CAMARGO6.
1,2,3,6.UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO - SE - BRASIL; 4,5.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL.
Introduction: Acute pancreatitis is an inflammatory disease of the pancreas that involves a complex interplay of many mediators. It is believed that hydrogen sulphide (H2S) can mediate this process. In this study, the role of H2S was investigated in two experimental models of AP in mice.
Methods and Results: AP was induced in male Swiss mice (20-30 g, n=8/group) by the common bile duct obstruction
(CBDO) or by the injection of two doses of L-arginine (4 g/kg, i.p., 1 h apart). Mice were treated with the H2S donor,
Lawesson´s reagent (30 mmol/kg, i.p.), the inhibitor of H2S synthesis, propargylglycine (50 mg/kg, i.p.) or vehicle, 1 h before and every 12 h after induction of pancreatitis. After 24 h of CBDO or 72 h of L-Arginine injection, mice were euthanized and serum amylase concentrations, as well as pancreatic and lung myeloperoxidase activity (MPO) were measured. The experiments were approved by the Instituition´s Ethic Committee (01/12).
Results: In the model of CBDO-induced acute pancreatitis, the treatment with Lawesson´s reagent, but not propargylglycine, reduced (P<0.05) the serum amylase concentration, when compared with the vehicle group (33,547±1,925; 7,955±2,421 or 34,704±3,293 U/dL for vehicle, Lawesson´s reagent or propargylglycine-treated groups, respectively). The MPO activity was also inhibited by treatment with Lawesson´s reagent in pancreas (4.6± 1.6 UMPO/mg of tissue; P<0.05) or lung (8.3±0.5 UMPO/mg of tissue; P<0.05), when compared with the vehicle group (13.9±3.2 and 19.2±1.9 UMPO/mg of tissue, for pancreas and lung, respectively). Treatment with propargylglycine did not affect MPO activity in these tissues. In the L-Arginine-induced pancreatitis model, the donation of H2S by treatment with LR significantly reduced the MPO activity in pancreas (3.1±0.4 UMPO/mg of tissue, P<0.05), when compared with vehicle-treated group (7.0±1.4 UMPO/mg of tissue), without affecting the serum amylase concentrations or lung MPO activity. The treatment with propargylglycine did not alter the parameters evaluated in this model.
Conclusions: These data suggest that H2S has a protective role in acute pancreatitis in rodents and further studies are necessary to clarify its therapeutical potential to treat acute pancreatitis.
Financial Support: CNPq and FAPITEC/SE.
A MARINE PSEUDOPTERANE DITERPENE INHIBITS THE INFLAMMATORY RESPONSE INDUCED BY LPS IN MACROPHAGES
YISETT GONZÁLEZ1; DEBORAH DOENS2; RICARDO SANTAMARÍA3; CARLOS M RESTREPO4; MARLA RAMOS5; LUCIANA BARROS DE ARRUDA6; RICARDO LLEONART7; MARCELINO GUTIÉRREZ8; PATRICIA L. FERNANDEZ9.
1,2,3,4,5,7,8,9.INDICASAT, PANAMA - PANAMA; 6.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL.
Introduction: The inflammatory response is initiated by the activation of innate immune cells such as macrophages that play a role in the recognition of microbial or host molecules through pattern recognition receptors (PRRs). These receptors are essential at the beginning of the innate response and in triggering the subsequent adaptive response. Toll like receptors (TLRs) belong to the PRR family and its role in the development of inflammatory response has been widely described. The diversity of the terrestrial and marine flora and fauna in Panama ensures the existence of abundant resources for searching new molecular structures with a variety of biological activities. Marine organisms have been described previously as sources of bioactive compounds. Diterpenoids isolated from corals have anti- inflammatory activity; but their mechanisms of action are unknown. This work proposes the characterization of the anti-inflammatory activity of a diterpenoid (compound 1) isolated from the coral Pseudopterogorgia acerosa in macrophages. Methods and Results: To characterize the anti-inflammatory effect of compound 1 murine macrophages were stimulated with LPS in the presence or absence of different concentrations of compound 1 and the expression and production of TNF, IL-6, IP-10, iNOS was determined by Real Time PCR and ELISA respectively. Compound 1 inhibited the response of macrophages to LPS in a concentration dependent manner with an IC50 ranging from 2.4 to 14 microM. This effect was also observed in human macrophages differentiated from PBMC. We then evaluated if this effect is dependent on the inhibition of NFkB activation. Macrophages were stimulated with LPS at various time points and the activation of NFkB was evaluated in the nuclear extracts by an ELISA-based assay. Phosphorylation and degradation of IkBa was also assessed by using Western Blot. We observed that compound 1 inhibits the activation of NFkB through the inhibition of IkBα degradation. We also evaluated the expression of CD80 and CD86 by flow cytometry in cells stimulated with LPS in the presence of compound 1. We observed a reversion in the expression of both molecules when compound 1 was present in the culture. Conclusions: Our results show that compound 1 inhibits the in vitro response of macrophages to LPS by a mechanism that involved the inhibition of IkBa degradation and the subsequent activation of NFkB.
Financially supported by National Secretariat of Science from Panama (SENACYT).
A POSSIBLE ROLE FOR LOCAL HEPCIDIN EXPRESSION IN COLON DURING TNBS-INDUCED COLITIS IN RATS
ÉRICA MARTINS FERREIRA GOTARDO; CAMILA HENRIQUE MOSCATO; RENATA BORTOLIN GUERRA TOMÉ; THALITA ROCHA; MARCELO LIMA RIBEIRO; ALESSANDRA GAMBERO.
UNIVERSIDADE SÃO FRANCISCO, BRAGANÇA PAULISTA - SP - BRASIL.
Introduction: Inflammatory cytokines produced during inflammation, notably interleukin-6 (IL-6), have been hypothesized to contribute to systemic iron deficiency observed in patients with inflammatory bowel disease. IL-6 through Jak/Stat-3 signaling can result in hepcidin synthesis, a major regulator of iron homeostasis. Therefore, we investigated hepcidin expression in rat colon during colitis that was induced by trinitrobenzene sulfonic acid (TNBS) administration; we also investigated changes in local IL-6 production and iron levels during this process, as well as systemic alterations. Methods and Results: We evaluated colitis status using macroscopic scoring and measurement of myeloperoxidase (MPO) activity. The colonic levels of hepcidin and IL-6 were measured by ELISA. Hepcidin-25 expression and iron deposition were analyzed by immunohistochemistry and Prussian blue reaction, respectively. Phosphorylation of Stat-3 was assessed by Western blot. Additionally, the ability of lipopolysaccharide (LPS) and IL-6 to induce gene hepcidin expression in HT-29 cells. Repeated intracolonic TNBS resulted in macroscopic lesions and increased MPO activity in the colon tissue (2.2±0.6 and 18.2±4.8 U/g for control and colitis group respectively, p<0.05, n=5). The IL-6 levels were increased in colon (8.2±1.7 and 14±0.7pg/mg protein of IL-6 for control and colitis group respectively, p<0.05, n=5). Hepcidin levels were increased in colon (10.2±4.0 and 71.0±8.4 pg/mg protein for control and colitis group respectively, p<0.01, n=5) and immunohistochemistry demonstrated a predominant hepcidin-25 expression in colonocytes. Iron deposition in colon was also observed during colitis.The phosphorylation of Stat-3 was also observed in the colon samples. Systemic alterations in iron homeostasis and hepcidin levels were not detected. HT-29 cells expressed higher levels of hepcidin after IL-6 and LPS stimulation (3.8±0.6, 11.6±2.6 and 6.6±0.9Hepcidin/β-actin for control,IL-6 and LPS respectively, p<0.05, n=6). Conclusion: The colonic inflammation increased local hepcidin expression and iron sequestration. Iron accumulation in immune cells can result in pro- inflammatory phenotype and consequent increased cytokine release. But, low level of iron is also an important local antimicrobial mechanism. Additional studies will be necessary to determine whether hepcidin expression in colon is a defensive or pathological response to intestinal inflammation.
Financial Support: FAPESP
A PROTEIN FRACTION ISOLATED FROM THE LATEX OF CALOTROPIS PROCERA ATTENUATES THE PROGRESSION OF IRINOTECAN-INDUCED INTESTINAL MUCOSITIS IN MICE BY MODULATING THE INFLAMMATORY RESPONSE
TAMIRIS DE FÁTIMA GOEBEL DE SOUZA; FLAVIO DA SILVEIRA BITENCOURT; KAROLINE SABOIA ARAGÃO; PATRICIA BASTOS LUZ; INGRID SAMANTHA TAVARES DE FIGUEIREDO; CONCEIÇÃO DA SILVA MARTINS; ROBERTO CESAR PEREIRA LIMA-JUNIOR; MARCIO VIANA RAMOS; GERLY ANNE DE CASTRO BRITO; RONALDO ALBUQUERQUE RIBEIRO; NYLANE MARIA NUNES DE ALENCAR.
FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Intestinal mucositis (IM) is a side effect of irinotecan (CPT-11), which is used for the treatment of colorectal cancer. The incidence of IM associated with severe diarrhea is estimated to be approximately 25% in patients. However, the clinical management of these side effects is still partially ineffective. Calotropis procera is a plant found in Africa, Asia, and South America. In Brazil, it is abundant in the northeast and has been shown to have antiinflammatory effects in animal models. The objective of the present study was to evaluate the antiinflammatory effect of a protein fraction of the latex of Calotropis procera in IM induced by CPT-11. Methods and Results: Swiss mice (n=10, 23±2g) were treated for 4 days with saline (Sal, 5 mL/kg, i.p.) or CPT-11 (75mg/kg, i.p.). In other experimental groups, CP (1, 5 and 50mg/kg/day, i.v.) was administered for 6 days, 30 min before the CPT-11. On the 7th day, we evaluated the total leukocyte count (x103/mL) and diarrhea (by scores). After sacrifice, the duodenum was collected for measurement of myeloperoxidase activity (MPO), morphometric analysis (villi/crypt), TNF-α and IL-1β levels (pg/mL), in vitro contractility (% contraction in relation to KCl 60mM) and immunohistochemistry for COX-2, TNF-a, IL-1β, iNOS, and NF-κB. ANOVA/Bonferroni or Kruskal Wallis/Dunn was used as statistical tests. P<0.05 was accepted. CP attenuates diarrhea scores and MPO activity at 5mg/kg (diarrhea: 1[0-2]; MPO: 4.05±1.07) and 50mg/kg doses (diarrhea: 1[0-3]; MPO: 5.57±1.50) when compared with CPT-11 (diarrhea: 3[2-3]; MPO: 24.45±3.0, respectively). In addition, CP decreased over contractility (5mg/kg: 165.1±57.7), cytokines TNF-a (5 mg/kg: 3.31 ± 3.0; 50 mg/kg: 9.79 ± 5.6) and IL-1β (5 mg/kg: 143.5 ± 41.5; 50 mg/kg: 182 ± 65.7) and villi/crypt ratio (5 mg/kg: 2.79±0.17) increased in comparison with only CPT-11 treated mice (contractility: 906.1±225.4, IL-1b: 806.1±247.6 and villi/crypt: 1.63±0.17). CP a dose of 5 mg/kg reduced immunohistochemistry labeling for COX-2, TNF-a, IL-1β, iNOS, and NF-κB and the expression of iNOS in the duodenum in animals with mucositis. However, CP did not change leukopenia induced by CPT-11 at doses tested. Conclusion: These results suggest that the antiinflammatory and antidiarrheal effects of Calotropis procera are produced by the inhibition of inflammatory mediators that are important in CPT-11- induced intestinal mucositis. Financial support: CNPq and CAPES.
A PROTEOLYTIC FRACTION FROM VASCONCELLEA CUNDINAMARCENSIS LATEX DISPLAYS ANTI- INFLAMMATORY EFFECTS IN A MOUSE MODEL OF INFLAMMATORY BOWEL DISEASE
MARINA PASSOS PIZZITOLA; ANA CANDIDA ARAUJO SILVA; ARIADNE DUARTE BRAGA; KATIA MICHELLE FREITAS; CARLOS EDMUNDO SALAS; MIRIAM TERESA PAZ LOPES.
UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Our previous results demonstrated that a proteolytic fraction (P1G10) from V. cundinamarcensis latex displays, among others, gastric protective/healing activities (Phytomed. 15(4):237-244, 2008), and anti-inflammatory effects on models of edema and inflammatory tumor (not published). In this study, we investigated the effect of P1G10 against colon lesions induced by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) in mice, model compatible with Inflammatory Bowel Diseases (IBD) in humans. Methods and Results: Colon lesions were induced by intra-colonic administration of TNBS (1.5 mg/0.1 mL 50% ethanol) to fasted and anesthetized male Swiss mice (30-35 g; n=10/group). After 24 h, mice were treated once a day v.o. with saline (TNBS and Sham groups), P1G10 (0.3-30 mg/kg) or P1G10 with proteolytic activity inhibited by iodoacetamide (P1G10-IAA - 0.3 mg/kg). Mice were monitored for body weight and killed 96 h after TNBS administration for evaluation of the macroscopic damage (presented as score) and measurement of some inflammatory parameters. Statistical Analysis: ANOVA, Student-Newman-Keuls post-test. Protocol was approved by Local Ethics Committee (#177/2013). Comparing to the TNBS group (5.71 ± 1.07), only 0.3 (1.12 ± 0.52, p < 0,05) and 3 mg/kg (1.43 ± 0.50, p < 0,05) were effective in reducing colon lesions, returning the score next to Sham values (0.44 ± 0.17). Animals treated with these doses presented a significant gain of weight (approximately 10%). At higher doses, P1G10 did not reduce the colonic damage, maybe due to the high proteolytic action on these doses. However, P1G10IAA (3.80 ± 0.58) presented a non-significant effect comparing to TNBS group (4.75 ± 0.48) and was different (p < 0,05) from P1G10 (1.43 ± 0,57) suggesting that the proteolytic activity is important for its anti-inflammatory effect. P1G10 reduced TNF-α level (59.44 ± 15.51 vs. 81.10 ± 28.49 pg/g tissue - control) and MPO activity (0.93 ± 0.37 vs. 8.34 ± 2.33; x10-4 D.O./mg tissue) while increased VEGF levels (717.80 ± 32.35 vs. 590.60 ± 28.49 pg/g tissue – control). P1G10IAA did not alter TNF-α levels but increased significantly VEGF levels (809.60 ± 53.31 pg/g tissue, p < 0,05). NAG levels were not altered by any treatment, comparing to TNBS group. Conclusion: The results suggest an anti-inflammatory effect of P1G10 on colon lesions induced by TNBS through modulation of cytokines and neutrophil activity/infiltration. Financial support: FAPEMIG, CAPES and CNPq.
A SULPHATED POLYSACCHARIDE FRACTION FROM SEAWEED AGARDHIELLA RAMOSISSIMA DIMINISHED NEUTROPHIL MIGRATION IN INFLAMMATORY EXPERIMENTAL MODELS.
EULINA GABRIELA DO NASCIMENTO DIAS1; JALLES ARRUDA BATISTA2; TARCISIO VIEIRA DE BRITO3; RAFAEL DA SILVA PRUDÊNCIO4; RENAN OLIVEIRA SILVA5; ANA LUCIA PONTE FREITAS6; MARCIA RUBIA MELO7; REGINA CÉLIA MONTEIRO PAULA8; MARCELLUS HENRIQUE LOIOLA PONTE DE SOUZA9; LUCIANO DE SOUSA CHAVES10; JAND-VENES ROLIM MEDEIROS11; ANDRÉ LUIZ DOS REIS BARBOSA12.
1,2,3,4,5,11,12.UNIVERSIDADE FEDERAL DO PIAUI, PARNAIBA - PI - BRASIL; 6,7,8,9,10.UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Several studies have investigated new drugs that may contribute to avoiding or minimizing excessive inflammatory process. Marine natural products have been the focus of discovery for new products of chemical and pharmacological interest. The aim of this study was to evaluate the anti-inflammatory effect of the sulphated polysaccharide (PLS) extracted from the red seaweed Agardhiella ramosissima. Methods: To evaluate the sulphated polysaccharide (PLS) actions on the carrageenan-induced neutrophil migration into the plantar tissue or peritoneal cavity we performed the myeloperoxidase (MPO) and peritonitis assays respectively. In the protocols mice (n=6) were pre-treated with indomethacin (IND; 10 mg•kg-1, i.p.) or sulphated polysaccharide (PLS; 30 mg•kg-1, i.p.) fraction. Results: The group treated with sulphated polysaccharide (PLS) showed a decreased myeloperoxidase (MPO) activity (2.76 ± 1.17 UMPO/mg of plantar tissue) when compared this group with carrageenan group (18.97 ± 2.53 UMPO/mg of plantar tissue). Adding to this, the same dose of sulphated polysaccharide reduces the total leukocyte (4.31× 106 ± 0.71 × 103 cells/ml) and neutrophil (1.90 × 103 ± 0.49 × 103 cells/ml) counts in comparison carragenaan group (total leukocyte: 8.22 × 106 ± 0.57 × 103 cells/ml / neutrophil: 4.63 × 103 ± 0.35 × 103 cells/ml). Conclusion: In conclusion, the PLS reduces the inflammatory response by modulating neutrophil migration into the site of inflammatory process induced by carrageenan agent.
ADENOSINE RECEPTOR ROLE IN ADIPOSE TISSUE INFLAMMATION IN AN EXPERIMENTAL MODEL OF OBESITY
CAROLINE CANDIDA DE OLIVEIRA1; ÉRICA MARTINS FERREIRA GOTARDO2; CÍNTIA RABELO E PAIVA CARIA3; SIMONE COGHETTO ACEDO4; PATRÍCIA FERNANDES ZANOTTI NAKAMITSU5; ALESSANDRA GAMBERO6.
1,4,5.UNICAMP, CAMPINAS - SP - BRASIL; 2,3,6.SÃO FRANCISCO UNIVERSITY, BRAGANÇA PAULISTA - SP - BRASIL.
Introduction: Obesity has been correlated with the presence of systemic inflammation characterized by adipokines production and macrophages infiltration in adipose tissue. Adenosine levels increase during inflammation and modulate inflammatory responses. Activation of A1 receptors has pro-inflammatory effects while A2A receptor activation results in antiinflammatory actions, such as inhibition of TNF-α production, which is produced by adipose tissue and involved in the establishment of insulin resistance. The characterization of the actions mediated by these receptors may contribute to the understanding the adenosinergic system participation in obesity. Methods and Results: Swiss mice were fed with commercial chow (CN) or high fat diet (HFD) for 12 weeks. In the last two weeks, mice were treated with N⁶-cyclopentyladenosine(CPA, selective A1 agonist, 0.05 and 0.1 mg/kg/day), CGS 21680 (selective A2A agonist, 0.1 and 0.5 mg/kg/day) or 5'-N-ethylcarboxamidoadenosine (NECA, non-selective agonist, 0.1 mg/kg/day). Body weight, glucose blood levels and insulin tolerance test (ITT) were evaluated. Adipose tissue biopsies were obtained for adipokines measurements using ELISA. CPA treated mice presented no reduction in body weight, adiposity or fasting glucose blood levels, but an improvement in insulin tolerance was observed (3.75±1.05, 0.64±0.26 and 1.57±0.44 kITT for CN, HFD and CPA 0.05, respectively, p<0.05, n=6). No changes were observed in IL-10, TNF-α and leptin adipose tissue levels. After CGS administration, mice presented no alterations in body weight or adiposity. Glucose blood levels were reduced after CGS treatment (123.40±10.80, 224.20±38.37, 144.50±6.02 mg/dL glucose for CN, HFD and CGS 0.1, respectively, p<0.05, n=6) as well as, mice was sensible to insulin (3.46±1.21, 1.85±0.68, 3.80±0.43 for CN, HFD and CGS 0.5, respectively, p<0.5, n=6). IL-10 and leptin levels were not modified but TNF-α levels in adipose tissue were reduced (0.50±0.25, 2.14±0.35, 1.21±0.60 for CN, HFD, CGS 0.5 and CGS 0.1, respectively, p=0.09, n=6). After NECA administration, there were no modifications in all parameters evaluated. Conclusions: The selective A2A agonist, CGS 21680, suggests that adenosine A2A receptors has an important role on inflammation control during obesity, since it was observed an improvement in the metabolic parameters and adipose tissue TNF-α reduction. Financial support: CAPES and FAPESP.
ANALOGUE OF KAURENOIC ACID OBTAINED BY ASPERGILLUS TERREUS BIOTRANSFORMATION INHIBITS INFLAMMATION AND PAIN IN MICE
DANIELA CRISTINA DE MEDEIROS1; SANDRA S. MIZOKAMI2; THIAGO H. HAYASHIDA3; RUBIA CASAGRANDE4; SÉRGIO R. AMBRÓSIO5; NILTON S. ARAKAWA6; WALDICEU A. VERRI JR7.
1,2,3,4,6,7.UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA - PR - BRASIL; 5.UNIVERSIDADE DE FRANCA, FRANCA - SP - BRASIL.
Introduction: Kaurenoic acid, a diterpene present in several genera of plants such as Sphagneticola, Mikania and Xylopia, underwent microbial transformation by the fungus Aspergillus terreus. The main analogue was the compound ent-7-a-hidroxy-kaur-16-en-19-oic acid (7-β-hydroxi-kaurenoic acid). Kaurenoic acid presents anti-inflammatory and antinociceptive activity, thus, the anti-inflammatory and analgesic effects of 7-β-hydroxi-kaurenoic acid (7-β) were evaluated. Methods: Male swiss mice (25-30 g, n = 6 per group) were treated orally (po.1, 3 and 10 mg/kg) with 7-β- hydroxi-kaurenoic acid or vehicle (saline) 30 min before inflammatory stimulus. The writhing response was evaluated during 20 min after ip injection of acetic acid (0.8%) or phenyl-p-benzoquinone (1890 µg/kg). Mechanical and thermal hyperalgesia and paw edema were evaluated 1-5 h after carrageenan (300 mg/paw) injection and myeloperoxidase activity at 5 h. Rota-rod test was performed in naive mice. This study was approved by the ethics committee of Universidade Estadual de Londrina (37748.2011.09). Results: 7-β-hydroxi-kaurenoic acid (1, 3 and 10 mg/kg) inhibited acetic acid (up to 58.31%, reduced from 91.5 ± 2.9 writhings to 58.1 ± 6.8; 53.0 ± 5.6 and 38.8 ± 6.3; respectively) and PBQ-induced writhing (up to 63.71%, reduced from 81.6 ± 5.4 to 57.6 ± 3.9; 43.0 ± 6.1; and 42.0 ± 7.4; respectively). 7-β-hydroxi-kaurenoic acid (1, 3 and 10 mg/kg) reduced carrageenan-induced mechanical (up to 85.71%, reduced from 4.3 ± 0.5 to 3.6 ± 0.5; 1.7 ± 0.4 and 0.6 ± 0.3; respectively) and thermal hyperalgesia (up to 52.59%, reduced from 8.3 ± 1.1 to 13.7 ± 1.3; 21.3 ± 3.0 and 20.6 ± 2.5; respectively), paw edema (up to 58.98% reduced from 0.5 ± 0.08 to 0.4 ± 0.04; 0.4 ± 0.06 and 0.2 ± 0.04; respectively), and myeloperoxidase activity (up to 56.58%, reduced from 49933.0 ± 6436.8 to 41749.1 ± 4748.3; 23918.4 ± 3148.7 and 55845.7 ± 9094.2 neutrophil recruitment, respectively). There was no alteration in the rota-rod test indicating no muscle relaxant effect. Conclusion: 7-β-hydroxi-kaurenoic acid presents anti-inflammatory and analgesic activity. Further studies are necessary to determine its mechanism of action.
Financial Support: CAPES, CNPq, SETI/Fundação Araucária, MCTI and Parana State Government.
ANALYSIS OF ANTI-NOCICEPTIVE EFFECT OF A SULPHATED POLYSACCHARIDE FRACTION FROM RED SEAWEED AGARDHIELLA RAMOSISSIMA.
AMANDA MOREIRA FONTENELE1; JALLES ARRUDA BATISTA2; EULINA GABRIELA DO NASCIMENTO DIAS3; TARCISIO VIEIRA BRITO4; RAFAEL SILVA PRUDÊNCIO5; RENAN OLIVEIRA SILVA6; MARCELLUS HENRIQUE LOIOLA PONTE SOUZA7; REGINA CÉLIA MONTEIRO PAULA8; LUCIANO SOUSA CHAVES9; MÁRCIA RUBIA MELO10; ANA LUCIA PONTE FREITAS11; JAND-VENES ROLIM MEDEIROS12; ANDRÉ LUIZ REIS BARBOSA13.
1,2,3,4,5,6,12,13.UNIVERSIDADE FEDERAL DO PIAUÍ- UFPI, PARNAÍBA - PI - BRASIL;
7,8,9,10,11.UNIVERSIDADE FEDERAL DO CEARÁ - UFC, FORTALEZA - CE - BRASIL.
AMANDA MOREIRA FONTENELE(A); JALLES ARRUDA BATISTA(A); EULINA GABRIELA DO NASCIMENTO DIAS(A); TARCISIO VIEIRA BRITO(A); RAFAEL SILVA PRUDÊNCIO(A); RENAN OLIVEIRA SILVA(A); MARCELLUS HENRIQUE LOIOLA PONTE SOUZA(B); REGINA CÉLIA MONTEIRO PAULA(D); LUCIANO SOUSA CHAVES(C); MÁRCIA RUBIA MELO(C); ANA LUCIA PONTE FREITAS(C); JAND-VENES ROLIM MEDEIROS(A); ANDRÉ LUIZ REIS BARBOSA(A*).
(A)LAFFEX – Laboratory of Experimental Physiopharmacology, Biotechnology and Biodiversity Center Research (BIOTEC), Federal University of Piauí.
(B)LAFICA – Laboratory of Pharmacology of Inflammation and Cancer, Department of Physiology and Pharmacology, Federal University of Ceará.
(C)Laboratory of Proteins and Carbohydrates of Marine Algae, Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, CE, Brazil.
(D)Laboratory of Polimers, Departament of Organic and Inorganic Chemistry, Federal University of Ceará, Fortaleza, Brazil.
INTRODUCTION: Several studies have investigated new drugs that may contribute to avoiding or minimizing excessive nociceptive process. Marine natural products have been the focus of discovery for new products of chemical and pharmacological interest. The aim of this study was to evaluate the antinociceptive effects of the sulfated polysaccharides (PLS) extracted from the red seaweed Agardhiella ramosissima. METHODS: In all assays the mice (n=6) were pre-treated with PLS (30mg.kg-1). After 30min the behavior injection stimuli were administered. In the writhing test, 0.6% acetic acid (10 mL•1 kg body weight), the intensity of nociception was quantified by counting the total number of abdominal writhing and extension paw over 20 min [56] . In the formalin test, 20 microliters of 2.5% formalin was administered (i.pl) the right hind paw of rats. The licking time was recorded from 0 to 5 min (first phase, neurogenic) and 20 to 25 minutes (stage 2 inflammatory) after formalin injection. The hot plate test each mouse was placed on a plate twice heated (51 ± 1 ° C), separated by an interval of 30 min. The first trial the animal familiar with the testing procedure, and the second assay served as the control for the reaction time (paw licking or jumping). RESULTS: In the antinociceptive tests, the pre-treatment with PLS (30 mg.kg-1) reduced (82.0%) the number of writhes acetic acid-induced in comparison to that in the control group (acid acetic+saline group). In the licking time formalin-induced the PLS (30 mg·kg-1), showed a significant anti-nociceptive effect, reducing the licking time, only in the second phase (80.54%) in comparison to the control group (saline+formalin group). In the hot-plate assay the PLS did not show any anti-nociceptive effect in all intervals. CONCLUSION: In accordance with our results we can infer that PLS produce anti-nociceptive effect by modulating a peripheral and inflammatory mechanisms rather than a central-acting.
ANNEXIN A1 IN CYSTIC FIBROSIS AIRWAY WOUND PATHOLOGY
ALEXANDER J DOYLE; R J FLOWER.
WILLIAM HARVEY RESEARCH INSTITUTE, QUEEN MARY UNIVERSITY LONDON, LONDON - REINO UNIDO.
Introduction- Cystic fibrosis (CF) occurs when a genetic mutation impairs epithelial cell function, resulting in the lack of bacterial clearance from the airways. This prompts a non-resolving inflammatory response leading to lung damage (Cell 140, 871-882). Annexin A1 (AnxA1) is an endogenous protein with pro-resolving activity up-regulated by glucocorticoids (GCs). The underlying problem in CF is mucus hypersecretion and airway remodelling. Mutation of the cftr gene is associated with a decrease in extracellular AnxA1 and increased inactive cleavage product, 33kDa AnxA1(Mol Cell Proteomics 4, 1591-1601). It is unclear what contribution these paradoxical attributes of AnxA1 make to CF airway pathology.
Methods- Healthy human bronchial epithelial cells (HBECs) and the cystic fibrosis equivalent (Δ508-deletion; CFBEC), were used to characterise differences in AnxA1 expression. Western blots (WB) of lysates were used to determine AnxA1 phosphorylation and transglutaminase 2 (TG2) concentration. To assess the contribution of AnxA1 to airway remodelling and wound healing in CF, 24h scratch assays were performed on cultured HBECs and CFBECs treated with the AnxA1 peptide 3µM Ac2-26 or 3nM dexamethasone (Dex).
Results- CFBECs retain two-fold more AnxA1 protein within cells, but 50% less phosphorylated (activated) protein is detected. This deficit is not corrected by treatment with Dex (3nM). Analysis of the supernatants by WB reveal increased cleavage of AnxA1 in favour of the 33kDa form in CFBECs. This is accompanied by the expression of ~78kDa molecular weight bands believed to be AnxA1 multimers linked by the enzyme TG2. WB analysis reveals a two-fold increase in TG2 levels in CFBECs compared to HBECs (n=3). Ac2-26 significantly (p<0.01) increases wound closure in HBECs but not in CFBECs. Blocking TG2 decreases 24h wound closure significantly(p<0.05) in HBECs but not in CFBECs (n=4-10 ±SEM).
Conclusion- CF and healthy cells show a clear discrepancy in AnxA1 profile. The disparity points to an impairment of AnxA1 phosphorylation or its translocation through the membrane. The increased TG2 activity may directly contribute to disease pathology by facilitating aggregate formation and promoting inflammation. TG2 is vital for wound healing and may prolong Ac2-26 activity, but in disease states this abundant TG2 may be a barrier to effective treatment.
This work is supported by Novartis. We thank Dr. Emma Hickman for guidance.
ANNEXIN A1 MEDIATES PPAR-PAN EFFECTS DURING INFLAMMATION RESOLUTION
JOSÉ ROBERTO SANTIN1; ISABEL MACHADO2; IVAN PITTA3; MAURO PERRETTI4; SANDRA FARSKY5.
1,2,5.UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL; 3.FEDERAL UNIVERSITY OF PERNAMBUCO, RECIFE - PE - BRASIL; 4.WILLIAN HARVEY INSTITUTE, LONDON - REINO UNIDO.
Introduction: LYSO-7 is a PPAR-pan agonist and COX inhibitor, which inhibit the development of inflammation. Although PPAR agonists improve the resolution of inflammation, the role of PPAR pan and the mechanisms of actions are not fully understood. Annexin-A1 (ANXA1) is a modulator of development and resolution of inflammation, and its secretion and action is induced by different modulators of the process. Here we investigated the participation of ANXA1 on LYSO-7 actions on resolution of inflammation.
Methods and Results: Gastric ulcer was induced in male Balb/c wild type (WT) or ANXA1-/- mice (n=6) (acetic acid, 20%). After 24 h, vehicle; benzafibrate (BZF) or LYSO-7 (50 mg/kg, p.o., once a day, 7 days) were administered. BZF or LYSO-7 treatments reduced the lesion, and the percentage of reduction was lesser in ANXA1-/- mice. Incubations of neutrophils with LYSO-7 (10 μM; 24 h) enhanced the apoptosis of neutrophils (ANXAV/PI labeling. Flow cytometry) obtained from WT mice, but not from ANXA1-/- mice. Enhanced phagocytosis of apoptotic neutrophils by peritoneal macrophages from WT mice treated with LYSO-7 (1, 5 or 10 μM, 1 h before) and reduced levels TNF-α and elevated levels of IL-10, TGF-1β or VEGF in the supernatant was observed. These effects were not detected if macrophages were obtained from ANXA1-/- mice.
Conclusions: Data obtained show that LYSO-7 actions on resolution of inflammation are mediated by secreted ANXA1, showing a relation of both modulators of the process. The mechanism involved may be related to the action of PPAR agonists on secretion and functions of ANXA1.
Financial support: Fapesp (2010/17175-0; 2011/01848-8).
ANTI-INFLAMATORY EFFECT OF RUTIN AND ISOFLAVONE (5’, 3’, 4’-TRIIDROXI - 6’’,6’’ - DIMETILPIRANO [2’’, 3’’ : 7,6] ISOFLAVONE) FROM POLYGALA MOLLUGINIFOLIA A. ST.-HIL. & MOQ.
FÁBIO ARRUDA SILVA; MARCUS VINICIUS PEREIRA DOS SANTOS NASCIMENTO; ANA BEATRIZ GOBBO LUZ; DALILA VENZKE; MOACIR GERALDO PIZZOLATTI; EDUARDO MONGUILHOTT DALMARCO.
FEDERAL UNIVERSITY OF SANTA CATARINA, FLORIANÓPOLIS - SC - BRASIL.
Introduction: The genus Polygala have the largest number of species in the family Polygalaceae. In Brazil there are 110 species. Until today, had been identified alkaloids, coumarins and flavonoids, which have attributed a significant in vitro and in vivo biological activities. In the present study, we aimed to analyse the anti-inflammatory effect of Polygala molluginifolia majority compounds, Rutine (Rut) and 5,3’,4’-triidroxi-6’’,6’’-dimetilpirano[2’’,3’’:7,6]isoflavona (Iso1) administered by intraperitoneal route (ip.) in the mouse model of pleurisy induced by carrageenan (Cg).
Methods and results: Female swiss mice (18-25 g) were housed under standard conditions (22±2°C) and were used through the experiments (CEUA PP 00757). The pleurisy model was performed as previously described by Br. J. Pharmacol. 118:811-819, 1996. The pleurisy was induced by a single intrapleural (ipl.) injection of 0.1 mL of carrageenan (Cg, 1%), N=5 per group. The pleural fluid were collected 4h after pleurisy induction for determination of the adenosine-deaminase (ADA) activity, nitric oxide products (NOx), tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1b) levels, as well as TNF-a and IL-1b mRNA expression were also evaluated. Dexamethasone was used as anti-inflammatory reference drug. In preliminary experiments, we obtained a curve dose and time response of Poligala molluginifolia majority compounds, that were able to significantly reduced the leukocyte influx and exudate concentrations (p<0.01). For this reason, in the present study we used the following doses: Rut (10 mg/kg) and Iso1 (5 mg/kg), i.p, 0.5 h before Cg. The Rut was able to reduce the ADA activity (% of inhibition: 60.6±4.5), TNF-a and IL-1b levels (% of inhibition: 42.1±4.5, 60.3±3.1, respectively) (p<0.01). The Iso1, was able to reduce all inflammatory parameters analysed, ADA activity and NOx levels (% of inhibition: 52.0±9.0 and 55.2±2.2, respectively), TNF-a and IL-1b levels (% of inhibition: 35.0±7.0 and 53.3±6.4, respectively) (p<0.01). Moreover, both compounds decreased the mRNA expression from TNF-a and IL-1b (p<0.01).
Conclusions: These results provide evidence the Rut and Iso1 have an important anti-inflammatory actions, since these compounds were effective in reducing the ADA activity and NOx levels, decreased the release of pro- inflammatory cytokines (TNF-a and IL-1b), as well as, their mRNA expression.
Financial Support: CAPES and CNPq.
ANTI-INFLAMATORY PROPERTIES OF METHOTREXATE LOADED LIPID-CORE NANOCAPSULES
ANTONIO LUIZ BOECHAT1; CATIÚSCIA PADILHA DE OLIVEIRA2; ANDREA MONTEIRO TARRAGÔ3; ADRIANA MALHEIRO4; SILVIA STANISÇUASKI GUTERRES5; ADRIANA RAFFIN POHLMANN6.
1,3,4.UNIVERSIDADE FEDERAL DO AMAZONAS, MANAUS - AM - BRASIL; 2,5,6.UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL, PORTO ALEGRE - RS - BRASIL.
Introduction. Methotrexate (MTX) has been used at the last tree decades the treatment of various rheumatic conditions and remains the cornerstone of Rheumatoid Arthritis (RA) treatment. MTX is carefully used because his important side effects. By the other hand, drug delivery systems such nanocapsules formulations could improve drug efficiency, reducing side effects and dose regimens. Objective. The aim of this investigation was evaluated the anti- inflammatory properties of MTX loaded nanocapsules. Methodology. Adjuvant Arthritis was induced in Lewis rats (AIA) and the effect on oedema formation, TNF-α and IL1-β levels of four different treatments was evaluated: a) arthritis group ; b) MTX; c) Placebo Lipid-core Nanocapsules (LNC) and d) MTX loaded Lipid-core Nanocapsules (MTX-LNC). mononuclear cells were collected and culture from synovial fluid from RA patients during articular infiltrations procedures. MTX, MTX-LNC were tested at different doses on cultured mononuclear cells. Quantification of TNF-α, IL6, INF-γ, IL17A and IL10 cytokines was performed using cytometric bead array (CBA) technique with flow cytometer. For in vivo experiments the MTX-LNC dose used was only 75% lower of MTX dose regimen. Results. Formulations (LNC and MTX-LNC) were prepared using the methodology of self-assembling and presented pH values of 5.8 ± 0.2 and 4.8 ± 0.5 to LNC and MTX-LNC, respectively. These formulations presented nanometric and monomodal profile, with mean volume-weighted diameters Dv[4.3] and Span, analyzed by laser diffraction, of the 172 ± 21 nm and 1.5 ± 0.2 for LNC and 175 ± 17 nm and 1.6 ± 0.2 for MTX-LNC. Two-Way ANOVA statistics and Tukey test shows that MTX-LNC has the same effect of MTX on arthritis inhibition at 28th day of experiment (p<0.0001). This effect is achieved early at 21th day (p<0.0001). The MTX-LNC was able to reduce both TNF-α and IL1-β serum levels at the last day of experiment (p=0.0041 and p=0.0178, respectively) using post-test for linear trends. The effect of MTX-LNC on cytokine production from mononuclear synovial cells was high comparing conventional MTX using non- linear regression analysis for: TNF-α (r2=0.90 and r2=0.29, p=0.0005), IL-6 (r2=0.81 and r2=0.11, p=0.0034), IL17A (r2=0.81 and r2=0.16, p=0.0175), INF-γ (r2=0.89 and r2=0.68, p=0.020) and IL10 (r2=0.67 and r2=0.28, p=0.049). Conclusion: These results shows that MTX-LNC is high potential drug-delivery system for the treatment of Rheumatoid Arthritis.
ANTI-INFLAMMATORY ACTIVITY OF AQUEOUS EXTRACT FROM MORINGA OLEIFERA SEED
LARISSA CARDOSO CORRÊA DE ARAÚJO; SANDRA CABRAL DA SILVA; FERNANDA VIRGÍNIA BARRETO MOTA; THIAGO HENRIQUE NAPOLEÃO; LUANA CASSANDRA BREITENBACH BARROSO COELHO; TERESINHA GONÇALVES DA SILVA; PATRÍCIA MARIA GUEDES PAIVA.
UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE - PE - BRASIL.
Introduction: Moringa oleifera tissues are used with several purposes in folk medicine. In order to understand the mode of action of this plant, this study evaluated the anti-inflammatory effect of aqueous extract from M. oleifera seeds in a carrageenan-induced pleurisy model. Methods and results: Albino swiss mice (Mus musculus) (n=6) (Committee for Ethics in Animal Research n° 23076.029506/2012-64) were used. The animals were pre-treated orally with the extract (125, 250 or 500 mg/kg), dexamethasone (0.5 mg/kg) or saline (NaCl 0.9%). After 1 h the animals were anesthetized and received 0.1 mL of carrageenan (1%) in pleural cavity through surgery. Four hours later, the animals were euthanized in a CO2 chamber and the pleural cavity was washed with heparinized PBS. The pleural fluid was collected to evaluate cell migration, cytokine and NO levels. Statistical analysis was performed by one-way ANOVA followed by Newman-Keuls test. Leukocyte migration in the treatments with extract at 125, 250 and 500 mg/kg was inhibited in 45.4%, 56.1% and 71.2%, respectively, in comparison with the control (11.1±1.1 x106 cells/mL). Dexamethasone inhibited the migration in 63.6% (4.1±0.4 x106 cells/mL). The NO level in control was 35.5±1.5 µM. The extract reduced NO levels and concentrations of 11.8±1.6, 4.68±1.7 and 3.10±0.6 µM were detected for doses of 125, 250 and 500 mg/kg, respectively. The value for dexamethasone was 2.54±0.1 µM. The extract at doses of 125, 250 and 500 mg/kg reduced TNF-α levels to 440.0±39.7, 237.1±7.0 and 167.3±36.1 pg/mL, respectively, when compared to control (469.4±21.2 pg/mL). The IL-1β levels for treatments at 125 mg/kg (529.4±9.4 pg/mL), 250 mg/kg (191.3±40.1 pg/mL) and 500 mg/kg (12.0±1.9 pg/mL) were also lower than in control (723.8±18.7 pg/mL); the values for dexamethasone were 215.53±58.14 pg/mL (TNF-α) and 356.4±2.0 pg/mL (IL-1β). These results are interesting since TNF-α acts stimulating the production of other proinflammatory cytokines, while IL-1β stimulates expression of adhesion molecules on endothelial cells. Conclusion: The M. oleifera seed extract promoted reduction of the leukocyte migration, levels of NO, TNF-α and IL-1β in a dose dependent manner. The data indicate that the anti- inflammatory activity of M. oleifera seeds aqueous extract is at least in part related to the regulation of the proinflammatory mediators NO, TNF-α and IL-1β. Financial support: CAPES, CNPq, FACEPE and MCTI.
ANTI-INFLAMMATORY ACTIVITY OF BUNCHOSIA ARMENIACA (CAV.) DC. (MALPIGHIACEAE)
MARCUS VINICIUS PEREIRA DOS SANTOS NASCIMENTO; GUSTAVO SILVA QUEIROZ; FABIO ARRUDA SILVA; MOACIR GERALDO PIZZOLATTI; INES MARIA COSTA BRIGHENTE; EDUARDO MONGUILHOTT DALMARCO.
FEDERAL UNIVERSITY OF SANTA CATARINA, FLORIANOPOLIS - SC - BRASIL.
Introduction: The specie Bunchosia armeniaca, native from Andes, is a plant from Malpighiaceae family, popularly known as “cafezinho”. In traditional medicine is used for treatment of endocrine, nutritional, metabolic disorders and also in the treatment of some kind of cancer. Although literature doesn’t have scientific information about this specie, other species from Malpighiaceae family presents several biological reports, highlighting their anti-inflammatory effect. Due to the lack of biological studies carried out with this specie, we decided evaluate the possible anti-inflammatory activity of this plant in the mouse model of pleurisy.
Methods and Results: Female Swiss mice (18-25g) were housed under standardized conditions (20 ± 2°C, with alternating 12-h periods of light and dark) and were allowed free access to a standard mouse chow and water. The procedure was approved by the Committee for Ethics in Animal Research of our University (protocol PP00757), and the experiments were performed in accordance with the norms of the Brazilian College of Animal Experimentation (COBEA). The induction and analysis of pleurisy were performed as described previously by Br. J. Pharmacol. 118:811-819, 1996. Briefly, different groups of animals (n=5) were treated with evans blue dye, 10 minutes after received the different doses of extracts or mixture of flavonoids and finally, 30 minutes after an intrapleural injection of carrageenan 1%. After 4h, the quantification of leukocytes influx, exudate, myeloperoxidase (MPO) activity, nitric oxide (NOx) and TNF-alpha levels were performed. The crude extract (200 mg/Kg) significantly reduced the leukocytes influx, exudation, MPO activity, NOx and TNF-alpha levels (% inhibition - leukocytes: 36.0±3.7; exudation: 25.7±2.4; MPO: 25.7±2.4; NOx: 40.3±4.3 and TNF-alpha: 43.5±13.0)(p<0.05). The mixture of flavonoids (1.0 mg/Kg) inhibited the same inflammatory parameters (% inhibition – leukocytes: 57.1±4.7; exudation: 21.3±3.1; MPO: 30.6±2.0; NOx: 51.0±3.6 and TNF-alpha: 63.6±13.5)(p<0.05).
Conclusion: Bunchosia armeniaca showed a significant anti-inflammatory action by inhibit the leukocyte influx and their activation induced by carrageenan. This affirmation is based in the ability of natural product in reduce the MPO activity, NO formation and TNF-alpha release in fluid leakage.
Financial Support: This study was supported by CNPq and CAPES.
ANTI-INFLAMMATORY ACTIVITY OF CARVACRYL ACETATE, A DERIVATIVE OF CARVACROL, IN MICE
NATHALIA SANTOS CARVALHO1; SAMARA RODRIGUES BONFIM DAMASCENO2; FRANCISCO RODRIGO DE AZEVEDO MENDES DE OLIVEIRA3; CAMILA DE FATIMA CARNEIRO BRITO4; IRISMARA SOUSA SILVA5; FRANCISCA BEATRIZ MELO DE SOUSA6; RENAN OLIVEIRA SILVA7; ANDRÉ LUIZ DOS REIS BARBOSA8; RIVELILSON MENDES FREITAS9; JAND-VENES ROLIM MEDEIROS10.
1,2,4,5,6,7,8,10.BIOTECHNOLOGY AND BIODIVERSITY CENTER RESEARCH (BIOTEC), FEDERAL UNIVERSITY OF PIAUÍ, PARNAÍBA - PI - BRASIL; 3,9.POST-GRADUATION PROGRAM IN PHARMACEUTICAL SCIENCES, FEDERAL UNIVERSITY OF PIAUÍ, TERESINA - PI - BRASIL.
Introduction: Several studies are conducted to identify novel therapeutic options with low adverse effects. Carvacrol is a monoterpenic phenol abundant in the essential oil fraction, renowned for its numerous therapeutic properties (Fitoterapia 75:801–804, 2004). The aim of study was to investigate the potential anti-inflammatory effects of carvacryl acetate (CC), a derivative of carvacrol. Methods and Results: The present study was approved by the local ethics committee (protocol no. 0066/10). The anti-inflammatory activity was evaluated using various phlogistic agents that induce paw edema and peritonitis in mice. The peritoneal exudate was collected for the determination of myeloperoxidase and pro-inflammatory cytokine levels. Pretreatment with CC (75 mg/kg) significantly reduced carrageenan-induced paw edema at the third (88.8%, p<0.001) and fourth hour (97%, p<0.001), when compared to vehicle-treated group. Likewise, CC (75 mg/kg) inhibited edema induced by histamine, serotonin, prostaglandin E2 and compound 48/80. The inhibition percentage of these edemas was 34.14% (p<0.001), 96.48% (p<0.001), 83.33% (p<0.001) and 37.50% (p<0.01) respectively. Pretreatment with CC (75 mg/kg) also significantly reduced the leukocyte recruitment (2720 × 103 ± 681.3 × 103 cells/ml) and neutrophil migration (1805 × 103 ± 400.7 × 103 cells/ml) to the peritoneal cavity, as compared to carrageenan group (total leukocyte: 13220 × 103 ± 2610 × 103 cells/ml; neutrophil recruitment: 11410 × 103 ± 2392 × 103 cells/ml). The levels of IL-1β and MPO were evaluated using the ELISA method, and the pre-treatment of animals with CC (75 mg/kg i.p) significantly reduced IL-1β (951.1 ± 143.3 pg/ml) and MPO (4.1 ± 0.6 U/ml) as compared to carrageenan group (IL1-β: 1995 ± 13.19 pg/ml; MPO: 7.6 ± 0.8 U/ml, p<0.001). Conclusion: Our data provide evidence that the carvacryl acetate-mediated anti-inflammatory effect and the mechanisms involved seems depends of inhibition of inflammatory mediators (histamine, serotonin and prostaglandins), as well as a decrease of mast cell degranulation, IL-1β and neutrophil migration.
ANTI-INFLAMMATORY ACTIVITY OF EPIISOPILOTURINE, AN IMIDAZOLE ALKALOID ISOLATED FROM PILOCARPUS MICROPHYLLUS
VALDELÂNIA GOMES DA SILVA; RENAN OLIVEIRA SILVA; SAMARA RODRIGUES BONFIM DAMASCENO; NATHALIA SANTOS CARVALHO; RAFAEL DA SILVA PRUDÊNCIO; JOSÉ ROBERTO DE SOUZA DE ALMEIDA LEITE; LEIZ MARIA COSTA VERAS; ANDRÉ LUIZ DOS REIS BARBOSA; JAND-VENES ROLIM MEDEIROS.
FEDERAL UNIVERSITY OF PIAUÍ, PARNAÍBA - PI - BRASIL.
Introduction: The long-term use Non-steroidal anti-inflammatory drugs (NSAIDs) has problematic side effects (J. Physiol Pharmacol. 59:117-133, 2008). Thus, natural compounds are an alternative form of therapy with fewer contraindications and side effects. The aim of this study was to investigate the anti-inflammatory effect of epiisopiloturine (EPI), an imidazole alkaloid found in the leaves of Pilocarpus microphyllus. Methods and Results: All experiments were approved by the local ethics committee (protocol 0066/10). Male Swiss mice (25−30 g) were initially treated intraperitoneally (i.p) with 2% DMSO, indomethacin 10 mg/kg (reference control), or EPI (0.1, 0.3, or 1 mg/kg test drug) after 30 min was injected in paw different phlogistic agents. In carrageenan-induced paw edema, the animals pretreated with EPI at 0.1, 0.3, and 1mg/kg showed maximal reductions in edema of 55.0, 70.0, and 85.0% respectively, compared to the carrageenan group. The administration of dextran sulfate (0.065±0.010 mL), serotonin
(0.225±0.008 mL), bradykinin (0.052±0.004 mL), histamine (0.074±0.009 mL) or prostaglandin E2 (0,035±0,013 mL) produced edema. Pretreatment with EPI (1 mg/kg) effectively inhibited paw edema induced by dextran sulfate
(54.08% inhibition), serotonin (46.66%), bradykinin (76.92%) and prostaglandin E2 (68.58% inhibition) (p < 0.05). However, did not inhibit paw edema induced by histamine. The results obtained showed that carrageenan produced a marked increase in myeloperoxidase activity (10.9±1.2 U/mg of tissue). This increase was reduced by treatment with EPI (3.8±0.9 U/mg of tissue). Carrageenan (500 μg/cavity) also induced cell migration into the peritoneal cavity 4 h after administration, with a total leukocyte count of (5.81±0.61) × 106cells/mL. However, the administration of EPI (1 mg/kg, i.p) 30 min before carrageenan reduced this peritoneal leukocyte count to (2.60±0.61) × 106 cells/mL and also reduced neutrophil migration to (1.26 ± 0.17) × 106 cells/mL, 73.8% lower than the carrageenan group (5.82 ± 0.47) × 106 cells/mL). Carrageenan induced a marked increase in TNF-α and IL1-β concentrations in the peritoneal fluid and pretreatment of animals with EPI (1 mg/kg, ip) reduced TNF-α and IL1-β levels significantly. Conclusion: Thus, EPI exhibited anti-inflammatory activity in mice by reducing inflammatory mediators (serotonin, bradykinin and PGE2), neutrophil migration, leukocyte count and cytokine concentration (TNF-α and IL1-β).
Financial support: CAPES - FAPEPI
ANTI-INFLAMMATORY ACTIVITY OF PHYLLANTHUS NIRURI SPRAY-DRIED STANDARDIZED EXTRACT IN ARTHRITIS MODEL IN RATS
GERLANE COELHO BERNARDO GUERRA1; GERLANE NOME DO MEIO - OPCIONAL GUERRA2; CÌNTHIA PORTO3; DANIEL OLIVEIRA4; TATIANE SOUZA5; PEDRO PETROVICK6; RAIMUNDO ARAÚJO JÚNIOR7; AURIGENA FERREIRA8; LUIZ ALBERTO LIRA SOARES9.
1,2,3,4,7,8.UFRN, NATAL - RN - BRASIL; 5.UFAM, MANAUS - AM - BRASIL; 6.UFRGS, PORTO ALEGRE - RS - BRASIL; 9.UFPE, RECIFE - PE - BRASIL.
Introduction: Animal models of arthritis aid in elucidating the pathophysiology and allow the study treatments for alleviating or inhibit the inflammatory processes involved. Phyllanthus niruri L., Euphorbiaceae is popularly known in Brazil as “stonebreaker”. Some species of Phyllanthus are widely used in folk medicine for different types of diseases. In this study, the antiinflammatory activity of a obtained Spray-dried extract from the aerial parts of Phyllanthus niruri was tested using a model of zymosan-induced arthritis.
Methodology and Results: The administration of zymosan in the joint cavity causes increased vascular permeability leading to local edema and severe cell influx predominance of polymorphonuclear cells. In the model of arthritis, the synovial fluid leukocyte infiltration was assessed by total leukocyte count in a Neubauer chamber, was measured in inflamed tissue markers of inflammation (myeloperoxidase) and oxidative stress (total glutathione), were assessed the damage and changes between groups by histopathological analysis and evaluated the expression of RANKL, RANK, OPG, COX-2, MMP-2 and MMP-9 by immunohistochemical technique. The extract reduced the leukocyte influx to articular cavity (p <0.001). the extract showed reduced levels of myeloperoxidase (p <0.05 and p <0.001) and increased the levels of total glutathione (p <0.05 and p <0.001). Additionally, the histological analysis of synovium revealed a reduction of inflammatory events assessed in inflamed tissue. Immunohistochemical evaluation suggests that the extract may have contributed to the reduction of expression for RANKL, RANK, OPG, COX-2, MMP-2 and MMP-9. Conclusion: The Spray-dried extract of Phyllanthus niruri exerts a beneficial effect in the acute phase of of zymosan-induced arthritis model rat is probably related to its antioxidant properties, due to its flavonoids content.
Financial support: CNPq.
ANTI-INFLAMMATORY AND ANALGESIC EFFECT OF CURINE, A BISBENZYLISOQUINOLINE ALKALOID ISOLATED FROM CHONDRODENDRON PLATYPHYLLUM (MENISPERMACEAE)
FAGNER CARVALHO LEITE1; JAIME RIBEIRO FILHO2; HERMANN FERREIRA COSTA3; PAULA REGINA RODRIGUES SALGADO4; ADRIANO FRANCISCO ALVES5; ANDREA SURRAGE CALHEIROS6; DIOGO VILAR DA FONSECA7; PATRICIA TORRES BOZZA8; MARCIA REGINA PIUVEZAM9.
1,3,4,5,7,9.UFPB, JOAO PESSOA - PB - BRASIL; 2,6,8.FIOCRUZ, RIO DE JANEIRO - PB - BRASIL.
Introduction: Curine is a bisbenzylisoquinoline alkaloid found as the major constituent from the root bark of Chondrodendron platyphyllum, a plant used to treat malaria, fever and pain. However, curine anti-inflammatory and analgesic effects remain to be elucidated. Aim of study: To investigate curine anti-inflammatory and analgesic effects on mice and to provide the scientific rationale for the folk medicine. Materials and Methods: We used protocols of inflammation and pain induced by different agents to investigate the effect of oral treatment with curine. A one-way ANOVA and a Dunnett test, as post-test, were used to determine the statistical significance in comparison to the control. Data are expressed as the mean ± standard error of the mean (SEM). A p<0.05 was considered significant. Results: Carrageenan injections significantly induced the formation of paw edema (0.17± 0.05), which was significantly inhibited by the pre-treatments with curine or indomethacin (0.11±0.04; 0.088±0.03, respectively). Similarly, zymosan injections induced the expressive formation of paw edema (0.09±0.03), which was inhibited by curine or indomethacin (0.043±0.02; 0.05±0.03, respectively). the writhing response was significantly inhibited by curine or indomethacin morphine (30.5±7.2; 27.8±7.0) compared to non-treated group (50.8±16.1). Mice treated with curine or indomethacin presented no difference in the licking time in the neurogenic phase compared to non-treated mice (61.9±28.1; 51.6±12.8; 62.5±18.0, respectively). However, the second phase was significantly inhibited by curine or indomethacin (158.8±39.38; 153.8±64.01, respectively) compared to non-treated group (274.7± 48.0). Carrageenan significantly decreased the heat withdrawal latency compared to the challenge with PBS in non-treated animals (10.40±1,786; 5.270±1.974, respectively). However, there were no significant differences between the carrageenan challenge and the PBS challenge in animals treated with curine (10.14±3.29; 8.72±4.06) or indomethacin (9.94±1.96; 9.3±5.7). LPS stimulated cells presented a consistent increase in COX-2 expression that was not affected by curine pre-treatments. However, the supernatants from curine treated cells presented lower concentrations of PGE2 compared to the supernatant of the non-treated cells (122.6±10.39; 276.9± 62.97). Conclusions: Curine displays interesting anti-inflammatory and analgesic effects and a potential for anti-inflammatory drug development.
ANTI-INFLAMMATORY AND ANTINOCICEPTIVE ACTIVITY OF THE ETHANOLIC EXTRACT OF THE BARK OF PHANERA FLEXUOSA (MORIC) L.P. QUEIROZ (CAESALPINIACEAE)
MAIANA FERRAZ ANDRADE1; KELLE OLIVEIRA SILVA2; ÉRIKA PEREIRA DE SOUZA3; RAFAEL SANTOS DANTAS MIRANDA DÓREA4; CÁSSIA MAVIONY FIUZA ANDRADE5; JOSELINE CEZÁRIO DUARTE6; JERSICA FIRMINO BRANDÃO7; APARECIDO ALMEIDA CONCEIÇÃO8; JULIANA TRINDADE CLEMENTE NAPIMOGA9; MARCELO HENRIQUE NAPIMOGA10; LUCAS MIRANDA MARQUES11; MARILUZE PEREIRA CRUZ12; REGIANE YATSUDA13.
1,2,3,4,5,6,7,8,11,12,13.FEDERAL UNIVERSITY OF BAHIA, VITÓRIA DA CONQUISTA - BA - BRASIL; 9.UNICAMP,FOP, PIRACICABA - BA - BRASIL; 10.INSTITUTE SÃO LEOPOLDO MANDIC, CAMPINAS - BA - BRASIL.
Introduction: Phanera flexuosa known as “escada de macaco”, is a common small tree in the Semi-arid region of Brazil. The stem bark is used by the population in the northeast of Brazil to treat sexual failure. This study evaluated anti-inflammatory and antinociceptive activity of the ethanolic extract of the bark of P. flexuosa (BBF), collected in the National Forest Contendas Sincorá (FLONA). Methods and Results:The anti-inflammatory and antinociceptive activity were evaluated using the writhing induced by acetic acid, formalin-induced hypernociception, Von Frey test, neutrophil migration and extravasation of Evans blue into the peritoneal cavity. The cytokines (TNF-α, IL-10 and IL-1β) were measured by ELISA from the peritoneal exudates. Male Balb/c mice were used at all tests with 6 mice per experimental group. Ethanol 10% (v/v) was the vehicle (VH). The statistical comparisons between groups were made using analysis of variance (ANOVA) followed by the Bonferroni’s or Dunnett test. Values were considered significance when p<0.05. BBF showed antinociceptive activity in the writhing tested induced by acetic acid (25 mg/kg – 14.20±30.46; VH 38±17.25) in the test hypernociception induced by formalin (12.5, 25, 50 and 100 mg/kg), and the von Frey test dose (25 mg/kg – 2.05±1.75; VH 9.02±1.40). The BBF showed anti-inflammatory activity by inhibiting the increase in vascular permeability (25mg/kg – 3.66±0.77; VH 7±1.96) and neutrophil migration (12.5, 25 and 50 mg/kg) in carrageenan-induced peritonitis. Higher levels of IL-10 were obtained with the BBF 12.5 mg/kg, and reduction of IL- 1β by BBF 12.5 and 25 mg/kg. Conclusion: The results suggest that the ethanolic extract of the bark of Phanera flexuosa (Caesalpiniaceae) has antinociceptive and anti-inflammatory activities, however, further studies should be conducted to elucidate the mechanisms involved and the possible bioactive compounds. Financial support: PIBIC/CNPq, CNPq Universal 2008/2010; Decit/SCTIE/MS (CNPq/FAPESB/SESAB); CAPES.
ANTI-INFLAMMATORY AND ANTINOCICEPTIVE EFFECTS OF A CARVACROL-DERIVED MONOTERPENE
RANGEL RODRIGUES BOMFIM1; IGOR OLIVEIRA PAIVA-SOUZA2; DENYSON SANTOS PEREIRA3; JANAINA PAREIRA MORAIS4; DAMIÃO PERGENTINO SOUZA5; ENILTON APARECIDO CAMARGO6.
1,2,3,4,6.FEDERAL UNIVERSITY OF SERGIPE, SÃO CRISTÓVÃO - SE - BRASIL; 5.FEDERAL UNIVERSITY OF PARAÍBA, JOÃO PESSOA - PB - BRASIL.
Introduction: Isopropoxy-carvacrol (IPC) is a new synthetic derivative, which was obtained from carvacrol and its pharmacological properties have not yet been investigated. The aim of this study was to analyze the antinociceptive and anti-inflammatory effects of IPC.
Methods and Results: Mice (25-30 g) and rats (150-230 g) were used in this study. Experiments were approved by the Instituition´s Ethics Committee (09/11). Animals were pre-treated (i.p.) with IPC at the doses of 10, 30 or 100 mg/kg or vehicle (Tween 80, 0.5%), 30 min before the injection of the phlogistic agents. The first phase of formalin (2%, 20 mL)- induced nociception was inhibited by IPC at the dose of 100 mg/kg (34±4 s, P<0.05), when compared with the vehicle- treated group (62±6 s). In the second phase, the pre-treatment with 30 or 100 mg/kg also reduced the formalin effects (56±22 s, P<0.05; 13±8 s, respectively, P<0.001), when compared with the vehicle-treated group (141±26 s). Injection of carrageenan (CAR, 3%, 20 mL) in mice paw reduced the threshold of intensity of stimulus of a magnitude of 6.6±0.3 g after 1 h and 6.7±0.3 g after 3 h. This was significantly reduced by 100 mg/kg of IPC (4.4±0.4 and 4.9±0.6 g, for 1 and 3 h respectively, P<0.05). The area under curve (0-4 h) of paw edema induced by injection of CAR (0.5%, 100 mL) in rats treated with vehicle (2.9±0.4 mL.h) was significantly diminished by the administration of IPC at the dose of 100 mg/kg (1.6±0.3 mL.h , P<0.01) or dexamethasone (1.4±0.1 mL.h, P<0.001), when compared with the vehicle group (2.9±0.4 mL.h). Mice ear edema or myeloperidase (MPO) activity induced by 1 mg/ear of TPA was measured in the presence of 0.3-3 mg/ear of IPC or vehicle (acetone), applied topically during the induction. IPC decreased the MPO activity in the ears (119±12, 69±6 and 118±14 UMPO/site, respectively for 0.3, 1 and 3 mg of IPC/ear, P<0.001), when compared with the vehicle group (229±12 UMPO/site).
Conclusion: IPC has anti-inflammatory and antinociceptive activities, suggesting that it can represents an alternative to develop new future therapeutic strategies.
ANTI-INFLAMMATORY AND ANTIPLATELET ACTIVITIES OF POLYSACCHARIDES FROM GENIPA AMERICANA LEAVES
JULIANA DA COSTA MADEIRA1; FRANCISCO DIEGO DA SILVA CHAGAS2; RACQUEL OLIVEIRA DA SILVA3; PEDRO HENRIQUE FERREIRA DE SOUZA BRINGEL4; ANA MARIA SAMPAIO ASSREUY5; MARIA GONÇALVES PEREIRA6.
1,3,4,5,6.INSTITUTO SUPERIOR DE CIÊNCIAS BIOMÉDICAS (ISCB), UNIVERSIDADE ESTADUAL DO CEARÁ - CE - BRASIL; 2.FACULDADE DE EDUCAÇÃO, CIÊNCIAS E LETRAS DO SERTÃO CENTRAL (FECLESC), UNIVERSIDADE ESTADUAL DO CEARÁ - CE - BRASIL.
Introduction: Polysaccharides of higher plants have shown immunomodulatory, anticoagulant and antiplatelet activities. Although hemostasis and inflammation are interrelated processes, few studies had described the modulatory effect of these molecules in both activities simultaneously. Here it was investigated the effect of polysaccharide fractions isolated from Genipa americana leaves in the model of rat peritonitis induced by zymosan and in the in vitro protocol of human platelet aggregation induced by Adenosine Diphosphate-ADP. Methods and Results: Total polysaccharides were fractionated by ion exchange chromatography on DEAE-cellulose. Wistar rats (150-200 g) received zymosan (1 mg/kg, intraperitoneal-i.p.) and were euthanized 4 h later for total and differential leukocyte counts of peritoneal fluid, protein content and aggregation 30 min before stimulation with polysaccharide fractions-PL-f 0.1 M or 0.25 M (1 mg/kg, intravenous-i.v.), acetylsalicylic acid-ASA (10 mg/kg, i.p.) or saline (100 ul; i.v.). Human Platelet Rich Plasma-PRP, previously incubated with PL-f (10 ul-0.01, 0.05, 0.1, 0.15 and 0.2 mg/ml) or with peritoneal fluid (10 ul) from rats pre-treated with polysaccharide fractions was activated with ADP (3 uM). All protocols were approved by the UECE Ethical Committee (n° 09553548-7/13). Data was expressed as Mean ± SEM (n=5). ANOVA and Bonferroni's test (p<0.05). PL-f 0.1 M, but not PL-f 0.25 M, reduced either total leukocyte (3120 ± 349) or neutrophil (2013 ± 309) migration induced by zymosan by 73% (total leukocytes: 820 ± 215) and 85% (neutrophils: 316 ± 142). ASA (positive control) reduced total leukocyte migration by 43% (1668 ± 187) and neutrophil by 48% (1034 ± 215). Neither PL-f nor ASA altered the increase of protein extravasation elicited by zymosan. PL-f 0.1 M, but not PL-f 0.25 M, reduced the ADP-induced direct aggregation by 36%, 48% and 38% at 0.05, 0.1 and 0.2 mg/ml, respectively. Additionally, the ADP-aggregating activity was inhibited about 31% and 48% by pre-incubation with peritoneal fluid of animals treated with ASA or PL-f 0.1 M, respectively. Conclusion: PL-f 0.1 M from leaves of G. americana inhibits neutrophil migration induced by zymosan and aggregation induced by ADP. The inhibitory activity on neutrophil migration and platelet aggregation could be associated with its anti-aggregating activity.
Financial Support: CAPES and FUNCAP.
ANTI-INFLAMMATORY AND ANXIOLYTIC EFFECTS HERISSANTIA TIUBAE (K. SCHUM) BRIZICKY (MALVACEAE) IN EXPERIMENTAL MODEL OF ASTHMA.
TALISSA MOZZINI MOZZINI MONTEIRO1; WEMERSON NEVES MATIAS2; MARIA DE FÁTIMA VANDERLEI DE SOUZA3; HERMANN FERREIRA COSTA4; PAULA REGINA RODRIGUES SALGADO5; MIRIAN STIEBBE SALVADORI6; EUGENE NALIVALKO7; MARCIA REGINA PIUVEZAM8.
1,2,3,4,5,6,8.UFPB, JOÃO PESSOA - PB - BRASIL; 7.SCHOOL OF BIOMEDICAL SCIENCES AND FARMACY, NEW SOUTH WALES - AUSTRÁLIA.
Introduction: Asthma is frequently associated with anxiety. Ideal anti-asthmatic drug should combine anti- inflammatory and anxiolytic properties, without having side effects of currently used anti-asthmatics and anxyolytics. Our preliminary data suggested that Herissantia tiubae (K. Schum) Brizicky (Malvaceae), used in folk medicine for treating fever and respiratory diseases, could possess such combination. Objective: to determine whether Herissantia tiubae extract (EHt) has anti-inflammatory and anxiolytic effects in a mice model of asthma. Methods: Ovalbumin (OVA)-sensitized BALB;c mice were treated with EHt (200, 100 and 50 mg/kg) or diazepam 1 h before each OVA challenge. After the last challenge, animals were subjected to anxiety test (elevated plus-maze), and respiratory rate was measured by means of whole-body plethysmography. Following euthanasia, bronchoalveolar lavage (BAL) fluid and mediastinal lymph node were analyzed for immune cell infiltration and IgE serum levels. Results: OVA-sensitized and EHt-treated (50 mg/kg) animals showed significant (p<0.05) reduction on absolute cell counts of inflammatory cells into BAL and also reduction of the amount of OVA-specific IgE titer as compared with non-treated OVA- sensitized animals (titer 1:512 and 1:1024 respectively). EHt treatment did not increased the amount of Treg cells in the mediastinal lymph nodes. In the elevated pluz-maze test, Ova-sensitized mice scored significantly higher on anxiety indices compared to non-sensitized mice. Diazepam 1 mg/kg i.p. reverted this behavioral changes. EHt (50 mg/kg) anxiolytic effect was comparable to diazepam. Higher dose of ETh (200 mg/kg) also reduced mean respiratory rate, suggesting potential sedative effect. Conclusions: Herissantia tiubae extract (EHt) possess both anti- inflammatory and anxiolytic properties.
Financial Support: CNPq/CAPES/INCT Cancer
ANTI-INFLAMMATORY AND HEPATOPROTECTIVE EFFECTS OF LPSF/GQ-02 ON NONALCOHOLIC FATTY LIVER DISEASE
AMANDA KAROLINA SOARES E SILVA1; MARIA GABRIELA DUQUE SPINOLA2; FABIANA OLIVEIRA DOS SANTOS GOMES3; AMANDA COSTA OLIVEIRA4; SHYRLENE MEIRY DA ROCHA ARAÚJO5; EDLENE LIMA RIBEIRO6; LAISE ALINE MARTINS DOS SANTOS7; BRUNA SANTOS SILVA8; CHRISTINA ALVES PEIXOTO9.
1,2,3,4,5,6,7,8.UNIVERSIDADE FEDERAL DE PERNAMBUCO – UFPE, RECIFE - PE - BRASIL; 9.CENTRO DE PESQUISAS AGGEU MAGALHÃES- CPQAM/FIOCRUZ, RECIFE - PE - BRASIL.
Introduction: Nonalcoholic fatty liver disease (NAFLD) defines a wide spectrum of liver diseases that extend from simple steatosis to nonalcoholic steatohepatitis. Although the pathogenesis of NAFLD remains undefined, it is recognized that insulin resistance is present in almost all patients who develop this disease. The Thiazolidinediones act as an insulin sensitizer and have been used in the treatment of patients with type 2 diabetes and other insulin- resistant conditions, including NAFLD. Hence, therapy of NAFLD with insulin-sensitizing drugs should ideally improve the key hepatic histological changes, but should also reduce cardiometabolic and cancer risk. The aim of our study was to evaluate the therapeutic effects of LPSF/GQ-02 on the liver the LDLR-/- mice after a diet rich in fat. Methods and Results: 40 male mice were divided into four groups: 1-received a standard diet, 2-fed with high-fat diet (HFD), 3–HFD+pioglitazone (20 mg/kg/day), 4–HFD+LPSF/GQ-02 (30mg/kg/day). The experiments were conducted for 12 weeks and in the last four weeks the drugs were administered daily by gavage. The liver was processed for optical microscopy, Oil Red O, Sirius red, immunohistochemistry IL-6, iNOS and western blotting for IκBα. The control group showed well-preserved tissue with low lipid content, collagen and low reactivity for IL-6, iNOS. The HFD group presented steatosis, inflammatory infiltrates, collagen deposition and a high reactivity for IL-6 and iNOS. The pioglitazone group was ineffective in reversing liver damage caused by high-fat diet. However, treatment with LPSF/GQ-02 improved organization of liver tissue with a significant decrease of lipid inclusions, collagen deposition and low reactivity for iNOS and IL-6. The western blotting analysis presented a significant increase in protein IkBα the group treated with LPSF/GQ-02 compared with the HFD group, indicating that LPSF/GQ-02 possibly acts on inflammation by inhibiting the activation of NFkB through its regulatory protein IκBα. Conclusion: This study showed that pioglitazone, a drug used to treat type 2 diabetes mellitus was unable to reverse the condition caused by the diet rich in fat. However LPSF/GQ-02, showed to be effective in reducing the accumulation of lipid and collagen, iNOS, IL- 6, also increasing the protein IkBα, suggesting a direct mechanism of action on the factors that influence inflammation and fat accumulation in the liver of LDLR-/- mice.
Financial support: CNPq and FACEPE.
ANTI-INFLAMMATORY AND IMMUNOMODULATORY PROPERTIES OF MONTANINE, AN ALKALOID ISOLATED FROM RHODOPHIALA BIFIDA
MIRIAN FARINON; VANESSA SCHUCK CLARIMUNDO; GRAZIELE PEREIRA RAMOS PEDRAZZA; MARCELI VILAVERDE DIELLO; JOSÉ ANGELO SILVEIRA ZUANAZZI; RICARDO MACHADO XAVIER; PATRÍCIA GNIESLAW DE OLIVEIRA.
UNIVERSIDADE FEDERAL DO RIO GRANDE DO SUL, PORTO ALEGRE - RS - BRASIL.
Introduction: Montanine (M) is an alkaloid isolated from Rhodophiala bifida, a plant used in folk medicine and never before tested in inflammatory diseases. Based on this, we evaluate the effect of M in vitro on lymphocytes and fibroblasts-like synoviocyte (FLS) and as an in vivo anti-inflammatory therapy in Antigen-induced Arthritis (AIA) and Collagen-induced Arthritis (CIA) models.
Methods and results: Male BALB/c mice had AIA induced with antigen methylated bovine serum albumin (mBSA) and were divided into 4 groups (n=6): vehicle (saline) and M (0.3; 1 and 3mg/kg - twice a day). Treatment started one day before intraarticular (ia) injection (ij) of mBSA. Was evaluated: paw nociception in 0, 3, 5 and 24h and leukocytes migration into the knee joints 24h after ia ij of mBSA. Male DBA/1J mice had CIA induced with bovine collagen type II and were divided into 3 groups (n=7): vehicle (saline) and M (0.5 and 1.5mg/kg – twice a day/10 days). Mice were monitored daily for signs of arthritis and treatment started after onset of disease. Was evaluated: articular score, paw nociception and histophatology of ankle joints and liver. Lymphocyte and FLS was treated with M 0.01-100mM for 24h viability. M 1mM was chosen for lymphocyte proliferation with ConA and LPS (n=7). FLS invasion in 24h was assayed in Matrigel kit and treated M 1mM (n=3). Statistical analysis: ANOVA or t-test. AIA: M inhibited leukocytes migration at doses 0.3; 1 and 3mg/kg (8.04±1.65x104, 4.16±0.99x104, 4.15±1.46x104 leukocytes/cavity, respectively) compared with vehicle (43.5±9.73x104 leukocytes/cavity) (P<0.01). Nociception was reduced compared with vehicle in all doses at 5 and 24h (P<0.01). CIA: M was not hepatotoxic. Compared with vehicle, M 0.5mg/kg ameliorates the articular score from day 3 to the end (P<0.01), reduced nociception at days 2 and 10 (P<0.05) and improved histophatological parameters (P<0.03). M 1.5mg/kg was similar to vehicle. In vitro, treatment was not toxic, maintaining cellular viability. M inhibited lymphocyte proliferation stimulated with ConA in 54.78% (P<0.01), although it had no effect on LPS stimulation. M 1mM decreased the number of invasive FLS in 48.6% (P=0.006).
Conclusion: M improve experimental arthritis attenuating joint damage and nociception in both models. Moreover, M decreased lymphocytes proliferation and fibroblasts invasion. Its indicate M as a potential anti-inflammatory treatment for immune-mediated arthritis.
Support: CAPES, FAPERGS, FIPE/HCPA.
ANTI-INFLAMMATORY EFFECT OF FRUCTOSE 1,6-BISPHOSPHATE IN EXPERIMENTAL MODEL OF ARTHRITIS
FLAVIO PROTASIO VERAS; RAPHAEL SANCHES PERES; LARISSA GARCIA PINTO; ANDRÉ LUIS LOPES SARAIVA; FERNANDO QUEIROZ CUNHA; JOSÉ CARLOS FARIAS ALVES FILHO.
UNIVERSIDADE DE SAO PAULO, RIBEIRAO PRETO - SP - BRASIL.
INTRODUCTION: Rheumatoid arthritis (RA) is an autoimune disease caracterized by a crohnic inflammation of the joints. The understanding of pathophysiology of RA is important for the development of new drugs to treat the disease. Taking acount the anti-inflammatory effects mediated by fructose 1,6-bisphosphate (F1,6BP), an intermediate of glycolysis, the aim of our study was to evaluate the effect of F1,6BP on antigen-induced arthritis (AIA) and investigate the possible mechanism involved in this phenomenon.
METHODS AND RESULTS: mBSA C57BL/6 immunized mice (n=5 per group) were treated with F1,6BP (10, 30 and 100 mg/Kg) 24 h and 30 min before the challenge with mBSA (30 µg/cavity). After 7h of the challenge, we evaluated neutrophil migration, articular hyperalgesia and cytokines of the joints by ELISA. To investigate the possible anti- inflammatory mechanism mediated by F1,6BP, we treated mBSA C57BL/6 immmunized mice with adenosine 2a receptor (A2aR) antagonist (1 mg/kg) three times per week during the immunization protocol. The treatment with F1,6BP reduced the development of arthritis, evidenced by a decrease of neutrophil migration (2.440 ± 1.410) and articular hyperalgesia (6.600 ± 0.5799) . Additionally, we observed a decrease of pro-inflammatory cytokines, such as TNF-α (3.341 ± 0.7145)), IL-17 (83.440 ± 8.217), KC (783.300 ± 65.730) and IFN-γ (110.900 ± 22.060); and an increase of anti-inflammatory cytokine IL-10 (269.300 ± 39.340) when compared to untreated mice. However, the effect of F1,6BP was abolished when mice were treated with A2aR antagonist. All animal care and experimental procedures were in accordance with the Ethics Committee.
CONCLUSION: These findings show that F1,6BP exhibits anti-inflammatory effect in experimental arthritis by an adenosine-dependent mechanism via activation of A2a receptor.
FINANCIAL SUPPORT: CAPES, FAPESP
ANTI-INFLAMMATORY EFFECT OF LIDOCAINE ON SILICA-INDUCED LUNG INFLAMMATION IN MICE
LIVIA LACERDA MARIANO; GRAZIELE GUEDES MONTEIRO; BIANCA TORRES CIAMBARELLA; TATIANA PAULA TEIXEIRA FERREIRA; MARCO AURELIO MARTINS; PATRICIA MACHADO RODRIGUES E SILVA.
OSWALDO CRUZ INSTITUTE-FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL.
1LIVIA LACERDA MARIANO; GRAZIELE GUEDES MONTEIRO; BIANCA TORRES CIAMBARELLA; TATIANA PAULA TEIXEIRA FERREIRA, MARCO AURÉLIO MARTINS, PATRICIA MACHADO RODIRGUES E SILVA1; (1).LABORATORY OF INFLAMMATION, OSWALDO CRUZ INSTITUTE-FIOCRUZ, RJ, BRAZIL.
Introduction: Silicosis is an occupational disease caused by crystalline silica particles inhalation, being characterized by an inflammatory process followed by an intense granulomatous fibrosis. Lidocaine is a local anaesthetic, which has significant anti-inflammatory properties as attested in the case of allergic diseases. In this study, we investigated the potential effect of lidocaine in a murine model of experimental silicosis. Methods and Results: Male Swiss-Webster mice were injected intranasally with silica particles (10 mg) and analyses made 8 and 28 post-silica. Lidocaine (1%) was nebulized daily, for 7 consecutive days according to two different protocols: 1) starting 6 h or 2) 21 days post- silica. Inflammatory parameters included morphology/ morphometry, leukocyte infiltration (H&E) and tissue collagen content (Sircol method). Cytokine and chemokine quantification was made by ELISA. Lung function was measured by invasive plethysmography (Buxco System, UK), in the absence or presence of methacholine. Silicotic mice showed an intense inflammatory infiltration, increase of the collagen deposition and granuloma formation. Levels of cytokines (TNFα, IFNγ) and chemokines (MIP-2, KC) were also detected at higher levels as compared to controls. Silicotic mice presented increased lung resistance and elastance as well as airways hyperreactivity to methacholine. In protocol 1, lidocaine inhibited lung alterations and granulomatous fibrosis. Values of granuloma area reduced from 14.83% ± 1.54% to 3.0 ± 0.95% (mean ± SEM; n=7, p<0.05), in silica and silica treated-mice, respectively. Cytokine and chemokine production was sensitive to lidocaine. No alteration was noted when lidocaine was given as protocol 2. Additionally, we observed that in in vitro system of macrophage activation by silica particles, treatment with lidocaine (0.1 μM – 1mM) reduced the release of nitric oxide, suggesting that macrophages can be crucial in silicosis. Conclusion: Our findings show that treatment with nebulized lidocaine although able to inhibit silicosis installation, in association with its anti-inflammatory properties, does not show clinical benefits for those patients in which silicosis has already been established.
Financial support: FIOCRUZ, PDTIS, CNPq, FAPERJ (Brazil).
ANTI-INFLAMMATORY EFFECT OF METHANOLIC EXTRATC OF CAULERPA MEXICANA IN MURINE MODEL OF NON-SEPTIC SHOCK INDUCED BY ZYMOSAN.
CÁSSIO RICARDO DE MEDEIROS SOUZA1; JÉSSICA TEIXEIRA JALES2; ALESSANDRA MARINHO MIRANDA3; HYLARINA MONTENEGRO DINIZ SILVA4; HANNALY WANA BEZERRA PEREIRA5; BÁRBARA VIVIANA OLIVEIRA SANTOS6; JANEUSA TRINDADE DE SOUTO7.
1,2,3,4,5,7.UFRN, NATAL - RN - BRASIL; 6.UFPB, JOÃO PESSOA - PB - BRASIL.
Introduction: Previous studies from our laboratory with Methanolic Extract (ME) of the green seaweed Caulerpa mexicana showed its high anti-inflammatory potential. The aim of this study was to understand more about this potential anti-inflammatory effect of C. mexicana, using its ME in a model of non-septic shock induced by zymosan. Methods and results: BALB/c male mice, 6 to 8 weeks old, received intraperitonially 1 g/kg of zymosan as non-septic shock inductor. To evaluate the effect of the ME of C. mexicana in this kind of shock, the animals were orally treated with different doses of the ME: 40, 4 and 0.4 mg/kg of animal. The treatments happened one hour before and 6 hours after stimulation with zymosan. The experiment was finalized 18 hr after zymosan inoculation. Every one hour after the inoculum of zymosan, the presence of clinical signs characteristic of the shock condition were evaluated: the bristling, prostration, diarrhea, mucous exudates in ocular and anal cavities. After 18 h, the animals were euthanized, the serum was collected for IL-6 determination, the peritoneal lavage was realized for measurement of IL-6 and total cellularity and liver, spleen, kidney and intestine were collected to investigate the weight, length (intestine) and appearance of organs. The treatment with ME of C. mexicana was able to significantly reduce the levels of IL-6 in the serum and peritoneal lavage supernatant and also reduced cell migration to the peritoneal cavity up to 67.55%. The clinical signs showed reduction up to 47.7% in the groups treated with the extract. Liver, spleen and kidneys showed a reduction in weight up to 13.21%, 9.98% and 12.31%, respectively. It was also observed a significant reduction in ceccum edema and 14.2% of reduction in the intestine length of treated animals. Conclusion: The treatment of mice with methanolic extract of C. mexicana presented promising results in reducing theIL-6 levels in serum and supernatants of peritoneal lavage, also reducing cell migration to the peritoneal cavity and tissue damage in the model of non-septic shock induced by zymosan.
ANTI-INFLAMMATORY EFFECT OF P-COUMARIC ACID ON LPS-INDUCED AIRWAY INFLAMMATION
GENILDA CASTRO NETA; JÉSU COSTA JÚNIOR; LAÍS COSTA AGRA; MARVIN PAULO LINS; LUCIA MARIA CONSERVA; EMILIANO OLIVEIRA BARRETO.
UNIVERSIDADE FEDERAL DE ALAGOAS, MACEIÓ - AL - BRASIL.
Introduction: p-Coumaric acid (CA) is a phenolic compound present in fruits and vegetables with a wide range of pharmacological activities, including antioxidant effects. However, their effects on acute airway inflammation are unknown. The aim of this work was to evaluate the effects of CA in a murine model of inflammation acute lung.
Methods and Results: Swiss male mice (n=5) were treated intraperitoneally with CA 1h before intranasal stimuli with lipopolysaccharide (LPS). 6h after instillation, the bronchoalveolar lavage (BAL) was collected to evaluate cellular infiltration in airway lumen. The reactive oxygen species (ROS) content in cells from BAL was measured by DCFH fluorescence assay. The in vitro assays were performed to evaluate the leukocytes adhesion on endothelial cells (tEnd.1) and expression of VLA-5 on leukocytes was determined by flow cytometry. Data were expressed as mean±SEM and were statistically analyzed using Student’s t test or one-way ANOVA. Mice exposed to LPS exhibited a marked increasing number of total leukocytes (from 7.6±0.5 to 39.5±8.5 x105 cells/BAL) in BAL mainly characterized by neutrophils (from 1.3±0.5 to 36.7±8.1 x105 cells/BAL) as compared to the vehicle-stimulated mice.CA, only at dose 10 mg/kg, attenuated significantly the total leukocyte (to 18.2±3.8 x105 cells/BAL) and neutrophil count (to 14.8±3.6 x105 cells/BAL). The cells of the BAL from the LPS-stimulated mice exhibited a ROS content 23-fold higher of than compared to cells from vehicle-stimulated mice, while mice treated with CA (10 mg/kg) and LPS-stimulated showed a reduction about to 59% on ROS content. CA (1 and 10 mg/kg) was unable to induce changes in VLA-5 expression on total leukocytes of the BAL after LPS-stimulation. In addition, in vitro pre-treatment with CA (50 µM and 100 µM) failed to inhibit leukocytes adhesion on the LPS-primed endothelial cells.
Conclusion: Thus, these results suggest that antioxidant activity of p-coumaric acid may contribute with the reduction the leukocytes recruitment in acute lung inflammation.
Financial support: CNPq, CAPES.
ANTI-INFLAMMATORY EFFECT OF PROTEIN FRACTION FROM COMBRETUM LEPROSUM MART. LEAVES WITH HIGH LECTIN ACTIVITY
JOSÉ CARLOS DA SILVEIRA PEREIRA; ROSUETI DIÓGENES DE OLIVEIRA FILHO; MAGDA LORENA TURBANO DOS SANTOS; JOÃO RONIELLY CAMPELO ARAÚJO; CARLOS CAMPOS CÂMARA; MICHELE DALVINA CORREIA DA SILVA.
UNIVERSIDADE FEDERAL RURAL DO SEMI-ÁRIDO, MOSSORÓ - RN - BRASIL.
Introduction: Alcoholic and hydro-alcoholic extracts from Combretum leprosum are evaluated in many studies exploring pharmacological properties, but few analyses of aqueous extracts are performed. Lectins are proteins that bind reversibly to carbohydrate through specific binding sites. Some lectins are described as anti-inflammatory. This work evaluated anti-edematous effect of protein fraction with high lectin activity obtained from C. leprosum aqueous extract.
Methods and with high ng thcaltion of let),Results: Leaf powder extract in 0.15 M NaCl was submitted to protein precipitations with ammonium sulfate (30%, 30-60% and 60-90% saturations). Protein fractions obtained after centrifugation were dialyzed and submitted to protein quantification and hemagglutinating activity (HA) assay that reveals lectin activity. Paw edema test was performed using Wistar rats (n=6/group). Protein fraction dialyzed that showed highest HA (30-60% salt saturation) named F2 was evaluated. F2 (10, 30 or 100mg/kg) or 0.15 M NaCl (control group) was administered intraperitoneally 30 minutes before carrageenan administration intraplantar (1mg/ml solution, 100μl/paw). Volume of paws was measured before (initial) and after injection of carrageenan (1, 3 and 6 hours) using an alternative plethysmometer. After experiments animals were anesthetized and euthanized by exsanguination. Results were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Bartllet) both to 5% and were approved. Statistical significance was evaluated by ANOVA (p <0.05) followed by Tukey’s test revealing significant difference between control and treated groups with 30 or 100mg/kg at the 6th hour after inflammatory stimulus that showed reduction of edema (values in mean±SEM) from 0.4281±0.0559 to 0.1505±0.0374 and 0.0651±0.0483, representing 64% and 84% reduction, respectively.
Conclusion: Protein fractions revealed high protein content and HA characterizing C. leprosum as excellent source for potential isolation of active lectins. F2 revealed anti-edematous effect significant statistically. Further investigations will be performed to better characterize protein content of F2 as well as its effect on inflammation.
Financial support: CNPq
ANTI-INFLAMMATORY EFFECT OF THE SULFATED POLYSACCHARIDE EXTRACTED FROM THE GRACILARIA BIRDIAE IN CROHN'S EXPERIMENTAL MODEL INDUCED BY TNBS.
JOSÉ SIMIÃO SIMIÃO DA CRUZ JÚNIOR1; JOSÉ PATRIOTINO REBELO PIRES NETO2; JALLES ARRUDA BATISTA3; TARCISIO VIEIRA BRITO4; RAFAEL SILVA PRUDÊNCIO5; RENAN OLIVEIRA SILVA6; RONALDO A. RIBEIRO7; MARCELLUS HENRIQUE LOIOLA PONTE SOUZA8; LUCIANO SOUSA CHAVES9; ANA LUCIA PONTE FREITAS10; JAND-VENES ROLIM MEDEIROS11; ANDRÉ LUIZ REIS BARBOSA12.
1,2,3,4,5,6,11,12.FEDERAL UNIVERSITY OF PIAUI, PARNAIBA - PI - BRASIL; 7,8,9,10.FEDERAL UNIVERSITY OF CEARA, FORTALEZA - CE - BRASIL.
JOSÉ SIMIÃO DA CRUZ JÚNIORA, JOSÉ PATRIOTINO REBELO PIRES NETOA, JALLES ARRUDA BATISTAA, TARCISIO VIEIRA BRITOA, RAFAEL SILVA PRUDÊNCIOA, RENAN OLIVEIRA SILVAA, RONALDO A. RIBEIROB, MARCELLUS HENRIQUE LOIOLA PONTE SOUZAB, LUCIANO SOUSA CHAVESC, ANA LUCIA PONTE FREITASC, JAND-VENES ROLIM MEDEIROSA, ANDRÉ LUIZ REIS BARBOSAA.
ALAFFEX – Laboratory of Experimental Physiopharmacology, Biotechnology and Biodiversity Center Research (BIOTEC), Federal University of Piauí.
BLAFICA – Laboratory of Pharmacology of Inflammation and Cancer, Department of Physiology and Pharmacology, Federal University of Ceará.
CLaboratory of Proteins and Carbohydrates of Marine Algae, Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, CE, Brazil.
INTRODUCTION: Inflammatory bowel disease (IBD) comprises two chronic bowel disorders: Crohn's disease (CD) and ulcerative colitis (UC), both are responsible for inflammation, leaving the intestine red, swollen and often with ulcers. Knowing in advance that the TNBS-induced colitis leads to inflammation of the intestinal mucosa causing alterations in the functioning of the organ, it becomes necessary check the possible anti-inflammatory activity of sulfated polysaccharide extracted from red seaweed Gracilaria birdiae on TNBS colitis. METHODS: TNBS-induces colitis by intracolonic instillation of a solution of 20 mg of TNBS in 50% EtOH (ethanol) in rats (n=6). Control groups received an equivalent volume of saline with 50% EtOH (ethanol). In the experiments involving TNBS-induced colitis, rats were treated with polysaccharide extracted from Gracilaria Birdiae (PLS: 30, 60, and 90 mg.kg-1, 500μl p.o.) and dexamethasone (1 mg·kg, s.c., positive control).The rats were killed on the third day and the abdomens were then opened, and the portion of distal colon was excised for the evaluation of macroscopic (Morris et al, 1989) and microscopes scores (Neurath et al.1995) and verification of wet weight of colons. Adding to this, samples of intestine were removed for the measurement of MDA and MPO concentration. RESULTS: The PLS (90mg.kg-1) significantly decreased the concentration of gastric mucosal MDA (23.95 ± 4.72 nmol/g of tissue), MPO (2.57 ± 1.41 umpo/mg of tissue) compared to TNBS group (MDA: 162.5 ± 25.63 nmol/g of tissue; MPO: 10.03 ± 2.55 umpo/mg of tissue). The PLS 90 also showed reduced scores macroscopic of lesion (2.8 ± 0.48 scores of lesion) and wet weight of samples of the colon (0.55 ± 0.052 mg of tissue) when compared this group with TNBS group (macroscopic scores: 18.00 ± 2.25; wet weight: 1.03 ± 0.03 mg of tissue). Thus, the analysis of the macroscopic scores, wet weight of colon, MDA and MPO findings revealed an excellent correlation, confirming the efficacy of the compound. CONCLUSION: This study showed that PLS 90 has anti-inflammatory activity for decreasing considerably criteria macroscopic, wet weight, MPO and MDA compared to the TNBS group.
ANTI-INFLAMMATORY EFFECTS OF ANACARDIUM OCCIDENTALE L. ETHYL ACETATE PHASE ARE RELATED TO IL-1 AND TNF-ALPHA
MARINA SUÊNIA ARAÚJO VILAR1; JACQUELINE ALVES LEITE2; ANNE KALIERY ABREU ALVES3; TÚLIO FLÁVIO ACCIOLY LIMA MOURA4; JOSÉ MARIA BARBOSA FILHO5; SANDRA RODRIGUES MASCARENHAS6.
1,2,3,5,6.UNIVERSIDADE FEDERAL DA PARAÍBA, JOÃO PESSOA - PB - BRASIL; 4.UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE, NATAL - RN - BRASIL.
Introduction: Anacardium occidentale L. (A. occidentale L.) is characterized by the presence of tannins in the bark, leaves, peduncle fruitful and chestnut husk. Studies describe various biological activities of this plant, as hypoglycemic, antimicrobial, antioxidant, anti-ulcer, anti-ophidian and anti-leishmania, but the possible role of A. occidentale L. in the inflammation had not been addressed previously Main: Evaluate A. occidentale L. ethyl acetate (AcEt) phase activity in experimental models of acute inflammation. Methods: Swiss mice (n=6) were treated with saline, carrageenan (2,5%), indomethacin (10 mg/Kg) in the presence of different AcEt phases concentrations (12,5 mg/Kg, 25 mg/Kg, 50 mg/Kg, 100mg/Kg). Paw edema was measured at 1, 2, 3, 4 and 6 hs after carrageenan administration. In addition to paw edema, four hours after challenge with i.p. carrageenan (500 µg/mL), peritoneal lavage (n=8) was collected undergoing total cell count and differential cell analysis by flow cytometry. Cytokines (IL-1, TNF-α, IL-6, IL-10) concentrations were assayed using ELISA kits (Bio Science). Results: Treatment with A. occidentale L AcEt phases at all concentrations used, reduced carrageenan-induced paw edema (P <0.05, P<0,01 and P<0.001). After induction of peritoneal inflammation, treatment with 50 mg/Kg and 100 mg/Kg A. occidentale L AcEt phases led to a 62% reduction in the neutrophil population in the peritoneal cavity. These concentrations also reduced IL-1 (P<0,01) and TNF-α (P<0,001) levels. Conclusion: A. occidentale L AcEt phases presented anti- inflammatory effecs, which might be related to IL-1 and TNF-α inhibition, but independent of IL-6 and IL-10 release. However, further studies are necessary to elucidate the mechanisms involved.
Financial support: CNPq and UFPB.
ANTI-INFLAMMATORY EFFECTS OF DIETHYLCARBAMAZINE ON LUNG INJURY INDUCED BY MONOCROTALINA IN MICE
EDLENE LIMA RIBEIRO1; INGRID TAVARES FAGROSO2; AMANDA KAROLINA SOARES SILVA3; MARIANA ARAGÃO MATOS DONATO4; FABIANA OLIVEIRA DOS SANTOS GOMES5; AMANDA COSTA OLIVEIRA6; BRUNA SANTOS SILVA7; CHRISTINA ALVES PEIXOTO8.
1,2,3,4,5,7.PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE - PE - BRASIL; 6.GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS, UNIVERSIDADE FEDERAL DE PERNAMBUCO, RECIFE - PE - BRASIL; 8.LABORATÓRIO DE ULTRAESTRUTURA, INSTITUTO AGGEU MAGALHÃES, FIOCRUZ, RECIFE - PE - BRASIL.
Background: Monocrotaline (MCT) is a pyrrolizidine alkaloid produced by Crotalaria genus, which causes liver damage, modest pulmonary fibrosis and immunotoxicity effects in animals and mainly employed to establish a model of pulmonary dysfunction. Clinical reports have described favorable results with the use of diethylcarbamazine (DEC) in bronchial asthma. This drug has an important anti-inflammatory role since it interferes with arachidonic acid metabolism. In the present study, we investigated the efficacy of oral DEC treatment in mice model of injury pulmonary induced monocrotaline.
Methods and Results: C57/BL6 male mice, were used in all experiments. MCT solution intraperitoneal injection
(600mg/kg) was administered once per week (7, 14 or 21 days). Six groups (n=10): control; MCT7; MCT14; MCT21,
MCT14/DEC14. (50mg/Kg per day of DEC from 1 to day 14), MCT21/DEC21 (from 1 to day 21). Bronchoalveolar lavage fluid were collected for imunoassay (Griess reaction) and lung tissues for light microscopy (H.E.), immunohistochemistry (COX-2, MCP-1, CD24, F4/80, IL-6, α smooth muscle actin and eNOS) and western blot (COX- 2, iNOS and NFkB). Pulmonary sections of the group control exhibited preserved morphological characteristics. MCT7 group revealed alveolar exudates, interstitial edema with thickening of the alveolar septae, perivascular edema, and inflammatory cellular Infiltrates; MCT14 group presented had an inflammatory infiltrate such as macrophages in plexiform lesion in the pulmonary arteries and emphysema; and MCT21 group showed a decrease of injury and the infiltration of PMNs. Nitrite and nitrate levels, were significantly increased in MCT7, MCT14 and MCT21 BALF comparing to the control group. In contrast, MCT14/DEC and MCT21/DEC groups showed significantly reduction of the nitrite and nitrate levels. MCT7, MCT14 and MCT21 groups revealed significant COX-2, MCP-1, CD24, F4/80, IL- 6, iNOS and NFkB higher expression, whereas these expression were significantly reduced after treatement with DEC (p<0,05). On the other hand, MCT7, MCT14 and MCT21 groups presented a reduced expression of eNOS and α smooth muscle actin , after treatment with DEC the expression of these proteins returned to normal levels (p<0,05).
Conclusion: In this study, treatment with DEC attenuated MCT-induced pulmonary dysfunction, reducing the lung damage .
Financial support: FACEPE and INBEB
ANTI-INFLAMMATORY EFFECTS OF DIETHYLCARBAMAZINE, AN ANTIFILARIAL DRUG, ON ACUTE LUNG INJURY INDUCED BY CARRAGEENAN IN MICE
LAISE ALINE MARTINS SANTOS; EDLENE LIMA RIBEIRO; KARLA PATRÍCIA SOUSA BARBOSA; INGRID TAVARES FRAGOSO; MARIANA ARAGÃO MATOS DONATO; BRUNA SANTOS SILVA; AMANDA KAROLINA SOARES SILVA; FABIANA OLIVEIRA SANTOS GOMES; SURA WANESSA SANTOS ROCHA; CHRISTINA ALVES PEIXOTO.
AGGEU MAGALHÃES RESEARCH CENTER (CPQAM/ FIOCRUZ), RECIFE - PE - BRASIL.
Introduction: Diethylcarbamazine citrate (DEC) is used in the treatment of lymphatic filariasis and Tropical Pulmonary Eosinophilia. New therapeutic possibilities have emerged for the use of this drug in Acute Lung inflammation (ALI). In the present study, we investigate the protective effect of DEC in an experimental model of acute inflammation, carrageenan-induced pleurisy. Methods and Results: Pleurisy was induced by an intra-thoracic injection of carrageenan (CAR, 1%.) in Swiss male mice (Mus musculus) pre-treated during three days by oral route with DEC (50mg/kg) or 0.9% isotonic saline (control group). The pleural exudates were collected 4h after CAR to evaluate leukocyte migration, myeloperoxidase activity (MPO) and nitric oxide (NO) production. Tissue samples from the lungs were processed for histological examination, immunohistochemistry (IL-1β, COX-2, iNOS) and Western blot analysis (COX-2, IL-1β, Phospho-IκB-α). All the experiments were realized under approval of Committee on Ethical Use of Laboratory Animals of Oswaldo Cruz Foundation (LW47/10). Each experimental group was performed with at least ten animals. Statistical analysis was realized using one-way analysis of variance (ANOVA) followed by a Tukey's post hoc tests, p-value < 0.05 were considered statistically significant. Injection of carrageenan elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavity that contained many polymorphonuclear leukocytes (PMNs), and increased production of NO. Our results demonstrate that DEC prevents cellular migration, and reduced MPO levels. DEC also inhibits NO production, and tissue expression of iNOS, COX-2 and IL-1β. Furthermore, treatment with DEC inhibits IκB-α phosphorylation, suggesting a possible inhibition of the NF-kB pathway. However more studies need to be performed in order to elucidate how DEC could inhibit this pathway. Conclusion: In this experimental model, our results suggest that treatment with DEC exerts a protective effect against pleurisy and offers a novel therapeutic approach for the management of acute lung injury.
Financial support: FACEPE
Disclosure of interest: None declared
ANTI-INFLAMMATORY EFFECTS OF N-ANTIPYRINE-3-CHLORO-4-4(FLUORANILINOMALEIMIDE) (NA-3CL-4F AM) IN MICE
GISLAINE FRANCIELI DA SILVA; FÁTIMA DE CAMPUS BUZZI; ROGERIO CORREA; VALDIR CECHINEL FILHO; NARA LINS MEIRA QUINTÃO.
UNIVALI, ITAJAI - SC - BRASIL.
GISLAINE FRANCIELI DA SILVA1; FÁTIMA DE CAMPOS BUZZI1,2; ROGÉRIO CORREA1,2; VALDIR CECHINEL FILHO1,2; NARA LINS MEIRA QUINTÃO1
1Post-Graduate Programe of Pharmaceutical Science, 2NIQFAR, CCS, Universidade do Vale do Itajaí, Santa Catarina, Brazil.
Introduction: Preliminary studies show that cyclic imides have an important anti-inflammatory and anti-hypersensitive activity in mice. A structural modification of the cyclic imide N-antipyrine-3, 4-dichloromaleimide (NA-3,4-DCM), produced the compound N-antipyrine-3-chloro-4-(4-fluoroanilinomaleimide) (NA-3Cl-4F AM). This study had the aim of evaluating the anti-inflammatory and anti-hypersensitive effects of the compound NA-3Cl-4F AM in paw-edema induced by carrageenan in mice. The IL-1β level and the neutrophil migration were also evaluated.
Methods and Results: Female Swiss mice (25-35g, N=6-8) were used. The animals were pretreated with ‘NA-3Cl-4F AM’ [70, 230 or 700 nmol/Kg, intraperitoneally (i.p.)] or saline. After 30 minutes, mice received an intradermal (i.d.) injection of carrageenan (300 µg/paw). The paw-edema was measured using a plethysmometer, and the difference between the right and left paws was indicative of edema. The mechanical hypersensitivity was evaluated using the von Frey Hair 0.6 g. The myeloperoxidase (MPO; indirect indicative of neutrophil migration) and IL-1β levels were evaluated in the hindpaw tissue collected 6 h and 4 h after the carrageenan injection. The compound was able to significantly reduce the paw-edema induced by carrageenan, with inhibition of 78± 1% for the dose of 700 nmol/kg [DI50 379 (356 - 403) nmol/kg]. The mechanical hypersensitivity was significantly inhibited, with inhibition of 74 ± 9%, and DI50 value of 0,1 (0,04 -0,24) nmol/kg for the dose of 700 nmol/kg. The pretreatment of mice with the compound also significantly reduced both IL-1β levels and MPO activity, with inhibition of 60 ± 3% and 50 ± 1%, respectively for dose of 700 nmol/kg.
Conclusion: These findings suggest that the compound NA-3Cl-4FAM presents important anti-inflammatory effects, once it was able to reduce the paw-edema induced by carrageenan in mice. The anti-hypersensitive effect is probably a consequence of the inflammation process reduction, related to the inhibition of neutrophil migration and consequently the reduction of IL-β release. Additional experiments are necessary to better delineate the mechanism of action of this compound, but it could be an interesting therapeutic tool for the treatment of inflammatory diseases.
FINANCIAL SUPPORT: CNPq, FAPESC-SC, ProPPEC/UNIVALI
ANTI-INFLAMMATORY POTENTIAL OF A POLYSACCHARIDE FRACTION OF THE GREEN MARINE ALGA CAULERPA RACEMOSA (FORSSKÅL) J. AGARDH
NATÁSSIA ALBUQUERQUE RIBEIRO; TICIANA MONTEIRO ABREU; ALEXIA NATHALIA BRIGIDO ASSEF; NORMA MARIA BARROS BENEVIDES.
UFC, FORTALEZA - CE - BRASIL.
Introduction: Marine algae are abundant sources of sulfated polysaccharides with various biological activities, thereby its biomolecules are great of commercial interest, especially in the pharmaceutical and food industries. In this study, we investigated the effects of the sulfated polysaccharide obtained from the green marine alga Caulerpa racemosa (CrII) on inflammation. Methods and Results: CrII (0.01, 0.1 and 1.0 mg/kg, i.v.) was evaluated for its potential anti- inflammatory using Wistar rats and all experiments were conducted only after approval by the ethics committee for animal research of the UFC. The data are presented as the mean± standard error (SEM) for six animals per group. Analysis of variance (ANOVA) was performed using Bonferroni’s test and Student’s t test for unpaired values. Values of p<0.05 were considered to be statistically significant. We used assays of cell migration into the peritoneal cavity induced by carrageenan (Cg - 700 mg/cavity), paw edema induced by Cg (700 µg/paw) or dextran (300 µg/paw). With respect to cell migration into the peritoneal cavity there was a significant reduction of neutrophils in all groups treated with the CrII. In the test paw edema induced by dextran or Cg, CrII was also able to significantly reduce the swelling at all doses used. The anti-inflammatory effect of CrII was confirmed by reducing the tissue levels of myeloperoxidase in the tissue of the Cg groups paws. In addition, we performed an enzyme inhibition assay Hemo-oxygenase-1 (HO-1), using the inhibitor ZnPP IX, in order to assess whether the anti-inflammatory effect of CrII was related to the expression of this enzyme. The results showed that the inhibition of the HO-1 is associated with inhibition of the anti- inflammatory response of CrII. Conclusion: Thus, CrII can be used as a tool for future investigations of processes related with inflammation associated with the hemoxigenase-1 pathway. This work was supported by Conselho Nacional de Desenvolvimento Científico e tecnológico (CNPq) and Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
ANTI-INFLAMMATORY, ANALGESIC AND ANTIOPHIDIC EFFECTS OF THE ESSENTIAL OIL FROM PIPER ALEYREANUM C.DC IN MICE
LEANDRO FLORES NASCIMENTO1; CATARINA NUCCI2; DANIELLA KARINE SOUZA LIMA3; FERNANDA ROCHA LAPA4; LAURO MERA SOUZA5; MARCELO LACOMINI6; VALDIR ALVES FACUNDO7; ADAIR ROBERTO SOARES SANTOS8.
1,7.FEDERAL UNIVERSITY OF RONDÔNIA, PORTO VELHO - RO - BRASIL; 2,3,4,8.FEDERAL UNIVERSITY OF SANTA CATARINA, FLORIANÓPOLIS - SC - BRASIL; 5,6.FEDERAL UNIVERSITY OF PARANÁ, CURITIBA - PR - BRASIL.
Introduction: Piper aleyreanum C.DC is a small tree belonging of the Piperaceae family and popularly known as “pimenta longa”, and is used in folk medicine as an analgesic, anti-inflammatory, gastroprotective, antidepressant and antidote for snake bite.
Objective: This study was designed to investigate the anti-inflammatory, analgesic, and antiophidic activities of the essential oils from the aerial parts of P. aleyreanum (EOPa) in mice.
Methods: Experiments were conducted using Swiss mice of both sexes (25–35 g) and all the procedures were approved by the CEUA (CEUA/UFSC protocol number PP00745).The anti-inflammatory, analgesic and antiophidic effects of orally administered of EOPa were evaluated in animals subjected to the Bothrops jararaca venom (Bjv, 1µg/paw) and pleurisy (1%, carrageenan) models. We also evaluated the effect of EOPa in temperature and oedema of the injected paw with Bjv and the locomotor activity the animals were performed in the open field test.
Results: The oral administration of EOPa(30–100 mg/kg, p.o.) given 1 h prior to carrageenan significantly decreased the total number of leukocytes (inhibition of 54±13%), including both neutrophils (inhibition of 66±10%) and mononuclear cells (inhibition of 60±8%), with mean ID50 values of 53.6, 21.7 and 43.5 mg/kg, respectively. In addition, the same doses of EOPa also reduced exudation (45±9% at dose of 3 mg/kg). Furthermore, EOpa (0.01-100 mg/kg, p.o.) significantly decreased the spontaneous pain (inhibition of 57±7%) and temperature (inhibition of 45±12%), but not paw edema induced by intraplantar injection of Bjv. In the open field model, EOPa did not show change in the locomotor activity.
Conclusions: These data show for the first time that EOPa has significant anti-inflammatory, analgesic and antiophidic actions in mice. Such findings are of interest because they support, at least partly, the use of Piper aleyreanum in popular medicine.
Financial Support: CNPq, CAPES, and FAPESC.
ANTIARTHRITIC MECHANISM OF ORALLY ADMINISTERED TRYPSIN
FLORA LUCENA; CARLOS TONUSSI.
UFSC, FLORIANÓPOLIS - SC - BRASIL.
ANTIARTHRITIC MECHANISM OF ORALLY ADMINISTERED TRYPSIN
FLORA LUCENA1; CARLOS TONUSSI1
1FEDERAL UNIVERSITY OF SANTA CATARINA
Introduction: Rheumatoid arthritis is an autoimmune disease that can cause severe physical incapacity. RA has no cure, and the current medical treatment can cause severe adverse effects, or may be highly expensive. In such context, the research for new drugs with lower side effects is needed.
Methods and Results: Female Wistar rats (180-200 g) received 50μL of CFA (0,5mg/mL) i.d. in the tail insertion region for immunization. After 7 days they received the same dose of CFA in the knee joint. Trypsin was given p.o. (2,95 mg/kg; 2 mL) 24 hs after the intra-articular CFA injection. Articular incapacitation was measured daily by counting the paw elevation time (PET; s) during 1-min periods of stimulated walk, during 7 days after intra-articular CFA injection. Articular edema (AE) was accessed by the difference between naïve and diseased knee-joint diameter (mm). TEP and AE values represent mean ± s.e.m taken in the 3rd day. Trypsin reduced the incapacitation and the diameter of inflammed knee (TEP: Tryp = 25 ± 4s; Sal = 44 ± 5s; AE: Tryp = 0,97 ± 0,04s; Sal = 1,08 ± 0,04). These effects could be observed throughout the 7-day period of evaluation. Animals that underwent subdiafragmatic vagotomy did not show the effects of trypsin administration (TEP: Vago + Tryp = 48 ± 5; Sham + Tryp = 21 ± 6; AE: Vago + Tryp = 1,05 ± 0,03; Sham + Tryp = 0,93 ± 0,05 ) unlike animals that received dexametasone (TEP: Sham + Dexa = 16 ± 4; Vago + Dexa = 14±1; AE: Sham + Dexa = 0,83 ± 0,02; Vago + Dexa = 0,82 ± 0,02). Likewise, trypsin had no effect in rats which received intrathecal injection of the neurotoxins, 6-OHDA and 5,7-DHT (TEP: 6-OHDA + Tryp = 42 ± 7; 5,7-DHT + Tryp = 48 ± 5; PBS + Tryp = 16 ± 1; AE: 6-OHDA + Tryp = 1 ± 0,03; 5,7-DHT = 1,06 ± 0,03; PBS+Tryp = 0,90 ± 0,02).
Conclusion: Oral trypsin may cause vagal activation followed by the activation of descending inhibitory noradrenergic and serotonergic pathways. Such mechanism seems to mediate its antinociceptive and anti-inflammatory effects.
Financial support: CNPq, CAPES.
ANTIINFLAMATORY EVALUATION OF CC COMPOUND IN MODELS OF INDUCED PAW EDEMA IN MICE
THIAGO HENRIQUE COSTA MARQUES1; MARIA LEONILDES BOAVISTA GOMES CASTELO BRANCO MARQUES2; RENAN OLIVEIRA SILVA3; NATHALIA SANTOS CARVALHO4; JAND-VENES ROLIM MEDEIROS5; DAMIÃO PERGENTINO DE SOUSA6; RIVELILSON MENDES DE FREITAS7.
1.IFPI/IFMA/UFPI, TERESINA - PI - BRASIL; 2.UESPI/UEMA/PUCRS, TERESINA - PI - BRASIL; 3,4,5,7.UFPI, PARNAIBA - PI - BRASIL; 6.UFPB, JOÃO PESSOA - PB - BRASIL.
Introdution: The potential anti-inflammatory activity of CC (a isolated substance with a chemical structure to define) was studied in models of induced paw edema in mice.
Methods and Results: Committee of Ethics in Animal Experimentation at UFPI: Nº 012/2011. Animals: Swiss male mice, Mus musculus (25-30g; 2-month-old). Models of induced paw edema: Carrageenan (J. Am. Pharma. Assoc. 46: 515-519, 1957) Prostaglandin (Prostaglandins Other Lipid Mediat. 80:123-135, 2006), 48/80 (Biol Pharm Bull. 25: 260- 263, 1997), Serotonin (Br. J. Pharmacol. 13: 65-70, 1958) and Hystamine (Br. J. Pharmacol. 13: 65-70, 1958). Levels of TNF-α and IL-1β were evaluated in peritoneal exudates (Immunopharmacol Immunotoxicol. 35: 1-8, 2012). Mieloperoxidase (MPO) activity in carrageenan model was performed (J. Invest. Dermatol. 78: 206-209, 1982). Statistical analysis: analysis of variance (ANOVA) and Student-Newman-Keuls as post hoc test by GraphPad Prism® 3.00 for Windows. Differences were significant when p<0.05. CC 75 showed significant decrease in IL-1β (62.8%) and TNF-α (60.1%) when compared with Negative Control. In prostaglandin model, CC 75 decreased significantly paw edema when compared with PGE2 (31.7%) 30 min after treatment. In 48/80 model, CC 75 no showed difference significantly in paw edema when compared with 48/80. In serotonin model, CC 75 decreased significantly paw edema when compared with PGE2, 30 min (54.8 %) and 60 min (62.5 %) after treatment. In histamine model, CC 75 decreased significantly paw edema, 30 min (31.7%), 60 min (29.4%), 90 min (73.5%) and 120 min (59.3%) after treatment when compared with PGE2. In bradykinin model, CC 75 showed decrease significantly paw edema by 47.37% after 30 minutes and by 67.86% after 60 minutes when compared with BRAD. In histamine model, CC 75 showed decrease significantly paw edema by 57.45% (t=30 min), by 82.14% (t=60 min), by 91.11% (t=90 min) and by 71.43% (t=120 min) when compared with HYST. In carrageenan model, CC 50 and CC 75 decreased significantly paw edema when compared with CG in third (34.21 and 47.37%, respectively) and fourth (61.11 and 62.96%, respectively) hours. CC 25 also decreased significantly paw edema (48.15%) when compared with CG in fourth hour. In MPO assay, CC 75 showed decrease significantly enzhime activity (47.02%) when compared with DMSO.
Conclusion: CC compound has anti-inflammatory activity in mice, and the mechanism of action may involve inhibition of IL-1β, TNF-α and MPO.
ANTIINFLAMMATORY AND ANTI-HIPERNOCICEPTIVE EFFECTS OF THE ETHANOLIC EXTRACT OF SALVIA LACHNOSTACHYS BENTH (LAMIACEAE) AND ITS ISOLATED COMPOUND FRUTICULINE A IN MICE
CANDIDA APARECIDA LEITE KASSUYA1; ANA CLAUDIA PICCINELLI2; ISABELLA CRISTINA DIAS3; DIANA FIGUEIREDO DE SANTANA AQUINO4; RENAN DONOMAE IWAMOTO5; REGIANE BATISTA LAURIANO STRAPASSON6; ÉLIDE PEREIRA DOS SANTOS7; MÁRIA ÉLIDA ALVES STEFANELLO8.
1,2,3,4,5.FACULDADE DE CIÊNCIAS DA SAÚDE, UNIVERSIDADE FEDERAL DA GRANDE DOURADOS,, DOURADOS - MS - BRASIL; 6,8.DEPARTAMENTO DE QUÍMICA, UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA - PR - BRASIL; 7.DEPARTAMENTO DE BOTÂNICA, UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA - PR - BRASIL.
Introduction: Despite of many species of Salvia in Brazil only few pharmacological and chemical studies had been. In folk medicine we have antiinflammatory uses. A previous phytochemical study with Salvia lachnostachys Benth showed the presence of oleanolic and ursolic acids besides fruticuline A, a rare compound isolated before from only other two species of Salvia (Microsc Res Tech. 75:1737-44, 2012). The present study aimed to evaluate the antiinflammatory and anti-hipernociceptive activities of the ethanolic extract of S. lachnostachys and its isolated compound fruticuline A in experimental models with mice. Methods and Results: Paw oedema was made with male mice (n=5 in each group) using a pletismometer with eight groups: control (saline solution), positive control (dexametasone 1mg/kg) Salvia extract (30, 100 and 300mg/kg) and fruticuline (0.3, 1, and 3mg/kg). All animals were treated orally and oedema was measured after 30 minutes, 1, 2 and 4 hours after carrageenan injection. Pleurisy test was made with female mice divided the same way as cited above with only an extra naïve group. All animals were treated orally and after 4 hours the pleural cavity was washed with phosphate-buffered saline and the total number of leukocytes was count with Turck solution in a Neubauer chamber. The protein exudation was evaluated by Bradford’s reaction and analyzed with ELISA. Mechanical allodynia was measured using electronic Von Frey and male mice were used. Only one dose of the extract (300mg/kg) and one of fruticuline (3mg/kg) were tested. Von Frey apparatus was applied to the hind paw before, 3 and 4 hours after intraplantar carrageenan injection. All parameters showed significant results. The reduction of paw oedema at 2 hours after carrageenan injection at the dose of Salvia (300 mg/kg) presented an inhibition of 83 ± 3% and fruticuline (3mg/kg) 81±2%, when compared to control group. The extract (100mg/kg) and fruticuline (3mg/kg) also decreased cell migration and protein extravasation at the doses of 300mg/kg for the extract and 3mg/kg for fruticuline, also in comparison to the control group. Salvia (300mg/kg) and fruticuline (3mg/kg) also decreased mechanical allodynia in 43 and 34%, respectively after 4 hours of carrageenan injection. Conclusion: In summary, S. lachnostachys extract and fruticuline A presented an important anti- inflammatory and anti-nociceptive activity, especially at the doses of 300mg/kg for the extract and 3mg/kg for the compound.
ANTIINFLAMMATORY PROFILE OF THE HYDROGEN SULPHIDE DONOR LAWESSON’S REAGENT ON ALENDRONATE-INDUCED GASTRIC DAMAGE IN RATS
LUCAS DUARTE NICOLAU1; RENAN OLIVEIRA SILVA2; SAMARA RODRIGUES DAMASCENO3; NATHALIA SANTOS CARVALHO4; NATÁLIA RODRIGUES COSTA5; KAROLINE SABÓIA ARAGÃO6; ANDRÉ LUIZ BARBOSA7; PEDRO MARCOS SOARES8; MARCELLUS HENRIQUE SOUZA9; JAND-VENES ROLIM MEDEIROS10.
1,2,3,4,5,7,10.UFPI, PARNAÍBA - PI - BRASIL; 6,8,9.UFC, FORTALEZA - CE - BRASIL.
Introduction: The discovery and development of bisphosphonates have been of great clinicalimportance for the prevention and treatment of bone diseases. Among the variousbisphosphonates used clinically, those with primary amino side chains, such as alendronate (ALD) and pamidronate, may have increased potential for causing gastricdamage.Hydrogen sulphide (H2S) is a well-known toxic gas, this toxicity is seen at concentrations well above those produced endogenously. This gas is synthesized endogenously from l-cysteine by two enzymes: cystathionine-
γ-lyase (CSE) and cystathionine-β-synthetase (CBS). Results from recent reports suggest that H2S protects against mucosal injury. NaHS and Lawesson’s reagent, both H2S donors, reduce the gastric damage induced byethanol and non-steroidal anti-inflammatory drugs (NSAIDs) in rats. Our objective was to investigate the protective effect of
Lawesson’s reagent, an H2S donor, against alendronate (ALD) induced gastric damage in rats.
Methods and results: Rats were pretreated with saline or Lawesson’s reagent (3, 9 or 27 μmol/kg) once daily during 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. Onthe last day of treatment, the animals were euthanized 4 h after ALD administration. Gastriclesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), pro- inflammatory cytokines (tumour necrosis factor [TNF]-α and interleukin [IL]-1β), and myeloperoxidase (MPO). ALD caused gastric damage (63.2±8.1 mm²), increased levels of TNF-α (2300.3±289.5 pg/mL), IL-1β (900.7±102.3 pg/mL), and MDA (121.1±4.3 nmol/g tissue); increased MPO activity (25.5±1.6 U/mg tissue) and reduced GSH level (180.3±21.9 µg/g tissue). ALD also increased CSE immune reactivity in the gastric mucosa. Pretreatment with Lawesson’s reagent attenuated ALD-mediated gastric damage (18.4±5.8 mm²); reduced TNF-α (1493±125 pg/mL), IL- 1β (621±19 pg/mL), and MDA formation (78.4±7.6 nmol/g tissue), lowered MPO activity (11.7±2.8 U/mg tissue) and increased the level of GSH (397.9±40.2 µg/g tissue) in the gastric tissue.
Conclusion: Our results showed that Lawesson’s reagent, a H2S donor, prevents ALD induced gastric damage. We propose that Lawesson’s reagent inhibits neutrophil infiltration and decreases damage secondary to the release of proinflammatory cytokines and elevations in oxidative stress.
Finantial Support: FAPEPI and CNPq, both in Brazil.
ANTINOCICEPTIVE AND ANTI-INFLAMMATORY EFFECTS OF A LECTIN FROM THE GREEN SEAWEED CAULERPA CUPRESSOIDES ON EXPERIMENTAL MODEL OF ZYMOSAN-INDUCED ARTHRITIS IN THE RATS TEMPOROMANDIBULAR JOINT
RENATA LINE DA CONCEIÇÃO RIVANOR1; HELLÍADA VASCONCELOS CHAVES2; TICIANA MONTEIRO ABREU3; DANIELLE ROCHA DO VAL4; JONAS CAVALCANTE LEMOS5; ALICE RAMOS DE FREITAS6; ANNYTA FERNANDES FROTA7; ISMAEL NILO LINO DE QUEIROZ8; IANNA WIVIANNE FERNANDES DE ARAÚJO9; VALDÉCIO SILVANO MONTEIRO10; KARUZA MARIA ALVES PEREIRA11; MIRNA MARQUES BEZERRA12; NORMA MARIA BARROS BENEVIDES13.
1,3,8,9,10,13.DEPARTAMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, , FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 2,6.FACULTY OF DENTISTRY, FEDERAL UNIVERSITY OF CEARÁ, SOBRAL - CE - BRASIL; 4,5,7,11,12.FACULTY OF MEDICINE, FEDERAL UNIVERSITY OF CEARÁ, SOBRAL - CE - BRASIL.
Introduction: Temporomandibular disorders (TDM) involving the temporomandibular joint (TMJ) are often associated with inflammation and secondary hyperalgesia, allodynia, referred pain and arthritis. Marine algae are sources of bioactive compounds, especially lectins, which currently have been shown their potential biological and pharmaceutical applications. This work aimed to investigate the effect of a lectin from the green seaweed Caulerpa cupressoides (CcL) in the model of arthritis induced by zymosan (Zy) in the temporomandibular joint of rats. Methods and Results: The CcL was obtained by extraction with Tris-HCl buffer 25 mM (pH 7.5) and purified by ion exchange chromatography on DEAE-cellulose. Male Wistar rats (160-220 g; n=6) (CEPA 80/10), were submitted to pretreatment with CcL (0.1; 1 or 10 mg/kg; i.v.), 30 min before the intra-articular injection of Zy (2 mg/40 μL) into the left TMJ. Control group received saline. Mechanical hypernociception in the TMJ was evaluated by measuring the threshold of force intensity that needed to be applied to the TMJ region until the occurrence of a reflex response of the animal. The force threshold value was recorded before the i.art. injections of either Zy or vehicle and after 4h. After 6 h, the rats were sacrificed under anesthesia and exsanguinated. The superficial tissues were dissected, the TMJ cavity was washed to collect the synovial fluid (total cell counting and myeloperoxidase (MPO) assay and TMJ tissues were excised to perform histological changes. Pretreatment with CcL (0. 1; 1 or 10 mg/kg) promoted a reduction (p<0.05) of facial hyperalgesia (79.5; 71.3 e 78.7%, respectively). The injection of Zy resulted in a significant increase in the number of leukocytes (22.850±4.777) in the synovial fluid. CcL (0.1; 1 or 10 mg/kg) decreased leukocyte migration in the synovial fluid (35.6; 36 e 97.5%, respectively), and also reduced MPO activity (42.6; 52.9 e 97.35 %, respectively) compared to group Zy (483.4±177.2). However, only 10 mg/kg was significant either assays. Inflammatory cell influx was observed in the synovial membrane, besides thickness in synovial membrane in the treated group with Zy. CcL (10 mg/kg) reduced (p<0.05) intense inflammatory process in synovial membrane. Conclusion: Lectin from the green seaweed Caulerpa cupressoides showed antinociceptive and anti-inflammatory effects in arthritis induced by Zy in TMJ rats considered an important therapeutic agent.
Financial support: CAPES and FUNCAP
ANTINOCICEPTIVE AND ANTI-INFLAMMATORY EFFECTS OF RIPARIN IV ON ANIMAL MODELS
MARILIA LEITE DIAS1; NAYRTON FLAVIO MOURA ROCHA2; ALYNE MARA RODRIGUES CARVALHO3; LEONARDO FREIRE VASCONCELOS4; EMILIANO RICARDO VASCONCELOS RIOS5; STANLEY JUAN CHAVEZ GUTIERREZ6; JOSE MARIA BARBOSA FILHO7; FRANCISCA CLEA FLORENCO SOUSA8.
1,2,3,4,5,8.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FACULTY OF MEDICINE UFC, FORTALEZA - CE - BRASIL; 6.DEPARTMENT OF BIOCHEMISTRY AND PHARMACOLOGY UFPI, TERESINA - PI - BRASIL; 7.LABORATORY OF PHARMACEUTICS TECHNOLOGY UFPB, JOAO PESSOA - PB - BRASIL.
Introduction: Riparin IV (RipIV) is an analogue of Riparin I that presents antinociceptive activity in different animal models. Considering similarity structural between two riparins, the aim of the present study was to investigate the antinociceptive and anti-inflammatory effects of RipIV. Methods and Results: Mice Swiss, male weighing 25-32g, were used (6-8 animals/group). RipIV was used at the doses of 25 and 50mg/kg, by gavage. Data were analyzed using One-Way ANOVA and Student Newman-Keuls test post hoc. Firstly, the acetic acid-induced abdominal writhing, formalin test, hot plate test were performed. Indomethacin 10mg/kg (p.o.) or morphine 7.5mg/kg (i.p.) were used as standard drugs. Results showed that RipIV decreased significantly the number of writhes (40.46% and 42.84%, respectively), when compared to the vehicle group (3% tween 80 in distillated water), as well as indomethacin (66.69%). In the formalin test, RipIV decreased paw licking time at both doses, only at the second phase of the test (25mg/kg: 56.03%; 50mg/kg: 53.36%). Morphine reduced paw licking time at both phases. At the hot plate test, RipIV showed no statistical difference when compared to the vehicle group. Animals that receiving morphine presented an increase at the response time to heat. At a second moment, the tests of mechanical inflammatory hipernociception induced by carragenann (Cg) and prostaglandine E2 (PGE2) were performed. Animals were pre-treated with RipIV at both doses 1h before Cg (0.1%) and PGE2 (100µg/ml) application (20µl/paw) and assessed 30, 60 and 180min after intraplantar injection. Pre-treatment with RipIV-25mg/kg was able to decrease the intensity of hypernociception observed at 60 and 180min after intraplantar injection of Cg and PGE2, and pre-treatment with RipIV-50mg/kg decreased the mechanical inflammatory hipernociception at all times observed, when compared to the animals treated with vehicle. After 3h of Cg injection, subplantar tissue was collected to quantify the scores of edema and inflammatory infiltrate. Animals pre-treated with RipIV at both doses showed no statistical difference when compared to the vehicle group, but animals pre-treated with indomethacin presented a significant reduction at both scores analyzed. Conclusion: The results indicate that RipIV presents an antinociceptive activity, probably due to prevention of nociceptors sensibilization, with no anti-inflammatory activity. Financial support: CNPq/CAPES/FUNCAP
ANTINOCICEPTIVE EFFECT OF EPIISOPILOTURINE, AN IMIDAZOLE ALKALOID ISOLATED FROM PILOCARPUS MICROPHYLLUS, IN MICE
SAMARA RODRIGUES BONFIM DAMASCENO1; VALDELÂNIA GOMES SILVA2; NATHALIA SANTOS CARVALHO3; RENAN OLIVEIRA SILVA4; RAFAEL DA SILVA PRUDÊNCIO5; LEIZ MARIA COSTA VÉRAS6; JOSÉ ROBERTO DE SOUZA DE ALMEIDA LEITE7; ANDRÉ LUIZ DOS REIS BARBOSA8; JAND-VENES ROLIM MEDEIROS9.
1,2,3,4,5,8,9.FEDERAL UNIVERSITY OF PIAUÍ - LABORATORY OF EXPERIMENTAL PHYSIOPHARMACOLOGY, PARNAÍBA - PI - BRASIL; 6,7.FEDERAL UNIVERSITY OF PIAUÍ - BIOTECHNOLOGY AND BIODIVERSITY CENTER RESEARCH, PARNAÍBA - PI - BRASIL.
Introduction: The inflammatory process triggers formation of inflammatory neuromediators that activate nociceptors when released, facilitating pain transmission and peripheral inflammatory responses (Manual Ther. 4: 196−202, 1999). Several analgesics drugs are associated with important side effects. Thus, studies are conducted to identify therapeutic options to develop of new drugs. The aim of this study was to investigate the antinociceptive activity of epiisopiloturine, an imidazole alkaloid found in the leaves of Pilocarpus microphyllus. Methods and Results: Each experimental group (swiss mice, n=5) was pretreated with 2% DMSO, Epiisopiloturine (Epi) (1 mg/kg, ip), or morphine (5 mg/kg, sc, reference control). Protocol was approved in the local ethics committee (Protocol 0066/10). In the abdominal writhing test, 0.6% acetic acid (10 ml/kg, ip) was administered 30 min after pretreatment with epiisopiloturine or morphine, and the number of constrictions was recorded over 20 min. In the hot plate test the control reaction time recorded the response latency on a hot plate (55±1°C); measurements were performed before (zero time) and 30, 60, 90, and 120 min after treatment. In the formalin test, thirty minutes after pretreatment with Epi or morphine, 2.5% formalin (20μL) was administered into the right hind paw and licking time was recorded from 0 to 5 min and 20−25 min after. To examine the involvement opioid, mice were pretreated with naloxone (3 mg/kg, sc; opioid antagonist). After 30 min, the animals were treated with Epi or morphine. 60 min after, 2.5% formalin (20μL) was administered into the right hind paw. Licking time was recorded from 0 to 5 min. To exclude the possibility that epiisopiloturine caused motor relaxation and sedation was performed rota-rod test. Pretreatment with Epi decreased the number of acetic acid-induced writhing (12.5±1.5 Epi group versus 35.1±2.3 acetic acid group), decreased paw licking time in the formalin test (27.4±4.7 s and 3.8±2.9 s of the Epi group versus 65.5±20.2 s and 35.8±12.3 s formalin group, 1ª and 2ª phases respectively), however Epi did not increased the latency time of the animals on the hot plate and did not impair motor performance of mice evaluated in the rota-rod test. Pretreatment with naloxone reversed the antinociceptive effect of Epi. Conclusion: In summary, epiisopiloturine presents antinociceptive mechanisms that are partially mediated by the activation of the opioid system.
Financial support: CNPq.
ANTIOXIDANT CAPACITY AND ANTI-INFLAMMATORY ACTIVITIES OF HEXANE AND ETHANOL EXTRACTS FROM ANNONA MURICATA LEAVES
BEATRIZ DE SOUSA E LIMA1; MICHELLE APARECIDA FREITAS DE ANDRADE2; CAMILA FREITAS BEZERRA3; JOANNA DE FREITAS ROCHA4; ALBERT LAYO COSTA DE ASSIS5; NEUZA FÉLIX GOMES6; ERIKA FREITAS MOTA7; DIANA CÉLIA SOUSA NUNES PINHEIRO8; DIRCE FERNANDES DE MELO9.
1,2,3,4,5,6,7,9.UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA - CE - BRASIL; 8.UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Annona muricata L. is a tree of Annonaceae family widely distributed in all tropical regions of the world, produces fruit known as soursop (graviola) and its leaves are used in traditional medicine. This study aimed to evaluate the antioxidant and anti-inflammatory activities of hexane (HE) and ethanolic (EE) soursop leaves extracts.
Methods and Results: Phytochemical analyses were performed to quantify total polyphenols, yellow flavonoid and anthocyanins in HE and EE. Antioxidant capacity was determined by ABTS●+ and DPPH methods. To evaluate the anti-inflammatory activity, ear edema model induced by xylene or TPA were performed in Swiss mice treated with EE or HE (10, 100 and 1000 mg/kg) by gavage (7 consecutive days) or topically (single dose after the challenge). The use of animals was approved by CEPA/UFC (protocol no. 101/11). On the 7th day, ear thickness was measured before xylene and TPA application and 1h (xylene) or 6h (TPA) after, using a micrometer. Dexamethasone (2.5 mg/Kg) and Indomethacine (5 mg/Kg) were used as a positive control in the ear edema assay. Edema formation due to the inflammatory challenge was expressed as the percentage inhibition when compared to untreated animals. Results were analyzed using ANOVA followed by Tukey’s test or Kruskal-Wallis (p<0.05). The extracts showed wide variety of phytochemical compounds and high antioxidant capacity, mainly EE. Pretreatment via i.g. with extracts was able to inhibit the edema induced by xylene, (61.72% (47.71±15.52) for EE 10 and 57.08% (55.57±14.57) for HE 100), but no changes were observed on TPA-induced edema. The topical application inhibited the xylene-induced edema (78.89% (31.71±14.04) for HE1000, while for EE1000 was 67.95% (61.5±29.36)). As for the TPA-induced edema, the inhibition was dose-dependent for HE (10, 100 and 1000 mg / kg) 84.39%(45.14 ± 29.33), 68.68% (90.58 ± 40.57) and 65.96%(98.43±20.22), respectively. Ethanolic extract (10, 100 and 1000 mg /kg) inhibited the TPA-edema by 75%(72.28±41.46), 56%(128.14±48.23) and 51% (140.57±69), respectively.
Conclusion: The hexane and ethanol extracts of soursop leaves exhibit high antioxidant capacity and a significant anti-inflammatory activity.
Financial Suport: CAPES; CNPQ; FUNCAP
BAICALEIN PRELIMINARY INFLAMMATORY ASSESSMENT IN EXPERIMENTAL PLEURISY
CLIOMAR ALVES DOS SANTOS; SARA MARIA THOMAZZI.
UNIVERSIDADE FEDERAL DE SERGIPE, ARACAJU - SE - BRASIL.
Introduction: The baicalein flavonoid (5,6,7-triidroxiflavone), the main component extracted from Scutelaria baicalensis Georgi (Lamiaceae), presented anti-inflammatory, antithrombosis e antioxidant effects. A hypothesis of this work consists in the improvement of the inflammation in the pleural cavity.
Methods and Results: Mice (n=6/group) were pretreated, p.o., with 10, 30, or 100 mg/kg of baicalein, vehicle, or dexamethasone (2 mg/kg). In non-allergic pleurisy, the animals, under anesthesia, were submitted at carrageenan (1%, 300 μg/cavity) intrapleural injection, after 4 h were anesthetized, and collected pleural lavage with PBS. In allergic pleurisy, animals were challenged with Complete Adjuvant Freund and ovalbumin (2 mg/kg, intraplantar, i.pl.), after 14 days received ovalbumin (12 μg/cavity, i.pl.) and after 4 h were euthanized. In both protocols, further on lavage, blood, and lung were collected. In non-allergic pleurisy, baicalein inhibited (p<0.001) leukocytes (leuk) migration in the blood at the 30 and 100 mg/kg doses, when compared with the vehicle group (9.39 x 106 leuk/mL) and the polymorphonuclear (PMN) cells at 30 and 100 mg/kg (p<0.001, vehicle group 7.27 x 106 leuk/mL) and inhibited the leukocytes migration in the thoracic cavity at 30 and 100 m/kg (p<0.001, vehicle group 7.95 x 106 leuk/cavity) and PMN cells at 30 and 100 mg/kg (p<0.001, vehicle group (6.08 x 106 leuk/cavity). Baicalein reduced lavage and lung MPO activity at 30 and 100 mg/kg (p<0.001, vehicle group 24.20 x 106 leuk/mL in lavage and 65.39 x 106 leuk/mg of lung). The oedema index not showed changed. In allergic pleurisy, baicalein inhibited the leuk migration in the blood at the 30 and 100 mg/kg (p<0.05, vehicle group 10.04 x 106 leuk/mL) and the PMN cells at 10, 30, and 100 mg/kg (p<0.05, vehicle group 6.86 x 106 leuk/mL) and inhibited the leukocytes migration in thoracic cavity at 10, 30, and 100 mg/kg (p<0.001, vehicle group 11.25 x 106 leuk/cavity) and PMN cells at 30 and 100 mg/kg (p<0.001, vehicle group 8.08 x 106 leuk/cavity). Baicalein reduced lavage and lung MPO activity at 100 mg/kg (p<0.001, vehicle group 31.47 x 106 leuk/mL in lavage and 80.35 x 106 leuk/mg of lung). Baicalein reduced the oedema index in allergic pleurisy at 100 mg/kg (p<0.05).
Conclusion: Baicalein reduces inflammatory cells to the cavity, lung and blood in allergic and non-allergic pleurisy and reduce oedema in the lung at the allergic pleurisy in mice.
Financial support: CAPES
BENEFICIAL EFFECTS OF OMEGA-3 FISH OIL SUPPLEMENTATION ON CYCLOPHOSPHAMIDE-INDUCED ALTERATIONS IN MICE
RAQUEL DAL SASSO FREITAS; MARIA MARTHA CAMPOS.
PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO GRANDE DO SUL, PORTO ALEGRE - RS - BRASIL.
Introduction: Hemorrhagic cystitis (HC) is an inflammatory and painful alteration commonly associated to the use of the chemotherapy agent cyclophosphamide (CYP) (Clin Pharmacokinet. 44:1135-1164, 2005; J Expl Integ Med. 2:93- 94, 2012). The supplementation with omega-3 fatty acids has been clinically used for the management of cancer patients (Am J Clin Nutr 83:1505-1519, 2006; Braz J Oncology 55:279-287, 2009). The beneficial effects of omega-3 are likely related to the production of lipid pro-resolution mediators, such as resolvins and protectins. These mediators are derived from the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) - both exclusively found in fish oils (Am J Pathol. 177:1576-1591, 2010; Front Immunol 3:1-6, 2012). This study evaluated the effects of diet supplementation with omega-3 fish oil in the mouse model of CYP-induced HC. Methods and Results: Male Swiss mice were used (N=8/group). The experimental protocols were approved by the Animal Ethics Committee (12/00303). Three separate groups of mice received 8% (1.4 g of EPA and 0.96 g of DHA), 10% (3.3 g of EPA and 2.2 g of DHA) or 20% (6.6 g of EPA and 4.4 g of DHA) of fish oil, added to the regular chow during 21 days. Negative control groups received the same percentages of corn oil. At the 22nd day, the animals received a single i.p. injection CYP (300 mg/kg). The behavioral tests were performed 30 min after CYP injection and each animal was observed for 2 min, every 1/2 h, for 4 h. Following the behavioral assessments, Von Frey test was performed using a 0.4 g-filament, for mechanical threshold determination. Six h after, the animals were euthanized and had their bladders removed and scored for inflammatory changes. None of the tested diets containing omega-3 fish oil was able to significantly affect the inflammatory bladder changes elicited by CYP, including hemorrhage and edema. Nevertheless, it was possible to observe a significant reduction of nociceptive behavior and frequency of response to mechanical stimulation following supplementation with 20% fish oil, when compared to corn oil, with inhibition percentages of 53±4% and 51±24%, respectively. Conclusion: Our data suggests that diets rich in omega-3 might be used as adjuvant for patients under chemotherapy with cyclophosphamide, by preventing painful alterations related to HC.
Financial Support: FINEP/PUCRSINFRA #01.11.0014-00, CAPES and CNPq.
BIOLOGICAL ACTIVITIES OF ARTINM REQUIRE OLIGOMERIZATION TO BE REPRODUCED BY ITS RECOMBINANT FORMS: YEAST EXPRESSED LECTIN AS A POSSIBLE AGENT FOR THERAPEUTIC APPLICATIONS
NERRY TATIANA CECILIO1; FERNANDA CAROLINE CARVALHO2; ANDRÉ L.V.Z FERNANDES3; YAN LIU4; TEN FEIZI5; EBERT SEIXAS HANNA6; JOSÉ CÉSAR ROSA7; MARIA HELENA SOUZA GOLDMAN8; MARIA CRISTINA ROQUE BARREIRA9.
1,2,3,7,9.SCHOOL OF MEDICINE OF RIBEIRAO PRETO/USP, RIBEIRAO PRETO - SP - BRASIL; 4,5.IMPERIAL COLLEGE LONDON, LONDON - REINO UNIDO; 6.INVENT BIOTECHNOLOGICAL, RIBEIRAO PRETO - SP - BRASIL; 8.SCHOOL OF PHILOSOPHY, SCIENCE AND LITERATURE OF RIBEIRAO PRETO/USP, RIBEIRAO PRETO - SP - BRASIL.
Abstract: ArtinM is an immunomodulatory plant lectin that induces Th1 biased immunity, initiated by recognition of N- glycans expressed on the surface of APCs. ArtinM acts on macrophages and neutrophils, through the binding to N- glycans attached to TLR2 and CXCR2. Because ArtinM administration to mice confers protection against intracellular pathogens, its potential pharmaceutical application is envisaged and led us to produce recombinant ArtinM in large scale through expression in S. cerevisiae (yArtinM) and E. coli (bArtinM).
Methods and Results: Comparison of native ArtinM (jArtinM), yArtin and bArtin in terms of mass spectrometry analysis of tryptic peptides and sugar recognition specificity, as determined by glycoarray, has demonstrated that these lectin preparations share structural and functional features. Surprisingly, the jArtinM biological properties were not reproduced by bArtinM. Otherwise, they were exhibited by yArtinM, as demonstrated by its ability of inducing: (a) neutrophil haptotaxis in vitro (129.2 ± 35.54 neutrophils/field); (b) neutrophil migration in vivo (1x107neutrophils/peritoneal cavity): (c) mast cell degranulation (50% β-hexosaminidase release); (d) NB4 cells growth inhibition (40% of growth inhibition). In order to investigate the molecules features accounting for the distinct biological properties, we examined the proteins oligomerization state. The native jArtinM is well known as a homotetramer composed by 16kDa subunits, as revealed by the detection of a 64kDa protein on gel filtration analysis. Otherwise, gel filtration of bArtinM provided a 13kDa MM and of yArtinM a 26kDa MM, which were considered as lectin monomers and dimers, respectively. This idea was reinforced by assaying the agglutination of type O human erythrocytes, which was determined by 97.6nM jArtinM and 5.56 μM yArtinM; no agglutination was triggered bArtinM, used in concentrations as high as 31μM.
Conclusion: Protein oligomerization is required for exerting the ArtinM biological activities. Because yArtinM reproduces the biological activities assigned to jArtinM, we postulate that yArtinM is the most likely useful recombinant lectin for pharmaceutical application. Financial support: FAPESP, CAPES, CNPQ and FAEPA
BLOCKADE OF THE CXCL-ELR+CHEMOKINES / CXCR2 AXIS DURING NEUTROPHILIC PHASE ACCELERATES WOUND CLOSURE IN MICE
LUCÍOLA DA SILVA BARCELOS1; TIAGO BRUNO REZENDE DE CASTRO2; MARIA CECILIA CAMPOS CANESSO (IC)3; BRIGIDA ALMEIDA GOMES SCHIRMER4; DANIEL CISALPINO5; FRANCESCO COLOTTA6; RICCARDO BERTINI7; AMANDA PROUDFOOT8; MAURO MARTINS TEIXEIRA9.
1,2,3,4,5,9.UNIVERSIDE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 6,7.DOMPÉ RESEARCH AND DEVELOPMENT, MILANO - ITÁLIA; 8.MERCK SERONO GENEVA RESEARCH CENTRE, GENEVA - SUÍÇA.
Introduction: The exacerbation of inflammatory response has been considered to be one major problem in non- healing wounds. CXCL1 and CXCL2 chemokines, ligands for CXCR2, stimulate both recruitment of neutrophils and angiogenesis during wound healing. Our aim was to evaluate the effects of Evasin-3, a CXCL1/2 binding protein isolated from salivary gland of the tick Rhipicephalus sanguineus, and Meraxin, a non-competitive allosteric inhibitor of CXCR2, on skin wound healing in mice.
Methods and Results: Excisional wounds were created on the dorsum of C57Bl/6 mice removing the entire thickness of the skin with a circular punch. Wound area was measured with a digital caliper for monitoring the closure. Accumulation of macrophages and neutrophils was evaluated by assaying the activity of N-acetyl-b-D- glucosaminidase and myeloperoxidase, respectively. Animals received daily i.p. injection of Evasin-3 or Meraxin at different doses. Control group received PBS vehicle. Animals were euthanized at varied time-points and wounds collected for analysis (n=5-8 animals/group). In a kinetics experiment, neutrophil infiltration peaked at 12h after surgery and return to baseline after day 3. CXCR2 mRNA expression peaked at day 3 and angiogenesis at day 7. Then, all treatment schedules started just after wounding and last until day 3. Evasin-3 treatment faster closure of wounds in all doses evaluated when compared with the control group (C 38±6.5% vs Ev 52±5%, and C 61±6% vs Ev 85.4±1.3%, at days 7 and 10, p<0.01, p<0.001, respectively). Wounds from these treated animals showed an increase in macrophage content at day 3 post wounding (C 0.3±0.01 vs Ev 0.4±0.02, p=0.01 and angiogenesis at day 7 (C 97±15 vs Ev 212±50 capillaries/mm2, p<0.05), but have no alteration in neutrophil content. Interestingly, evasin-3 had no effects on wound closure when treatment lasted till day 7. Likewise, animals treated with Meraxin also displayed faster wound closure when compared to control group (C 78±2% vs Mer 88±2%, p<0.001, at day 10), while only at lower doses, and showed an increase in macrophage content at day 3 (C 0.1±0.01 vs Mer 0.2±0.02, p<0,05), although no alteration in neutrophil content or angiogenesis.
Conclusion: Our data suggest that pharmacological blockade of the CXCL-ELR+ chemokine/CXCR2 axis during neutrophilic, but not angiogenic, phase accelerates wound closure in mice and may have therapeutical potential to treat non-healing wounds.
CARVACRYL ACETATE, A DEVATIVE OF CARVACROL, REDUCES NOCICEPTIVE ACTIVITIES IN MICE.
IRISMARA SOUSA SILVA1; SAMARA RODRIGUES BOMFIM DAMASCENO2; MAISA DE SOUSA DOS SANTOS3; NATHALIA SANTOS CARVALHO4; CAMILA DE FÁTIMA CARVALHO BRITO5; FRANCISCA BEATRIZ DE MELO SOUSA6; RENAN OLIVEIRA SILVA7; FRANCISCO RODRIGO DE ASEVEDO MENDES DE OLIVEIRA8; DAMIÃO PERGENTINO DE SOUSA9; ANDRÉ LUIS DOS REIS BARBOSA10; JAND-VENES ROLIM MEDEIROS11.
1,2,3,4,5,6,7,10,11.BIOTECHNOLOGY AND BIODIVERSITY CENTER RESEARCH (BIOTEC), FEDERAL UNIVERSITY OF PIAUÍ, PARNAÍBA - PI - BRASIL; 8.POST-GRADUATION PROGRAM IN PHARMACEUTICAL SCIENCES, FEDERAL UNIVERSITY OF PIAUÍ, TERESINA - PI - BRASIL; 9.DEPARTMENT OF PHYSIOLOGY, FEDERAL UNIVERSITY OF PARAÍBA, JOÃO PESSOA - PB - BRASIL.
Introduction: Currently several analgesics drugs are associated with important side effects, low efficacy and specificity, medicinal plants are natural products known to be a significant source of new chemical substances with potential therapeutic effects (J. Ethnopharmacol. 100: 131–134, 2005). The aim of the present study was to investigate the role of carvacryl acetate, a derivative of carvacrol, in the analgesic processes induced by intraplantar and intraperitoneal injection of different phlogistic agents. Methods and Results: The analgesic effects of carvacryl acetate were evaluated in classic models: writhing test, hot plate test, formalin, glutamate and capsaicin-induced paw licking and rota-rod test. The present study was approved by the local ethics committee (protocol no.0066/10). In test of abdominal writhing, the pain was induced by the intraperitoneal injection of acetic acid (0,6%)(n=6), carvacryl acetate (75 mg/kg, i.p.) decreased significantly the number of writhing, induced by acetic acid 26.75 ± 8.18, compared 10.33 ± 4.24. In test hot plate, (55 ± 1 °C) (n=6), carvacryl acetate (75 mg/kg, i.p.), significantly increased latency time on the plate in all the evaluated times, as compared to time zero. The formalin test (n=6) occurs in a biphasic pattern, the action nociceptive was induced by injecting 2.8% formalin (20 μl) administered intradermally (i.d.) into the right hind paw, carvacryl acetate, (75 mg/kg, i.p.), reverted significantly the licking time in both the neurogenic (63.99%) and inflammatory (93.30%) phases. The carvacryl acetate (75 mg/kg, i.p.) promoted accentuated decrease in licking response of the paw, 53.27% (p < 0.001), as compared to group capsaicin (33.08 ± 1.27s).The glutamate group (56.05 ± 11.21) also caused pronounced anti-nociception (24.71 ± 9.45 s) versus the carvacryl acetate group. In rota- rod test (n=10), animals treated by gavage with carvacryl acetate (75 mg/kg), did not affect the number of falls or time of stay on the rotating bar, when compared with the vehicle (untreated) in rotarod test. Conclusion: Carvacryl acetate presents anti-nociceptive effect, centrally and peripherally, through the involvement of capsaicin and glutamate pathways and decreased inflammatory mediators.
Financial support: CNPq, FAPEPI, UFPI.
CHARACTERIZATION OF CARRAGEENAN-INDUCED ACUTE MUSCLE INFLAMMATION IN RATS
ANA CARLA ARAÚJO SOUZA; DENYSON SANTANA PEREIRA; FABIULA FRANCISCA ABREU; JANAINA PEREIRA MORAES; LUCINDO JOSÉ QUINTANS-JUNIOR; JOSIMARI MELO DESANTANA; ENILTON CAMARGO.
UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO - SE - BRASIL.
Introduction: Muscle inflammation (MI) causes pain and impairs motor function. Experimental models of MI are mostly directed to nociceptive evaluation at later stages. No experimental model of acute MI has been properly developed. This study was designated to evaluate the acute inflammation induced by carrageenan (CAR) in the gastrocnemius muscle, in an attempt to develop an appropriate model of acute MI. Methods and Results: Male Wistar rats (n=6/group) were used and the experiments were approved by the Instituition´s Ethic Committee (02/12). Gastrocnemius muscle injury was induced by the administration of CAR (3%, 100 µL). Injection of saline (SAL) was used as control. The injection of CAR increased (p<0.001) the myeloperoxidase (MPO) activity after 6 (5.8±1.2 UMPO/mg) or 24 h (7.8±0.9 UMPO/mg) post-induction, when compared with SAL group (0.2±0.1 and 0.4±0.2 UMPO/mg, respectively). In the same way, it augmented (p<0.001) the muscle edema after 6 (7.7±0.1 mg/g) or 24 h (7.5±0.2 mg/g) post-induction, when compared with SAL group (5.9±0.1 and 6.0±0.3 mg/g, respectively). Lipid peroxidation (LP) was observed after 6 (60.0±6.8 pmol of MDA/mg, p<0.05), but not 24 h (42.0±14.4 pmol of MDA/mg) after CAR injection, when compared with SAL group (22.7±1.8 and 21.2±5.1 pmol of MDA/mg, respectively). As a control, the pre-treatment of animals with dexamethasone (2 mg/kg, s.c., 30 min before induction) reversed (P<0.001) the increase in MPO activity and edema, but did not affect the LP. Also, the injection of CAR induced histological alterations in the muscle, mainly involving the neutrophil infiltration. The injection of CAR (24 h) also increased the concentrations of interlukin-1β (708±101 pg/mL; p<0.01) in muscle, when compared with SAL group (108±27 pg/mL). Besides these alterations, CAR injection induced (p<0.001) mechanical hyperalgesia (reduction of withdrawal threshold of stimulus applied to the rat paw with electronic von Frey) after 6 (-21.0±4.3 g) or 24 h (-22.8±3.5 g), when compared with SAL group (-2.3±1.8 and -2.6±3.5 g, respectively). Grip strength was also impaired by the CAR injection (675.4 ±41 and 679±45.5 g for 6 (p<0.01) and 24 h (p<0,05) respectively, when compared with SAL group (965±76.5 and 948±66.5 g for 6 and 24 h respectively). Conclusion: These results indicate that CAR injection in the gastrocnemius muscle induces acute inflammation, accompanied by hypernociception, which consists in a suitable model to be used for future studies.
CHARACTERIZATION OF CYTOSKELETON INVOLVEMENT IN THE ANTINOCICEPTIVE EFFECT OF CROTALPHINE
ANA CAROLINA DE ALMEIDA; VANESSA PACCIARI GUTIERREZ; SANDRA COCCUZZO-SAMPAIO; YARA CURY.
BUTANTAN INSTITUTE, SÃO PAULO - SP - BRASIL.
Introduction: Crotalphine (CRF), a peptide first identified and isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, induces potent and long lasting antinociceptive effect. These properties depend on the presence of previous sensitization and are mediated by the release of endogenous opioid peptides, mainly dynorphin-A and activation of kappa and/or delta opioid receptors (KOR and DOR, respectively). This effect also involves activation of the L-arginin/NO/cGMP pathway and opening of ATP-sensitive K+ channels. The cytoskeleton is a set of elements closely related to cell signaling, including the signaling induced by opioid receptors. The aim of this work is to evaluate the role of cytoskeleton main elements in CRF antinociceptive effect.
Methods and Results: Latrunculin B (LB: 0.05 µg/paw) was used to disrupt actin microfilaments; nocodazole (NDZ: 1.0 µg/paw) was used to disrupt microtubules; acrylamide (ACR: 1.0 µg/paw) was used to disrupt intermediate filaments. Male Wistar rats were injected by intraplantar (i.pl.) route with prostaglandin E2 (PGE2, 100 ng/paw), cytoskeleton inhibitors and CRF (0.6 ng/paw). After 4 hours from PGE2 administration, nociceptive threshold was determined by the paw pressure test. All cytoskeleton inhibitors prevented CRF antinociceptive effect (Baseline: 68.61±4.1 g; PGE2: 44.6±4.3 g; PGE2+CRF: 99.6±6.0 g; PGE2+LB+CRF: 46.3±4.8 g; PGE2+NDZ+CRF: 49.0±4.2 g; PGE2+ACR+CRF: 43.0±2.7 g). Based on these results, we then evaluated the role of cytoskeleton on KOR expression (immunohistochemistry) in dorsal root ganglion and dynorphin-A release (ELISA) by the plantar tissue in PGE2- treated rats. The cytoskeleton inhibitors did not change the expression of KOR (naïve: 0.2±0.07; PGE2: 0.39±0.08; PGE2+LB: 0.6±0.2; PGE2+NDZ: 0.7±0.5; PGE2+ACR: 0.5±0.4). In contrast, NDZ and ACR decreased dynorphin-A release in PGE2-treated rats (naïve: 0.5±0.2 ng/ml; PGE2: 1.4±0.5 ng/ml; PGE2+LB: 1.4±0.9 ng/ml; PGE2+NDZ: 0.34±0.14 ng/ml; PGE2+ACR: 0.33±0.3 ng/ml).
Conclusion: Actin microfilaments, microtubules and intermediate filaments are key elements for the antinociceptive effect of CRF. Microtubules and intermediate filaments are also relevant for dynorphin-A release mediated by PGE2 administration.
Financial support: São Paulo Research Foundation - Fapesp (Process number 2013/07467-1 and 201210105-1) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Capes (Process number 421337/2010).
CHECKING NOVEL ANTI-INFLAMMATORY DRUGS WITH NOVEL INFLAMMATION MODELS
DOMINIK HERMANN SCHREIBER1; GERHARD ERKEL2; MARKUS KLOTZ3; JASMIN CLASOHM4; KARL- HERBERT SCHÄFER5.
1,3,4,5.UNIVERSITY OF APPLIED SCIENCES KAISERSLAUTERN, ENTERIC NERVOUS SYSTEM GROUP, ZWEIBRÜCKEN - ALEMANHA; 2.TECHNICAL UNIVERSITY KAISERSLAUTERN, BIOTECHNOLOGY AND SYSTEMS BIOLOGY GROUP, KAISERSLAUTERN - ALEMANHA.
Introduction
Although inflammatory bowel disease (IBD) is currently the major gastrointestinal autoimmune disease, treatment strategies are still far from being optimal. Effective new therapeutics which produce less side effects are needed. In order to test new experimental substances for their therapeutic potential in IBD, we established an in vitro intestinal perfusion system where the in vivo situation is simulated. Six new compounds, fungal derivatives were tested both in the perfusion model as well as on isolated myenteric plexus from the enteric nervous system (ENS).
Methods and Results
ENS testing can help to specify effects on neuroinflammation which play an important role in vivo. BALB/c intestines were used for perfusion and ENS isolation. Surgery was performed under anesthesia. A segment of the jejunum with the adhering cannulated mesenterial root was used for in vitro mesenterial perfusion. Acute inflammation in the small intestine was induced by the application of a cytokine mix during a five hours perfusion period. Mucosal integrity of the perfused segments was demonstrated using scanning electron microscopy. Two of the test substances, galiellalactone and dehydrocurvularin as well as dexamethasone proved to be effective. The substances were added either to the cell culture medium or the perfusion fluid. qRT-PCR, whole genome microarray, immune-histology (iNOS, COX-2) and DIGE analysis were performed on the perfused tissue. iNOS expression was significantly decreased in immunehistochemical stainings of treated tissue. Anti-inflammatory effects on gene expression level could be observed after treatment; mRNA expression in comparison to induced control±SD in qRT-PCR for e.g. galiellalactone, 4 µg/ml, n=5, normalized to GAPDH: iNOS 7.6%±2.7, IL-1β 38.8%±11.6, TNF-α 56.6%±10.0. DIGE analysis revealed 1.9% of spotted proteins to be regulated for e.g. galiellalactone. For isolated ENS cells similar but more pronounced effects could be observed on transcriptional level as well as in immunehistochemical stainings.
Conclusion
The assessment of drug effects in our in vitro model in combination with testings on ENS can help to evaluate the potential of new drugs intended for the treatment of IBD and accelerate drug development. Galiellalactone and dehydrocurvularin proved to be interesting candidates for further studies.
We thank the Stiftung Rheinland-Pfalz für Innovation and the IBWF e.V. Kaiserslautern for funding this work.
CHRESTA MARTII REDUCES THE ZYMOSAN-INDUCED TEMPOROMANDIBULAR JOINT INFLAMMATORY HYPERNOCICEPTION DEPENDENT FROM TNF-Α AND HEMOXYGENASE-1 PATHWAY
ALICE RAMOS DE FREITAS1; LORENA VASCONCELOS VIEIRA2; JONAS CAVALCANTE LEMOS3; DANIELLE ROCHA DO VAL4; CHRISTIANE AGUIAR NOBRE5; ANTONIO ALFREDO RODRIGUES E SILVA6; MIRNA MARQUES BEZERRA7; GERARDO CRISTINO FILHO8; VICENTE DE PAULO TEIXEIRA PINTO9; KARUZA MARIA ALVES PEREIRA10; MARIA BERNADETE DE SOUSA MAIA11; GERLY ANNE DE CASTRO BRITO12; HELLÍADA VASCONCELOS CHAVES13.
1,2,3,4,5,6,7,8,9,10,12,13.FEDERAL UNIVERSITY OF CEARÁ - SOBRAL CAMPUS, SOBRAL - CE - BRASIL; 11.FEDERAL UNVIERSITY OF PERNAMBUCO, RECIFE - PE - BRASIL.
Introduction: The genus Chresta are known by the population of Brazil northeastern and used to treat gastric disorders and other inflammatory conditions (Journal of Ethnopharmacology. 142:206–212, 2012). This study aims to investigate the mechanism involved in the anti-inflammatory and antinociceptive effects of hydroalcoholic extract from Chresta martii (EHA) in the zymosan-induced temporomandibular joint (TMJ) inflammatory hypernociception, assessing the involvement of TNF and the possible role of Heme-oxygenase-1(HO-1) pathway. Methods and Results: Male Wistar rats (160-220 g) (n = 6) were pretreated with EHA (100, 200 or 400 mg / kg, p.o.) 60 min before intra-articular injection (i.art.) Zy (2 mg/40μL) into the left TMJ. The Zy group received saline (p.o.) 60 minutes before induction of TMJ inflammatory hypernociception (Journal of Biomedicine and Biotechnology. 2011, 2011:707985). Indomethacin (5 mg/kg) was used as a positive control. In another series of experiments animals were pretreated with ZnPP-IX (3 mg/kg s.c.) a specific inhibitor of HO-1 prior to EHA (400 mg/kg). The parameters evaluated were: mechanical hypernociception, total cell count, measurement of myeloperoxidase activity (MPO), histopathology, and dosage of TNFα. EHA (400 mg/kg) increased (p < 0.05) the inflammatory hypernoniception threshold (90.6 ± 2.0), and reduced (p <0.05) the inflammatory infiltrate (1780 ± 390.3), MPO activity (32.82 ± 3.01), cell influx in the synovial membrane [1.5(1-2)], and the dosage of TNF in periarticular tissue (2.34 ± 2:34) and in the trigeminal ganglia (3:58 ± 1.78) compared to Zy group (61.0 ± 1.36), (28063 ± 7738), (96.01 ± 8.79), [3(1-4)], (36.55 ± 4.24), (47.09 ± 6:56), respectively. The antinociceptive effect of EHA was not observed (p < 0.05) when administered ZnPP-IX (68.38 ± 6.99), compared to EHA 400 mg/kg (100.1 ± 5.77). Conclusion: These results suggest that the Chresta martii exerts an antinociceptive and antiinflammatory effects, at least in part, by reducing TNF-α levels, and its antinociceptive effect depends on the integrity HO-1 pathway. Financial Support: CAPES, CNPq, FUNCAP, UFC.
CISSAMPELOS SYMPODIALIS EXTRACT REDUCES PRO-INFLAMMATORY CYTOKINES AND NO PRODUCTION ON MURINE MACROPHAGES
MARIA TALITA PACHECO DE OLIVEIRA; THERESA RACHEL DE OLIVEIRA RAMALHO; JACQUELINE ALVES LEITE; MARCIA REGINA PIUVEZAM.
UNIVERSIDADE FEDERAL DA PARAÍBA, JOÃO PESSOA - PB - BRASIL.
Introduction: Cissampelos sympodialis is a plant used in folk medicine to treat diseases like asthma, flu, bronchitis and rheumatism. The alcoholic fraction of leaves (AFL) of this plant has shown potential anti-allergic and anti- inflammatory effect by inhibiting inflammatory cell recruitment, mucus production in the lungs and release of inflammatory mediators. Resident tissue macrophages produce low levels of inflammatory mediators however when they are exposed to danger signals they release cytokines, such as TNF-α, IL-1β and IL-6 as well as nitric oxide (NO). Given the above, the aim of this study was to evaluate the anti-inflammatory potential of the AFL pre-treatment in peritoneal macrophages stimulated with LPS. Methods: Female swiss mice were stimulated with thioglycolate and four days later, peritoneal lavage was performed with RPMI 1640 medium to obtain monocytes. Cells were transferred to culture plates and pre-treated with AFL (100, 50 and 25μg/mL) and, after one hour, it was added LPS (1μg/mL). Twenty four hours later, cell viability, NO and cytokine production were assessed by MTT assay, Griess method and ELISA respectively. Results: AFL (100, 50 and 25μg/mL) pre-treatment did not decrease the viability of the cells but inhibited significantly (P<0.05) NO production (65.6%, 62.2% and 61.1% respectively). AFL, at highest concentration, decreased significantly (P<0.05) IL-1β and TNF-α (33.9% and 55.6%, respectively) without interfered with IL-6 and IL- 10 production. Conclusion: These preliminary data corroborate with previous works where they showed the anti- inflammatory property of the plant in experimental models of inflammation supporting the idea of using the plant extract as a new phytotherapic to treat inflammatory process.
Financial support: CNPq/CAPES/INCT Cancer.
COMPARATIVE EFFECT BETWEEN WARIFTEINE AND METHYL WARIFTEINE IN EXPERIMENTAL MODEL OF ACUTE INFLAMMATION
ADRIANO FRANCISCO ALVES; HERMANN FERREIRA COSTA; LAERCIA PAIVA FERREIRA; MARCIA REGINA PIVEZAM.
UFPB, JOAO PESSOA - PB - BRASIL.
INTRODUCTION: Warifteine and methyl warifteine are alkaloids bisbenzilisoquinolinic of Cissampelos sympodialis, a plant popularly known as milona and used in folk medicine for the treatment of diseases of the respiratory, gastrointestinal and genitourinary systems. Methyl warifteine is the natural methylation of warifteine at carbon 7. AIM: The goal of this study was to compare the anti-inflammatory action of these molecules in experimental models of acute inflammation. METHODS: Swiss mice (n = 5) were treated with PBS, indomethacin (10mg/kg), warifteine (2 mg / kg or 10 mg / kg) or methyl warifteine (2 mg / kg or 10 mg / kg) one h after the administration of phlogistic agents (carrageenan, prostaglandin E2 (PGE2 ) or bradykinin). Edema was measured at 1, 2, 3, 4, 6 and 24 hs for carrageenan, 15, 30 and 60 min for PGE2 and 15 and 30 min for bradykinin. The vascular permeability was assessed by intraperitoneal injection of acetic acid, and the amount of protein was measured by spectrophotometry. RESULTS: Warifteine treatment (2.0 mg / kg or 10mg/kg) inhibited paw edema induced by carrageenan at 4 and 6 h (P <0.05 and P <0.0001 respectively) and PGE2 at 30min (P <0.0001) but it did not reduce the bradykinin edema formation. Warifteine, also decreased significantly (P <0.05) vascular permeability. On the other hand the methylation of the molecule did not interfere with the edema formation or vascular permeability. CONCLUSION: These preliminary results demonstrated that warifteine has anti-inflammatory property however the natural methylation of the molecule did not improve its effect in the experimental models tested.
Financial Support: CNPq and Capes.
COMPARATIVE EFFECTS OF NAPROXEN AND ITS H2S-RELEASING DERIVATIVE ATB-346 ON ALVEOLAR BONE LOSS AND INFLAMMATION IN RATS WITH LIGATURE-INDUCED PERIODONTITIS
AGATHA RIBEIRO SILVA1; BRUNO S. HERRERA2; LEILA S. COIMBRA3; SIMONE A. TEIXEIRA4; SORAIA K. P. COSTA5; JOHN L. WALLACE6; LUIS C. SPOLIDORIO7; MARCELO N. MUSCARÁ8.
1,4,5,8.DEPT. OF PHARMACOLOGY, INSTITUTE OF BIOMEDICAL SCIENCES, SÃO PAULO - SP - BRASIL; 2,3,7.ARARAQUARA SCHOOL OF DENTISTRY, SAO PAULO STATE UNIVERSITY, ARARAQUARA - SP - BRASIL; 6.DEPT. OF MEDICINE, FARNCOMBE INSTITUTE, MCMASTER UNIVERSITY, HAMILTON - CANADÁ.
Introduction: Periodontitis is a chronic inflammatory disease that damages the structures responsible for attachment of the teeth to alveolar bone, which may result in tooth loss. Non-steroidal antiinflammatory drugs (NSAIDs) are widely prescribed for control of a variety of inflammatory conditions, although gastric effects limit their use. In experimental periodontitis, NSAIDs also decrease the associated alveolar bone loss. H2S-releasing NSAIDs result in antiinflammatory compounds with improved gastric safeness. We decided to compare the effects of naproxen with its
H2S-releasing derivative ATB-346 on ligature-induced periodontitis in rats.
Methods and Results: All animal procedures were approved by the local ICB-USP Ethics Committee. A ligature was placed around the lower first molars of anesthetized male Wistar rats; sham animals had the ligature immediately removed. After ligature placement, groups of animals (n=8) started to receive daily equimolar oral doses of naproxen (10 mg/kg), ATB-346 (16 mg/kg) or vehicle (1 ml/kg of 0.5% CMC) during 7 days; a non-treated group was also included. At the end of this treatment period, the animals were anesthetized and euthanized; samples of stomach and gingiva were collected for measurement of myeloperoxidase activity (MPO; a marker of granulocyte infiltration) and interleukin (IL)-1b contents. Bone loss was also evaluated in alveolar bone by both X-ray and histological analysis. Data were analyzed by one-way ANOVA followed by the Student-Neuman-Keuls test. Untreated and vehicle-treated animals showed similar bone loss (0.94±0.06 and 0.96±0.08 mm), which was significantly inhibited by either naproxen (0.74±0.06 mm, P<0.05) or ATB-346 treatment (0.61±0.05, P<0.01). The administration of ATB-346 resulted in marked inhibition of the ligature-induced response in the gingival tissues. Gastric MPO activity increased by more than 4-fold after 7-day treatment with naproxen (P<0.001) but was unaffected by ATB-346 in comparison with the sham group.
Conclusion: These results show that the presence of the H2S-releasing moiety in the ATB-346 compound does not impair the effects of the parent naproxen on periodontitis, but rather prevents the occurrence of deleterious effects on the gastric mucosa due to long-term prostaglandin inhibition.
Financial support: CAPES, CNPq, FAPESP.
COMPARATIVE INFLAMMATION: LONGER DURATION OF INFLAMMATION IN THE ORAL CAVITY THAN IN RAT PAWS
TAISSA IOLANDA CHECON FRADE1; IGOR MENEZES GONÇALVES REGO2; JULIANA OLIVEIRA SILVA3; GEOVANNI DANTAS CASSALI4; Y S BAKHLE5; JANETTI NOGUEIRA DE FRANCISCHI6.
1,2,3,4,6.UFMG, BELO HORIZONTE - MG - BRASIL; 5.IMPERIAL COLLEGE, LONDON - REINO UNIDO.
AIM: Carrageenan injected into the oral cavity of rats induced a rapid and reliable inflammatory response (Ladeira et al, 2011). We have now compared this response with that in rat paws and the effects of general anaesthesia (GA) on these responses. METHODS: Male Holtzman rats (150-200 g, n=5) were anesthetized with a mixture of ketamine- xylazine (62.4;15 mg/kg, 0.2 ml/100 g, i.p.) and then injected s.c. with either 500 mcg/0.1 ml l-carrageenan in the right cheek (RC), via the oral mucosa and 0.1 ml sterile physiological saline in the contralateral side (LC). In another group (n=5), rats were injected intraplantarly (i.pl.) in the right hind paw (RP) with the same dose of carrageenan and saline contralaterally (LP), without anesthesia. To assess inflammatory edema, cheek or paw thickness (in mm) was measured with digital Mitutoyo calipers at 0, 0.5, 1, 2, 3, 4, 5, 6, 24, 48, 72 and 96-h after carrageenan. Another group of animals was injected with carrageenan i.pl., and 1 h later anesthetized with the same dose of the anesthetic mixture. Mean (+ SEM) thickness for each group was calculated and analyzed with one-way ANOVA; P<0.05. RESULTS: Carrageenan significantly increased the cheek thickness by 0.5-h (RC=5.8+0.2; LC=4.8+0.20), reaching a maximal response by 2-h after carrageenan (RC=6.3+0.10; LC=4.8+0.20), whereas edema in the paws was maximal by 3 h of injection (RP=7.0+0.20; LP=5.2+0.20). The maximal response in the cheeks was maintained from 2-6 h, while that in the hind paws decreased over this time, returning to baseline by 24 h. Cheek edema only returned to basal levels 96 h after carrageenan injection. Histological examination of cheek tissues at 3, 6 and 24 h, showed a mononuclear cell infiltrate only at 24 h. Strikingly, a reduced dose of anesthetic mixture (50%) was associated with an increased inflammatory response in the cheeks at 2-h of carrageenan (RC=8.5+0.4; LC=6.7+0.50, P<0.05). However, paw edema was significantly reduced 3-h after carrageenan (RP=6.0+0.03; LP=5.1+0.10) by treatment with the anesthetic mixture, given at 1-h. CONCLUSIONS: Carrageenan induced a longer lasting inflammatory response in the oral cavity than in the rat paw, but this difference was not due to the anesthetic which exhibited antiinflammatory effects in both.
Ladeira PO, Bakhle YS, Francischi JN – A rapid and reliable model of oral oedema to screen for new antiinflamamtory drugs. Inflammation Res 60:S200-201, 2011
Financial Support: CNPq, CAPES
CRITICAL ROLE OF 5-LIPOXYGENASE AND HEME OXYGENASE 1 IN WOUND HEALING
ARIANE RENNÓ BROGLIATO1; ANDREA MOOR2; SHANNON KESL3; RAFAEL F GUILHERME4; JANAINA LIMA5; MARC PETERS-GOLDEN6; CLAUDIO CANETTI7; LISA GOULD8; CLAUDIA F BENJAMIM9.
1,4,5,9.INSTITUTE OF BIOMEDICAL SCIENCES/UFRJ, RIO DE JANEIRO - RJ - BRASIL; 2.5DEPARTMENT OF SURGERY, UNIVERSITY OF SOUTH FLORIDA, TAMPA - ESTADOS UNIDOS; 3,8.DEPARTMENT OF SURGERY, UNIVERSITY OF SOUTH FLORIDA, TAMPA - ESTADOS UNIDOS; 6.DIVISION OF PULMONARY AND CRITICAL CARE MEDICINE, UNIVERSITY OF MICHIGAN, ANN ARBOR - ESTADOS UNIDOS; 7.INSTITUTE OF BIOPHYSICS CARLOS CHAGAS FILHO, FEDERAL UNIVERSITY OF RIO DE JANEIRO/UFRJ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Chronic wounds affect a large number of patients especially elderly, patients with cardiovascular diseases and diabetes. The treatments available are often ineffective and represent high costs to the health systems. This scenario encourages studies based on understanding the mechanisms involved in tissue repair to point out new therapeutics targets. Lipid mediators produced by 5-lipoxygenase (5-LO) are important regulators of inflammation due their ability to activate pro and anti-inflammatory pathways but their role in wound healing is unexplored. Based on our preliminary data in which 5-LO knockout (5-LO-/-) mice showed accelerated wound healing, we aimed to investigate the mechanism involved in the beneficial effect of 5-LO absence on wound healing and its implications in the oxidative response.
Methods: Full thickness wounds were used to evaluate the wound healing and primary cultures of fibroblasts were used to explore the mechanisms involved in the cellular alterations after 5-LO inhibition.
Results: Besides the fast wound healing, 5-LO-/- mice showed up regulated Heme Oxygenase-1 (HO-1) and decreased inflammatory infiltrate in wound tissues. No changes were observed in production of collagen, cytokines, and alpha-smooth muscle actin (α-SMA) levels. In addition, the wound treatment with a specific HO-1 inhibitor (SnPPIX) prevented the accelerated wound healing in 5-LO-/- mice, suggesting the relevant contribution of HO-1 to wound healing in 5-LO-/- mice. However, the production of fibronectin, transforming growth factor-β I and III, and α- SMA expression were unchanged. The production of reactive oxygen species in fibroblasts induced by hyperbaric chamber was inhibited by AA861 incubation. HO-1 was strongly induced by AA861 in a dose-dependent manner which may explain the antioxidant response. Although this induction is independent of Nrf-2 and it is not correlated with the effect of COX-2 inhibitor (NS398).
Conclusion: Taken together, our results show that 5-LO inhibition performs an important modulation of wound healing and fibroblast function, including stimulation of an antioxidant response due to HO-1 induction. Over expression of HO-1 in wounds may facilitate early resolution of inflammation in 5-LO-/- mice improving the wound healing.
Financial support: CAPES, CNPQ, FAPERJ, USF (USA) and Department of Veterans Affairs or the United States Government.
CRUDE EXTRACTS OF PIPERACEAE SPECIES AFFECT PRO-INFLAMMATORY ACTIVITY BY HUMAN MONOCYTES
ANGELA CAROLINA FINATO1; THAIS FERNANDA DE CAMPOS FRAGA-SILVA2; CAMILA MARTINS MARCHETTI3; LIDIANE GASPARETO FELIPPE4; MARJORIE DE ASSIS GOLIM5; AMAURI ALVES DE SOUZA JÚNIOR6; MARIA SUELI PARREIRA DE ARRUDA7; JAMES VENTURINI8.
1.DEFACULDADE CIÊNCIAS - UNESP, BAURU - SP - BRASIL; 2.INSTITUTO DE BIOCIÊNCIAS - UNESP, BOTUCATU - SP - BRASIL; 3,4,5.INSTITUTO DE QUÍMICA - UNESP, ARARAQUARA - SP - BRASIL; 6.FACULDADE DE MEDICINA - UNESP, ARARAQUARA - SP - BRASIL; 7,8.FACULDADE DE CIÊNCIAS - UNESP, BAURU - SP - BRASIL.
Introduction: Several species of the Piper and Peperomia genera have been used in alternative medicine to treat vaginitis, gynecological and intestinal disorders. Furthermore other species of the large family Piperaceae have been identified as a source of several compounds with different properties: psychotropic, antioxidant, antimicrobial and cytotoxic effects. However, the influence of plant extract in the immune system modulation remains to be explored. In the present study we determined the influence of ethanol extract of leaves of two species of Piper (Piperaceae) and one Peperomia on the human monocytes activity.
Methods and Results: The ethanol extracts from the aerial parts of Piper fuligineum Kunth, Piper gaudichaudianum Kunth and Peperomia obtusifolia L. were obtained by maceration using ethanol as solvent and then filtered and dried. The first dilution of the extract was performed in dimethyl sulfoxide followed by subsequent dilution in sterile phosphate buffered saline solution. Sample of blood were collected from six healthy individuals for peripheral blood mononuclear cells (PBMC) isolation. The IC50 of each crude extracts were determinate by MTT assay in the PBMC culture. To access the influence of the Piper and Peperomia crude extracts on the inflammatory profile of monocytes, these cells were simultaneously cultivated with lipopolissacaride (LPS, 10 µg/ml) and the crude extracts, according to the IC50 as following: P. fuligineum - 8.5 mg/ml, P. gaudichaudianum - 10 mg/ml and P. obtusifolia - 43 mg/ml. After 24 hours of incubation, the cells were submitted to determination of peroxide hydrogen (H2O2) production and the supernatants were submitted to determination of TNF-α, IL-8, IL-6, IL-1β, IL-10 and IL-12p70 by flow cytometry. Our results showed that the three Piperaceae species displayed same profile of H2O2 and cytokines productions. LPS- stimulated monocytes showed high levels of H2O2, IL-1β and IL-10 than unstimulated monocytes. Crude extracts- stimulated monocytes showed high levels of IL-12 and lower levels of H2O2, TNF-α, IL-6, IL-10 and IL-8. The monocytes stimulated with crude extract plus LPS showed lower levels of H2O2, TNF-α, IL-6, IL-10, IL-1β and IL-8 and higher levels of IL-12p70 than LPS-stimulated and unstimulated monocytes.
Conclusion. The results suggest that crude extracts of P. fuligineum, P. gaudichaudianum and P. obtusifolia show an expressive anti-inflammatory activity with a potential immunotherapeutical approach.
CURINE INHIBITS EOSINOPHIL ACTIVATION AND AIRWAY HYPER-RESPONSIVENESS IN A MOUSE MODEL OF ALLERGIC ASTHMA
JAIME RIBEIRO FILHO1; ANDREA SURRAGE CALHEIROS2; ADRIANA VIEIRA DE ABREU3; KATHARINNE INGRID MORAES DE CARVALHO4; DIEGO DA DILVA MENDES5; CHRISTIANNE BANDEIRA MELO6; MARCO AURÉLIO MARTINS7; CELIDARQUE DA SILVA DIAS8; MÁRCIA REGINA PIUVEZAM9; PATRÍCIA TORRES BOZZA10.
1,2,3,4,5,7,10.FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL; 6.UFRJ, RIO DE JANEIRO - RJ - BRASIL; 8,9.UFPB, JOÃO PESSOA - PB - BRASIL.
Introduction: Allergic asthma is a chronic inflammatory airway disease with increasing prevalence around the world. Current asthma therapy includes drugs that usually cause significant side effects, justifying the search for new anti- asthmatic drugs. Curine is a bisbenzylisoquinoline alkaloid that modulates calcium influx in many cell types; however, its anti-allergic and putative toxic effects remain to be elucidated. Our aim was to investigate the effects of curine on eosinophil activation and airway hyper-responsiveness (AHR) and to characterize its potential toxic effects. Methods and Results: We used a mouse model of allergic asthma induced by sensitization and challenge with ovalbumin (OVA) to evaluate the anti-allergic effects of oral treatment with curine. The oral administration of curine significantly inhibited eosinophilic inflammation, eosinophil lipid body formation and AHR in animals challenged with OVA compared with animals in the untreated group. The curine treatment also reduced eotaxin and IL-13 production triggered by OVA. Verapamil, a calcium channel antagonist, had similar anti-allergic properties, and curine pre- treatment inhibited the calcium-induced tracheal contractile response ex-vivo, suggesting that the mechanism by which curine exerts its effects is through the inhibition of a calcium-dependent response. A toxicological evaluation showed that orally administered curine did not significantly alter the biochemical, hematological, behavioral and physical parameters measured in the experimental animals compared with saline-treated animals. Conclusion: In conclusion, curine showed anti-allergic activity through mechanisms that involve inhibition of IL-13 and eotaxin and of Ca++ influx, without inducing evident toxicity and as such, has the potential for the development of anti-asthmatic drugs.
Financial support: CNPq, CAPES, FAPERJ, PRONEX.
CYCLO-GLY-PRO ATTENUATES THE NOCICEPTIVE BEHAVIOR AND INFLAMMATORY RESPONSE IN MICE
FERNANDA LIMA TORRES DE AQUINO1; JAMYLLE NUNES SOUZA FERRO2; RENAN GUEDES BRITO3; PRISCILA LAÍSE DOS SANTOS4; JULLYANA DE SOUZA SIQUEIRA QUINTANS5; LUCAS COSTA DE SOUSA6; ALMAIR FERREIRA DE ARAÚJO7; WALDECY LUCCA-JUNIOR8; LUCINDO JOSÉ QUINTANS-JUNIOR9; BRUNO LOURENÇO DIAZ10; EMILIANO DE OLIVEIRA BARRETO11.
1,2,11.UNIVERSIDADE FEDERAL DE ALAGOAS, MACEIÓ - AL - BRASIL; 3,4,5,8,9.UNIVERSIDADE FEDERAL DE SERGIPE, SÃO CRISTÓVÃO - SE - BRASIL; 6,7,10.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL.
FERNANDA LIMA TORRES DE AQUINO(IC)1; JAMYLLE NUNES SOUZA FERRO(PG)1; RENAN GUEDES BRITO(PG)2; PRISCILA LAÍSE DOS SANTOS(PG)2; JULLYANA DE SOUZA SIQUEIRA QUINTANS2; LUCAS COSTA DE SOUSA(IC)3; ALMAIR FERREIRA DE ARAÚJO3; WALDECY LUCCA-JUNIOR2; LUCINDO JOSÉ QUINTANS-JUNIOR2; BRUNO LOURENÇO DIAZ 3; EMILIANO BARRETO1.
1Laboratório de Biologia Celular-UFAL; 2Departamento de Morfologia-UFS; 3Laboratório de Inflamação-UFRJ; Brazil.
Introduction: Cyclo(Gly-Pro) is a biologically active cyclic dipeptide that is found in several fungi. Recent studies have revealed that this dipeptide has neuroprotective activity in ischemic stroke. The present investigation was designed to evaluate the effect of cyclic dipeptide cyclo(Gly-Pro), called CGP, on nociception and inflammation. Methods and results: Anti-nociceptive actions of CGP were assessed in Swiss male mice (25-30g, n=5) using the formalin test and hot-plate test, while the hypernociception was evaluated using carrageenan-induced mechanical hyperalgesia. The neuronal activation patterns were evaluated in the periaqueductal gray area (PAG) by using Fos expression in the CGP-treated mice. The anti-inflammatory action was evaluated using compound 48/80, serotonin and PGE2-evoked paw edema. Data were expressed as mean±SEM and were statistically analyzed using one-way ANOVA. In formalin- induced paw licking test, one hour before stimulus, intraperitoneal injection of CGP (1 and 10µmol/kg) produced a significant inhibition in neurogenic phase from 65.6±6.7s to 40.8±3.5 and 33.7±4.3s and inflammatory phase from 329.5±24.5s to 125.8±26.5 and 108.0±9.5s, respectively. Furthermore, the latency response time during the hot-plate test at 1h was increased significantly (from 5.4±1.5 to 16.1±0.6s, respectively), whereas pretreatment with naloxone reduced (to 7.4±1.3s) the analgesic potential of CGP (1µmol/kg). The CGP induced an increase of 4.5 fold in the number of Fos positive cells in the PAG. Another set of experiments, CGP (1µmol/kg) reduced the carrageenan- induced mechanical hyperalgesia with inhibition of 51%. In addition, the paw edema evoked by C48/80, serotonin and PGE2 was reduced by the CGP in 51%, 26% and 66.5%, respectively. Conclusion: Together, these results may represent a possible therapeutic application of cyclic dipeptide CGP toward alleviating pain and damage caused by inflammation conditions.
Financial support: CNPq, CAPES
CYTOTOXIC EVALUATION OF HERISSANTIA TIUBAE EXTRACT AND ISOLATED SUBSTANCES ON TUMOR CELL LINES
ANA LUISA LIMA1; MARIANA TEXEIRA2; MARCIA REGINA PIUVEZAM3; MARIA FATIMA SOUZA4; WEMERSON MATIAS5.
1,3,4,5.UNIVERSIDADE FEDERAL DA PARAÍBA, JOÃO PESSOA - PB - BRASIL; 2.UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL.
INTRODUCTION: Secondary metabolites of plants have been the target of intensive studies as therapeutic possibilities for various diseases, including different types of cancer. Herissantia tiubae (Malvaceae), popularly known in Brazil’s Northeast, as “mela-bode” or “lava prato” is used in folk medicine for influenza and fever treatment. Flavonoids, diraminosídeo and tilirosídeo, isolated from this plant, as well as neolignan, otobobona-CE-1 and CE-2 from Cordia exaltata; diterpene, kolavelona from Casearea arborea; alkaloid, criptolepinona from Sida rhombifolia and flavonoid, methyl-escutelareina dimethoxy were targets of this study. OBJECTIVES: To evaluate the cytotoxic effect of isolated compounds and Herissantia tiubae extract (EHt) against tumor cells. METHODS: The tumor cell lines: Ehrlich, K562 and Lucena were used. For control, human peripheral blood mononuclear cells (PBMC) was employed. Cell suspensions were seeded in 96-well plates in the presence or absence of different concentrations of the isolated compounds (100, 50, 25, 10, 5, and 1μM) or extract (400, 200, 100, 50, 25 and 10μg/mL) and incubated at 370C for 72h. Cell viability was determined by MTT assay and cell death by Annexin-V and propidium iodide via flow cytometry. RESULTS: The isolated substances were not cytotoxic for the different tumor cells. However, the EHt (400, 200 or 100μg/mL) showed cytotoxic effect on K562 and Lucena, with cell death of 68.6%, 94.7% and 99.55%, 53.37%, 94.14% and 97.7% respectively. However, similar results were observed against normal PBMC. Concentrations at 100, 75, 50, 37.5, and 25μg/mL of EHt showed cell death on Lucena and K562 cells by apoptosis mechanisms rather than necrosis. CONCLUSION: The results demonstrate that isolated compounds showed no toxicity against tumor cells. However, EHt showed a cytotoxic effect against tumor cell lines and normal PBMC. Because, there are no reports of toxicity of EHt taken in vivo as a folk medicine, further in vivo experiments, using mice, should be performed to verify if this extract affects PBMC and/or subpopulations of cells from the immune system.
DASATINIB ATTENUATES LUNG INFLAMMATION AND FIBROSIS IN EXPERIMENTAL SILICOSIS
JOHNATAS DUTRA SILVA1; FERNANDA F CRUZ2; LUCAS F HORTA3; MIQUÉIAS L PACHECO4; ANDRÉ B SILVA5; ANA CLARA T RAMOS SILVA6; CASSIANO F GONÇALVES-DE-ALBUQUERQUE7; HUGO C FARIA NETO8; PATRICIA R M ROCCO9.
1,2,3,4,5,6,9.FEDERAL UNIVERSITY OF RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL; 7,8.FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL.
Introduction/Objectives: Dasatinib is a tyrosine kinase inhibitor that reduces inflammation in experimental lung injury. We hypothesized that therapy with dasatinib could present beneficial effects in silicosis, which is a lung disease associated with inflammation associated with fibrosis.
Methods: 40 female C57BL/6 mice were randomly assigned into two groups. In control (C) group, saline was intratracheally (i.t.) administered (50 µl), whereas silicotic (S) mice received silica (20 mg/50 μl). At day 14, C and S groups were further randomized into subgroups receiving DMSO 1% (100 µl, DMSO) or Dasatinib (1 mg/kg/dose, 100 μl, DAS) through gavage administration, twice a day, for 14 days. At day 28, lung mechanics (resistive and viscoelastic pressures, and static elastance), fraction area of granuloma, number of cells in alveolar septa and granuloma, fraction area of normal and collapsed alveoli; collagen fiber content, as well as levels of transforming growth factor (TGF)-b, interleukin (IL)1-b and tumour necrosis factor (TNF)-a in lung tissue were measured.
Results: At day 28, resistive (915%) and viscoelastic pressures (19%), lung elastance (27%) were higher in S compared to C group. Moreover, S resulted in higher number of neutrophils and mononuclear cells in alveolar septa and granuloma, greater alveolar collapse, collagen fiber content, and levels of TGF-b (69%), IL-1b (501%) and TNF-a (53%). Dasatinib reduced resistive (67%) and viscoelastic pressures (3.9%), lung elastance (18%), as well as neutrophil infiltration in granuloma and alveolar septa, alveolar collapse and amount of collagen. Moreover, dasatinib decreased significantly levels of TGF-b (48%), IL-1b (37%) and TNF-a (48%).
Conclusions: The therapy with dasatinib was effective at modulating the inflammatory and fibrotic processes thus improving lung mechanics in the current experimental model of silicosis.
Financial Support: Coordination Theme 1 (Health) of the European Community's FP7 (TARKINAID). Grant agreement number HEALTH-F4-2011-282095. CNPq, FAPERJ.
DEXAMETHASONE CAN IMPAIR THE ACTIVITY OF SOME ANTIMICROBIAL DRUGS AGAINST STAPHYLOCOCCUS AUREUS AND S. EPIDEMIDIS IN VITRO
MARCUS VINÍCIUS DIAS SOUZA1; PEDRO HENRIQUE FERREIRA MARÇAL2.
1.UNIVERSITY VALE DO RIO DOCE, GOVERNADOR VALADARES - MG - BRASIL; 2.FEDERAL UNIVERSITY OF JUIZ DE FORA, GOVERNADOR VALADARES - MG - BRASIL.
Introduction: Staphylococcus aureus and S. epidermidis are commensal of the human skin and nares, and are frequent pathogens in skin and soft tissues infections as well as bacteremia and sepsis. Currently, they are the main biofilm producers in clinical settings, the leading virulence factor in staphylococci. Dexamethasone (DEX) is often combined to antimicrobial drugs (AMD) in clinical protocols for staphylococcal diseases such as osteomielytes and endocarditis, to mitigate toxic effects of AMD and to keep the inflammatory process under control. DEX could theoretically impair the activity of AMD, and despite the large clinical use of such combinations, the effects of DEX on the activity of AMD remains poorly explored. Therefore, this study aimed to assess if dexamethasone decreases the in vitro antimicrobial activity of systemic drugs against planktonic cells and formed biofilms of clinical isolates of S. aureus and S. epidermidis. Methods and Results: The antimicrobial activity of several AMD was tested in triplicate in broth microdilution assays using 96-well polystyrene microtiter plates against 13 clinical isolates of S. aureus and S. epidermidis in log phase, combined or not to DEX (1mg/ml) in the following standard doses (mg/ml): meropenem (500), clindamycin (150), cloranfenicol (1000) and amoxycilin combined to potassium clavulanate (500/100). Formed biofilms (24h growth) were also exposed to the drugs in the same way. Possible antimicrobial or antibiofilm effects of DEX were assessed. Viability staining tests could not be performed due to the yellow color of some drugs, thus, following incubation, aliquots of all combinations were dispensed in Mueller-Hinton agar plates, and bacterial growth was observed after 24h. All AMD were effective against planktonic cells and biofilms of all strains, except amoxycilin (active for only 5 strains). DEX had no antimicrobial activity alone; however, bacterial grow and biofilm formation was seen in all strains in all combinations of DEX and AMD. Once AMD and DEX have different pharmacological targets, it is possible that chemical interferences are important for such results. Conclusions: DEX can impair the activity of the tested antimicrobials when combined in vitro against Staphylococci, what may influence clinical outcomes of the pharmacological treatment. More studies are being conducted for assessing safety in vivo. Acknowledgments: FAPEMIG, CAPES.
EFECT OF GLUTAMINE SUPLEMMENTATION ON INDUCED ULCERATIVE COLITIS IN RATS
MARIA DO SOCORRO DO MEDEIROS AMARANTE; ANA KEILA QUEIROZ DA SILVA; MARYANNE NUNES MELO; GERLANE COELHO BERNARDO GUERRA; CHRISTINA DA SILVA CAMILLO.
FEDERAL UNIVERSITY OF RIO GRANDE DO NORTE, NATAL - RN - BRASIL.
Introduction: Inflammatory bowel diseases include a set of inflammatory chronic disorders, as ulcerative colitis (UC) and Chron’s disease. UC treatment is based on anti-inflammatory drugs administration, however, these medicines causes undesirable side effects, leading to a search for new and alternative therapies. The goal of this study was to evaluate the effect of glutamine supplementation on clinical aspects, intestinal mucosa structure and inflammation markers in response to acetic acid induced UC.
Methods and Results: Acetic acid 10% solution was used to induce UC in adult Wistar rats. A set of animals received the standard therapy by sulfasalazine (SSZ) (500 mg/Kg/day), other set of animals received glutamine supplementation (1 g/Kg/day). The animals were euthanized (n=25) and the colon segment was exposed to macroscopic analysis. Colon fragments were used for histopathological evaluation by HE staining and determining of malonaldehyde (MDA) and myeloperoxidase (MPO) concentration in the tissue. Results are expressed as mean plus standard deviation. The differences between means was analyzed by ANOVA with significance level of 5% (p<0.05). SSZ-treated animals exhibited increased weight loss compared to those supplemented with glutamine, additionally these animals showed similar macroscopic colon damage. Histopathological analysis showed a protective role for glutamine on the colonic mucosa in relation to SSZ. The concentration of inflammatory markers MDA and MPO appeared reduced in the colon of animals supplemented with glutamine or SSZ compared to the untreated group.
Conclusions: These results suggest that the use of glutamine can reduce tissue damage observed in UC through the stimulation of cell proliferation. Thus, we may conclude that glutamine has benefic effects upon ulcerative colitis in rats.
EFFECT AND MECHANISM OF ESCULIN IN EXPERIMENTAL MODEL OF INFLAMMATORY BOWEL DISEASE
ALINE WITAICENIS FANTINATI; ALEXANDRE DA SILVEIRA CHAGAS; LUIZ CLAUDIO DI STASI.
DEPARTAMENTO DE FARMACOLOGIA, INSTITUTO DE BIOCIÊNCIAS, UNESP, BOTUCATU - SP - BRASIL.
Introduction: IBD is a chronic inflammatory condition of the gastrointestinal tract. It is described that reactive oxygen species are involved in the development of tissue injury and antioxidants compounds showed beneficial effects. Esculin (6,7-dihydroxycoumarin-6-o-glucoside) is a coumarinic derivative known as lipoxygenase inhibitor, hydroxyl radicals scavenger and gastroprotector. Furthermore, esculin is hydrolyzed by colonic bacteria in glucose and esculetin that has antioxidant and intestinal anti-inflammatory activity. Our aim was to evaluate the intestinal anti- inflammatory activity of esculin and to identify its mechanism of action.
Methods and Results: Anaesthetised rats (n=8) were treated daily (p.o.) with 25 mg/kg of esculin or 50 mg/Kg of sulphasalazine (reference drug) for 4 days and 2 hours prior to TNBS administration into the colon (preventive protocol) or 2 hours after this administration and daily until the 7th day (curative protocol). No treatments ameliorated the macroscopic parameters (score, extension of lesion, colonic weight/length ratio) in both protocols. In preventive protocol, esculin and sulphasalazine vs. control group, inhibited myeloperoxidase (MPO) activity (1348.2±114.6 and 1188.1±110.6, vs. 1792.5±173.6 U/g tissue) and reduced the colonic levels of IL-1β (938.1±116.5 and 940.7±67.4 vs. 1510.8±83.3 pg/mg protein). In curative protocol, esculin and sulphasalazine vs. control group, inhibited MPO activity (755.8±208.9 and 569.0±76.4 vs. 1250.9±177.3 U/g tissue), maintained glutathione levels (2006.7±238.2 and 1669.3±174.3 vs. 1127.1±77.1 nmol/g tissue) and reduced the colonic levels of TNF-α (31.3±1.1 and 21.85±2.5 vs. 47.1±3.0 pg/mg protein). Esculin also reduced the colonic levels of IL-1β (1525.6±226.5 vs. 2319.1±290.3 pg/mg protein control) and increased levels of IL-10 (572.9±43.6 vs. 382.1±50.8 pg/mg protein control).
Conclusion: Esculin showed intestinal anti-inflammatory activity with half of the dose of sulphasalazine and exerts its activity by inhibiting pro-inflammatory cytokine secretion and increasing both levels of anti-inflammatory cytokines and antioxidant defences.
Financial Support: FAPESP (11/50824-4; 11/50512-2)
EFFECT OF BETA-CARYOPHYLLENE IN THE LUNG INFLAMMATORY RESPONSE INDUCED BY MYCOBACTERIUM BOVIS (BCG)
MAGAIVER ANDRADE SILVA1; ELAINE CRUZ ROSAS2; SIMONE CAVALHER MACHADO3; TATIANA ALMEIDA PÁDUA4; PATRICIA PACHECO5; MARIA DAS GRAÇAS HENRIQUES6.
1,2,3,4,5.LABORATÓRIO DE FARMACOLOGIA APLICADO DE FAR-MANGUINHOS, FUNDAÇÃO OSWALDO CRUZ, FIOCRUZ,RJ, RIO DE JANEIRO - RJ - BRASIL; 6.LABORATÓRIO DE FARMACOLOGIA APLICADO DE FAR-MANGUINHOS; INCT/IDN/CDTS, FIOCRUZ, RJ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Mycobacterium tuberculosis complex (MTBC) members, the causative agents of human and animal tuberculosis, are a highly adapted pathogens that have evolved different strategies to subvert the immune and metabolic responses of host cells However, few studies have addressed the modulatory effects of the host response in the development of the disease. Therefore,we investigated the effects of the plant metabolite, β-caryophyllene (BCP), a natural agonist of endogenous cannabinoid 2 receptors(PNAS, 105:9099-9144, 2008) in the lung inflammatory reaction induced by bacillus Calmette-Guérin (BCG) infection.
Methods and Results:C57BL/6 mice were treated orally 1 hour before and 24h after BCG nasal instillation (BCG, 1x106 UFC; i.n) with BCP (50 mg/kg) and the effects were evaluated 48 hours after infection from bronchoalveolar lavage fluid (BALF). All protocols were approved by the Fundação Oswaldo Cruz animal Welfare Committee. BCP inhibited total leukocyte(6.60±1.49 to 1.67±0.25 x105/lung), mononuclear cells (2.45±0.32 to 1.56±0.28 x105/lung), neutrophil (4.00±1.16 to 0.10±0.03 x105/lung) and eosinophil (0.13±0.04 to 0.003±0.00 x105/lung) accumulation in BALF after BCG infection.We also showed that BCP was able to reduce the production of IL-10 (19.30±7.57 to 0,26±0.52 pg/mL), MCP-1 (1.20±0.047 to 0.00±0.00 pg/mL), IFN-γ(168.90±39.15 to 0.37±0.75 pg/mL)and IL-12 (12.68±5.17 to 0.42±0.34 pg/mL).BCP also inhibited the lipid bodies (LB) formation in total cells (27.96±0,42 to 9.50±0,49 LB/cell). The results are expressed as mean ± SEM using from 6 to 8 animals.
Conclusion:Our results indicate that BCP promotes in vivo a down regulation of the inflammatory response caused by Mycobacterium bovis BCG infection. However, further studies are needed to investigate its mechanism of action.
Financial Support: CAPES; Faperj; CNPq
EFFECT OF INFLIXIMAB ON SILICA-INDUCED PULMONARY FIBROSIS IN MICE.
BIANCA TORRES CIAMBARELLA; ANA CAROLINA ARANTES; TATIANA PAULA TEIXEIRA FERREIRA; PATRÍCIA BARBOSA JURGILAS; ANA LÚCIA PIRES; RENATO SÉRGIO BALÃO CORDEIRO; MARCO AURÉLIO MARTINS; PATRÍCIA MACHADO RODRIGUES E SILVA.
OSWALDO CRUZ INSTITUTE/FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: TNF-alpha has been described as a pleiotropic mediator known to induce leukocyte recruitment and NF-kappaB activation. We previously demonstrated the involvement of TNF-alpha in the airways hyperreactivity and granuloma formation in silicotic mice, indicating that it may be a potential therapeutic target in the disease. In this study we investigated the effect of a TNF-alpha neutralizing antibody infliximab (Remicade®) on the experimental silicosis in mice. Methods and Results: Male Swiss-Webster mice were instilled intranasally silica particles (10 mg) and saline (control). Infliximab (1.25 and 2.5 mg/kg) was given i.p. into mice once a day on day 7, 14 and 21 post- silica provocation. The parameters analyzed consisted of i) lung function (resistance and elastance) and airways hyperreactivity to increasing concentrations of methacholine (invasive whole body plethysmography); ii) lung tissue morphology and morphometry; iii) collagen deposition (Sircol method); iv) cytokine quantification (ELISA) and metaloprotease (MMP)-2 and -9 activity (zymography). Our data showed that silicotic mice had an increase in the basal levels of lung resistance and elastance together with hyperreactivity to aerosolized methacholine. A marked granuloma formation, mediator generation (MIP-1alpha, MIP-2, INF-gamma and TNF-alpha) and MMP-2 and -9 activation were also noted in the lung tissue of silicotic mice as compared to controls. Treatment with infliximab, at the dose of 1.25 mg/kg, inhibited lung function failure, airways hyperreactivity, leukocyte infiltration and fibrosis. Values of granuloma area reduced from 37.85% ± 3.88% to 20% ± 3.375% (mean ± SEM; n=7, p<0.05) in silicotic mice treated with vehicle and infliximab, respectively. Cytokine and chemokine generation as well as MMP-9 release were also markedly reduced by infliximab. The compound failed to alter the MMP-2 release in the silicotic mice. Conclusion: Altogether our findings show that treatment of silicotic mice with the TNF-alpha neutralizing antibody was effective to inhibit important features of the disease including lung function and tissue compromise. They also reinforce the proposition that TNF-alpha is an important therapeutic target in silicosis and indicated that treatment with infliximab seems to constitute a promising pharmacological tool for the treatment of chronic fibrotic diseases such as silicosis.
Financial support: FIOCRUZ/CNPq/FAPERJ.
EFFECT OF JMF2-1, A NON-ANESTHETIC LIDOCAINE ANALOGUE, ON PATHOLOGICAL CHANGES TRIGGERED BY HOUSE DUST MITE IN A MURINE MODEL OF ASTHMA
JAMIL ZOLA KITOKO; PRISCILLA CHRISTINA OLSEN; TATIANA PAULA FERREIRA; ANA CAROLINA ARANTES; MARCO AURÉLIO MARTINS.
OSWALDO CRUZ FOUNDATION, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Asthma is one of the most common chronic diseases in developed countries. In accordance to the need of pharmacologic advances in asthma treatment we have studied the antiasthmatic properties of JMF2-1. JMF2-1 inhalation prevented cardinal features of asthma in an experimental model of BALB/c mice sensitized and challenged with ovalbumin. We sought to evaluate here the effect of JMF2-1 inhalation in a more clinically relevant experimental model of asthma. Methods and Results: AJ mice were intranasally challenged with house dust mite (HDM) extract 3 times a week, for 3 weeks. JMF2-1 (2 %) was aerosolized during the last week of antigen challenge for 30 min and 1 h before HDM administration. Analyses were performed 24 h after the last challenge. Analysis of leucocyte infiltrate in broncoalveolar lavage fluid (BALF) showed that JMF2-1 reduced leukocyte infiltration (21.96 x105 ± 2.82, n= 7), including reduced eosinophil counts (6.94 x105 ± 1.23, n= 7), both generated by HDM exposure (respectively, 39.75 x105 ± 5.22, n= 7 and 22.67 x105 ± 5.22, n= 7). Flow cytometry analyses did not reveal changes in CD4 or CD8 T cells populations in BALF when compared to JMF2-1 treated HDM-challenged group. Using invasive whole body plethysmography, we found that JMF2-1 ameliorates airway hyperresponsiveness (AHR), as observed by reduction of resistance (3.76 ± 0.37, n= 6) and elastance (121.4 ± 14.54, n= 6), both increased by HDM challenge (respectively, 5.26 ± 0.59, n= 5 and 165.0 ± 17.56, n= 6). Lung sections stained with H&E showed peribronchiolar inflammatory pockets while PAS staining showed increased presence of mucus producing cells, both in HDM-challenged group (respectively, 4.86 ± 0.14, n= 7 and 3.16 ± 0.13, n= 6), but no reduction of this parameters was observed in mice treated with JMF2-1 (respectively, 4.0 ± 0.27, n= 8 and 3.09 ± 0.28, n= 6). All data are presented as mean ± SEM. License of FIOCRUZ’s Ethics Committee on Use of Laboratory Animals approval is LW-23/10. Conclusion: JMF2-1 inhalation reduced critical features of asthma triggered by HDM, such as eosinophil lung influx and AHR, as observed in the OVA-challenged model. In view of the fact that most of asthmatics are typically HDM allergic, these results open new perspectives for a future success of clinical application of JMF2-1, although more investigations are necessary, since several pathological changes remained unaltered in this model. Financial Support: CNPq, FAPERJ.
EFFECT OF LOW LEVEL LASER IN MYOBLAST DIFFERENTIATION SUBMITTED TO BOTHROPS JARARACUSSU VENOM
STELLA REGINA ZAMUNER1; ANA TEREZA FRANCO2; LUCIANA MIATO SILVA3; JOSE CARLOS COGO4; SILVIA FERNANDA ZAMUNER5.
1,2,3,5.UNINOVE, SÃO PAULO - SP - BRASIL; 4.UNIVAP, SÃO JOSE DOS CAMPOS - SP - BRASIL.
Introduction: Local myonecrose is a common consequence in envenoming caused by snakes of the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse the local myonecrose. Thus, there is an urgent need to find therapies that can complement antivenoms in the neutralization of local tissue damage. The low level laser therapy (LLLT) is being considered as an alternative treatment for muscle injury situations because its bioestimulation effect. Objective: This study analyses the effect of LLLT on myoblast differentiation submitted to injury by Bothrops jararacussu venom (BjssuV). Methods and Results: C2C12 myoblast were divided into tubes according to each experimental group and received the venom (12.5 mg/mL) or culture medium alone (control) and were centrifuged for 2 min. Cells were irradiated at the bottom of the tube for 13s with a laser at 635 and 830nm, 4 J/cm2 dose and power of 100 mW. Subsequently, the cells were plated on 13 mm coverslips in 24 well plates and incubated for 15, 30 and 60 min. After this time the cells supernatant were removed and immediately adding to cells DMEM medium, containing 2% horse serum to induce differentiation, and incubated at 37°C with 5% CO2 for 4 days. The differentiation of myoblasts was determined by morphological analysis of the formation of multinucleated myotubes. The results showed that untreated cells incubated with 2% horse serum exhibited an elongated and slim form. Cells incubated with the venom died, it occurred in all periods of incubation. However, cells that received the venom and were laser irradiated exhibited a similar morphology to the control cell, showing elongated cells and slim form, which characterizes the differentiation of C2C12 muscle cells.
Conclusion: BjssuV is toxic to muscle cells and LLLT protected against this effect and cause muscle differentiation. The use of LLLT should be considered as a potentially useful therapeutically approach for treatment of the local effects of Bothrops snakebites.
Financial Support: 2011/04660-0 and 2011/14376-7 São Paulo Research Foundation (FAPESP).
EFFECT OF LOW LEVEL LASER THERAPY (LLLT) ON BOTHROPS JARARACA VENOM-INDUCE MYOTOXICITY IN ENDOTHELIAL CELL
ANA BARUFI FRANCO1; STELLA REGINA ZAMUNER2; LUCIANA MIATO SILVA3; CATARINA TEIXEIRA4; SILVIA REGINA ZAMUNER5.
1,2,3,5.UNIVERSIDADE NOVE DE JULHO, SÃO PAULO - SP - BRASIL; 4.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL.
Introduction: Hemorrhage is a common consequence in envenoming caused by snakes of the genus Bothrops which occurs through the action of toxins that acts directly in the endothelial cells. Antivenom therapy and other first-aid treatments do not reverse the local injury and prevent systemic evolution. Thus, there is an urgent need to find therapies that can complement antivenoms in the neutralization of local and systemic damage. The low level laser therapy (LLLT) is being considered as an alternative treatment for endothelial injury situations because its bioestimulation effect. Objective: The present study was designed to investigate the effect of LLLT on endothelial cells tEnd submitted to injury by Bothrops jararaca venom (BjV). Methods: tEnd cell line was used. We analyzed cell viability by MTT assay and cell detachment by crystal violet. The cells were grown in culture medium DMEM supplemented with 10% of fetal bovine serum, incubated at 37°C with 5% CO2 for 48 hours for cell attachment, after that, the cells received BjV venom in the concentration (10 µg/mL). Cells were irradiated for 10 s immediately after the venom administration with a semiconductor laser at 660 and 780 nm, dose of 4 J/cm2 and power of 100 mW and were incubated for 30 and 60 minutes. The cells that did not receive venom served as control. Results: Our results showed an increase of 31% of cell integrity at 60 min, after LLLT by 660 nm. However, LLLT did not increase cell viability by tEnd endothelial cell at 660 and 780 nm, in 30 and 60 min after the venom administration. Conclusion: BjV causes alterations in the viability and integrity of endothelial cells. LLLT protect the cell integrity probably by acting the integrins of the cellular matrix, but not affect the viability caused by the venom. Financial support: PROSUP/Capes.
EFFECT OF RESVERATROL ON APOPTOSIS AND SUPEROXIDE PRODUCTION OF CANINE NEUTROPHILS
MARINES CASTRO1; BRENO FERNANDO MARTINS ALMEIDA2; MARIA FERNANDA CEREIJIDO BERSANI FINK3; ANELISE MARIA BOSCO4; PRISCILA PREVE PEREIRA5; MARIA RITA PACHECO6; VALÉRIA MARÇAL FÉLIX LIMA7; PAULO CESAR CIARLINI8.
1,6.FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS, JABOTICABAL - SP - BRASIL; 2,3,4,5,7,8.FACULDADE DE MEDICINA VETERINÁRIA, ARAÇATUBA - SP - BRASIL.
Introduction: After complete activation, the oxidative metabolism of neutrophils generates great amounts of reactive oxygen species (ROS). When the production of ROS is excessive, these substances react with cell components promoting apoptosis in vitro and in vivo, impairing the function of the cell and compromising the innate immune response. There is evidence that resveratrol (RV) inhibits ROS production and decreases apoptosis in a large variety of cells. However there are no studies on the antioxidant effect of RV in oxidative metabolism and apoptosis of canine neutrophils. This study aimed to examine the possible protective effect of different concentrations of RV on apoptosis and superoxide production of neutrophils stimulated with camptothecin (CAM) and phorbol 12-myristate 13-acetate (PMA), respectively.
Methods and Results: Neutrophil suspensions (106/mL) from 10 healthy adult dogs were incubated with different final concentrations of resveratrol (0, 30, 60 and 90 µM) for 4 hours. After this period, apoptosis and superoxide production rate were determined by capillary flow cytometry using Annexin V-PE and the probe hydroethidine, respectively. In presence of CAM (3 mM) and PMA (0,27 µM) the apoptotic index and superoxide production were increased in all treatments, confirming inducing effect of these drugs on canine neutrophils. The superoxide production, viability and apoptosis rate of neutrophils incubated with different concentrations of RV did not differ between the tested concentrations and the control without RV.
Conclusion: Besides the antioxidant capacity of resveratrol, in the tested concentrations, no protective effect was observed on apoptosis and superoxide production of canine neutrophils in the presence of stimulus.
Financial support: FAPESP (Proc. 2011/08081-4)
EFFECT OF THE HO-1 IN THE DEVELOPMENT OF BLEOMYCIN-INDUCED PULMONARY FIBROSIS IN INVARIANT NKT CELLS DEFICIENT MICE
FELIPE GRABARZ; MARISTELLA ALMEIDA LANDGRAF; TÁRCIO TEODORO BRAGA; NIELS OLSEN SARAIVA CÂMARA.
USP, SÃO PAULO - SP - BRASIL.
FELIPE GRABARZ(1); MARISTELLA ALMEIDA LANDGRAF(1); TÁRCIO TEODORO BRAGA(1); NIELS OLSEN SARAIVA CÂMARA(1)
(1). Laboratory of Immunobiology Transplantation, Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
Introduction: Pulmonary fibrosis is a potentially fatal disease caused as a result of defective healing of the lung in response to an injury of the alveolar epithelium. NKT cells can mediate a protective effect against fibrosis by producing and secreting IFN-Ϫ. It is also reported the protective role of the enzyme heme oxygenase-1, as its byproducts can act attenuating the inflammatory process, protecting from oxidative stress and demonstrate antiapoptotic effects. Currently, there is no therapeutic treatment available for this disease, which contributes to the increase of mortality among the patients due to ventilatory insufficiency. From these facts, our project aimed to clarify whether the pre-treatment of mice with HO-1 can compensate the absence of NKT cells in the progression of the disease and attenuate the development of the disease.
Methods and results: To induce pulmonary fibrosis, bleomycin sulfate (5mg/kg) was administrated intra-tracheally (i.t.) in C57BL/6 and Jα18-/- mice. The pre-treatment of HO-1 in mice was made 24 hours before bleomycin administration (n=5 per group). After 14 days of bleomycin administration, mice were euthanized and had lungs harvested and analyzed. Animals treated with hemin (HO-1 inductor) had fibrosis attenuated when compared to control. In the bronchoalveolar lavage assessment, fewer cells recruited to lung site were detected as well as less inflammation. Also, decreased levels of hydroxyproline and collagen deposition in lung tissue were measured. Moreover, the treatment with hemin impaired the synthesis of pro fibrotic cytokines preventing the fibrosis development.
Conclusion: In the experimental model of pulmonary fibrosis, hemin treatment provides an improvement in the course of the development of the disease impairing a fibrotic scenario. However, the cellular and molecular mechanisms of the disease still have to be elucidated.
Financial Support: FAPESP, CNPq.
EFFECT OF THE LECTIN FROM THE RED ALGA SOLIERIA FILIFORMIS (KÜTZING) P.W. GABRIELSON IN ACUTE INFLAMMATION MODELS CARRAGEENAN INDUCED.
TICIANA MONTEIRO ABREU1; NATÁSSIA ALBUQUERQUE RIBEIRO2; ALEXIA NATHÁLIA ASSEF3; HELLÍADA VASCONCELOS CHAVES4; MIRNA MARQUES BEZERRA5; ÉRIKA FREITAS MOTA6; NORMA MARIA BENEVIDES7.
1,2,3,7.DEP. OF BIOCHEMISTRY AND MOLECULAR BIOLOGY; UFC, FORTALEZA - CE - BRASIL; 4.FACULTY OF DENTISTRY; UFC, SOBRAL - CE - BRASIL; 5.FACULTY OF MEDICINE; UFC, SOBRAL - CE - BRASIL; 6.DEPARTMENT OF BIOLOGY; UFC, FORTALEZA - CE - BRASIL.
Introduction: Inflammation is a process that occurs as a natural response of multicellular organisms to agents foreign. This process can be controlled with anti-inflammatory drugs, which may cause side effects. Therefore, there is given greater emphasis to the study of natural compounds that have anti-inflammatory activity without causing many side effects. Among these compounds, are the lectins. Thus, the aim of this study was to investigate the anti- inflammatory effect of the lectin from the red alga Solieria filiformis (LSf) in acute inflammation models induced by carrageenan (Cg) in rats. Methods and Results: LSf was purified by extraction with Tris-HCl buffer 25 mM (pH 7,5), precipitation with ammonium sulfate (70%) and chromatographies in DEAE-cellulose and Sephadex G-100 columns. The use of animals in this study was approved by the Ethics Committee on Animal Research of the UFC (nº 02/11). The evaluation of the anti-inflammatory effect was performed in male rats Wistar (n=6) that were pretreated with LSf (1, 3 or 9 mg/kg;iv), 30 min before of the Cg injection. The models used were peritonitis and paw edema induced by Cg, and, in both models, was performed the dosage of myeloperoxidase (MPO) levels. To analyze the involvement of the hemoxygenase pathway in the anti-inflammatory activity, the paw edema model was performed with animals pretreated with zinc protoporphyrin IX (ZnPP;3 mg/kg;sc). Saline (NaCl 0,9%) and dexamethasone (1 mg/kg) were used as negative and positive controls, respectively. The data are presented as the means + S.E.M. Variance analysis (ANOVA) followed by the Bonferroni’s test was used to compare means, being considered significative p<0,05. LSf (1, 3 and 9 mg/kg) reduced the total leukocyte migration in 39,7; 67,9 and 76,2%, respectively, and neutrophils migration in 48,1; 75,5 and 80,7% , respectively. Moreover, the LSf was able to reduce the paw edema in the time from 1-5 h. Confirming these data, LSf also was able to reduce MPO levels. The ZnPP IX application changed the anti- inflammatory effect of the LSf, decreasing the capacity of this lectin in to reduce the edema. Conclusion: The LSf was able to reduce the acute inflammation induced by Cg, reducing the neutrophils migration in the peritonitis and in the paw edema models. Its mechanism of action may be related to activation of the hemoxigenase-1 pathway. Thus, the LSf showing itself as a promising molecule in controlling inflammation.
Financial support: UFC and CNPq.
EFFECTS OF A LECTIN FROM COLOCASIA ESCULENTA (TARO) ON HEMATOPOIETIC PROGENITORS FROM MOUSE BONE MARROW
ANNA CAROLINA NITZSCHE TEIXEIRA FERNANDES CORRÊA1; PATRICIA RIBEIRO PEREIRA2; FENANDA MARTINS BELTRÃO3; JULIA CRUZ MARIANI4; MARIA DE FÁTIMA BRANDÃO PINHEIRO5; JOAB TRAJANO SILVA6; GERLINDE AGATE PLATAIS BRASIL TEIXEIRA7; MAURICIO AFONSO VERICIMO8.
1,3,4,5,7,8.UFF, NITERÓI - RJ - BRASIL; 2,6.UFRJ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: In terms of chemical composition, the diversity of substances occurring in nature is vast and as the demand for biologically active products has intensified it is likely that natural products derived from plants may be useful for the development of new drugs. Recently we isolated a globulin with lectin activity from yam (Colocasia esculenta) named Tarina (TR). Objectives:Verify the effect of TR on the recovery of cyclophosphamide (CY) myelosuppressed animals thus it’s applicability as a bone marrow immunostimulatory agent. Methods and Results:In vivo - BALB/c mice received intraperitoneally the following treatments: S) Saline, C) CY-300 mg/kg, TC) TR-200 mg/kg + Cy-300 mg/kg and T) TR-200 mg/kg. Blood samples were obtained to determine the number of leukocytes on days 0, 2, 5, 7, and 12. In vitro - bone marrow cells (BM) extracted from normal BALB/c mice were cultivated in the presence of TR or with a combination of IL3 + erythropoietin. The number of blast colonies forming unit (CFU) of myeloid and erythroid progenitors was evaluated. To determine significance, analysis of variance (ANOVA) with Tukey post test was used. The profile of the in vivo circulating leukocytes are as follows - TR administration induced a transient increase (D0 - 9,81±0,6/106; D2 - 19,12±1,5/106; D5 - 12,66±2,0/106; D7 - 11,08±1,38/106; and D12 - 9,51± 0,9/106) and as expected CY treatment resulted in a profound reduction of leukocytes (D0 - 12,5±1,1/106; D2 - 1,16±0,4/106; D5 - 0,35±0,2/106; D7 - 4,87±0,8/106 and D12 - 4,37±0,8/106 while the group treated with TR + CY, showed a partial reduction in the number of leukocytes D0 - 10,5±0,7/106; D2 - 4,51±0.9/106; D5 - 5,25±0,7/106; D7 - 6,08±1,8/106 and D12 - 4,13± 0,9/106 with no significant alteration in the control group. In the in vitro assays, we found that regardless of treatment, the number of polymorphonuclear cells (PMN) were profoundly reduced on day 9 (TR – from 82 ± 7% to 8 ± 3% and Control – from 86 ± 10% to 0 ± 0%). However, on 19th day a significant increase in PMN cells was observed only in the cultures treated with TR (53 ± 9%) p <0.001. In the TR and IL3+EPO cultures CFU myeloid progenitors were observed. Conclusion:TR was able to maintain the viability of myeloid progenitors and stimulate the proliferation of the granulocyte lineage in a methylcellulose cultures suggesting that TR may act as a stimulator of bone marrow progenitors of the granulocyte lineage and substitute IL3. FOPESQ/UFF
EFFECTS OF ANASTROZOLE ON LIGATURE-INDUCED PERIODONTITIS IN OVARIECTOMIZED RATS
THAYANNE BRASIL1; IRACEMA MATOS MELO2; VILANA MARIA ADRIANO ARAUJO3; MARIANA VASCONCELOS GUIMARAES4; ANA CARLA SILVA SANTOS5; LUCIANA CARVALHO CANDIDO6; ANA PATRICIA SOUZA LIMA7; GERLY ANNE CASTRO BRITO8; RONALDO ALBUQUERQUE RIBEIRO9; VILMA LIMA10.
1,9,10.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 2,3,4,5,6,7.FACULTY OF PHARMACY, DENTISTRY AND NURSING, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 8.DEPARTAMENT OF MORPHOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Biosynthesis of estrogen is catalyzed by aromatase enzyme and its inhibition is important for breast cancer therapy. Patients who use aromatase inhibitors as Anastrozole (ANA) show a higher number of fractures and lower bone density. Periodontitis is characterized by inflammatory response and alveolar bone loss (ABL). The aim of this study was to investigate whether ANA affects the periodontitis in ovariectomized rats (OVX). Methods and Results: Initially, rats were divided into 3 groups, Normal (NOR; n=7), OVX (n=28) and Sham-OVX (S-OVX; n=7). After 14d, the OVX group was divided into 4 groups (n=7), submitted to ligature around the upper 2nd molar and receiving vo saline (SAL) and ANA (0.02, 0.1 or 0.5 mg/kg). After 11d, periodontitis was analyzed by macroscopy (mm2), histometry (mm), histology (scores) and myeloperoxidase activity (MPO). Serum dosages of estrogen, leukogram, weigth variation and liver, renal and spleen analysis were also performed. The data were presented as mean±standard error of the mean or median (and range). 14d of ovariectomy reduced estrogen and lasted until 11thd after ligature (d-14=47.5±3.4; d0=32.7±3.6; d11=31.7±4.6 pg/ml; p<0.05). Ligature caused intense ABL (NOR=0.0±0.0; S-OVX=5.3±0.4; p<0.05), being corroborated by histometry (NOR=0.08±0.01; S-OVX=0.3±0.05). OVX only or associated with ANA did not increase ABL (p>0.05) after 25d of OVX (Macroscopy: SAL=4.68±0.37; ANA0.02=5.15±0.2; ANA0.1=5.4±0.4; ANA0.5=5.08±0.4); histometry: SAL=0.3±0.03; ANA0.5=0.3±0.03). The ligature also caused cell infiltration and intense destruction of alveolar bone, cementum and periodontal ligament in S-OVX (p<0.05) [NOR=0(0-1); S-OVX=3(1-3)], but these parameters were not worse (p>0.05) in SAL or ANA [SAL=2(1-3); ANA 0.5=2(1-3)]. Although OVX only did not increase MPO when compared to S-OVX, ANA0.5 demonstrated an important (p>0.05) increase in MPO when compared to SAL (NOR=0.76±0.15; S-OVX=3.9±0.7; SAL=3.9±1.2; ANA 0.5=14.35±3.8). On the 11thd OVX combined or not with ANA caused leukocytosis, but did not cause any changes in liver, renal or spleen. SAL group showed more gain of weigth compared to S-OVX, but similarly to ANA weight curve (p<0.05). Conclusion: Although the reduction of estrogen-induced 25 days of OVX with the last ones 11 days of administration of ANA, have increased the early inflammatory response, it was not sufficient to increase alveolar bone loss in short term periodontitis. Financial support: Capes; CNPq.
EFFECTS OF CAFFEINATED AND DECAFFEINATED COFFEE INTAKE IN THE INFLAMMATORY ALTERATIONS ASSOCIATED WITH OBESITY
CÍNTIA RABELO E PAIVA CARIA; CAROLINE CANDIDA DE OLIVEIRA; ÉRICA MARTINS FERREIRA GOTARDO; SIMONE COGHETTO ACEDO; THALITA ROCHA; ALESSANDRA GAMBERO.
SÃO FRANCISCO UNIVERSITY, BRAGANÇA PAULISTA - SP - BRASIL.
Introdution: Coffee consumption has been associated with a lower risk of type 2 diabetes and chronic liver disease. Thus, we aim to study the effects of caffeinated and decaffeinated coffee intake up on inflammatory alterations associated with obesity in mice. Methods and Results: Swiss mice fed with a high-fat diet (HFD; obese group) for 12 weeks and free access to caffeinated (HCC group) or decaffeinated coffee (HCD group) in the drink water in the last 2 weeks were used. Final body weight, adiposity, insulin tolerance test (ITT), glucose tolerance test and blood levels of glucose, aspartate aminotransferases (AST) and alanine aminotransferases (ALT) was evaluated. Cytokines, adipokines and inflammatory markers (iNOS) were measured by ELISA or WB in adipose tissue samples (AT) and liver. Histopathological liver analysis was also performed. There were no alterations in body weight and adiposity in obese animals after coffee intake. In the HCC group glucose blood levels were reduced (172±8 for HFD and 136±6 mg/dl glucose for HCC; p<0.01, n=7-8), insulin tolerance was also reduced in HCC group (2.1±0.1 and 2.8±0.2 kITT for HFD and HCC group, p=0.08, n=8), as well as, glucose tolerance (45192±2485 for HFD and 33243±2916 mg/dl for HCC; p<0.05, n=5). The adiponectin level in AT was increased in HCC group (108±11 and 161±13 ng/mg protein of adiponectin for HFD and HCC, p<0.05, n=3) as well as leptin level (59670±5190 and 81894±1406 pg/mg protein of leptin for HFD and HCC; p<0.05; n=3). TNF-a and IL-10 levels in AT were not altered. The AST (814±86, 343±112 and 388±41 U/L for HFD, HCC and HDC; p<0.05, n=4) and ALT (697±59, 309±99 and 403±88 U/L for HFD, HCC and HDC; p<0.05, n=4) serum levels reduced after coffee intake in HCC and HDC groups. Liver iNOS expression (1.42±0.87 for HFD, 0.73±0.66 for HCC, and 0.93±0.89 iNOS/β-Actin for HDC; p<0.01 and p<0.05, n=3) and steatosis were reduced (82±3% for HFD, 59±9% for HCC, and 59±4% for HDC; p<0.05; n=4). But, TNF-a and IL-10 levels in liver were not modified by coffee intake. Conclusion: Coffee intake can improve liver alterations associated to obesity but only caffeinated coffee intake improves glucose homeostasis and modifies adipose tissue level of adipokines, suggesting an important role for caffeine but also for other coffee substances in controlling the inflammatory alterations induced by obesity.
EFFECTS OF PROTEOLYTIC FRACTION FROM VASCONCELLEA CUNDINAMARCENSIS LATEX IN ACUTE DAMAGE UVB-IRRADIATED SKIN HAIRLESS MICE
KÁTIA MICHELLE FREITAS; DALTON DITTZ; LUCÍOLA DA SILVA BARCELOS; MIRIAM TERESA PAZ LOPES.
UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Solar ultraviolet (UV) radiation causes skin damage including increases in skin thickness, edema, redness and photocarcinogenesis (Eur. J. Pharmacol, 661; 124, 2011). Previously, we demonstrated that latex Vasconcellea cundinamarcensis fraction (P1G10), rich in cysteine proteases, has angiogenic activity, mitogenic and wound healing effect (Phytomedicine, 15(4); 237, 2008). In this study, we investigated the possible effects on tissue repair of topical formulations containing P1G10 against UVB-induce damages in mice. Methods and Results: Hairless mice (n=49) were irradiated by UVB light (dose 2.4J/cm2 - Coler-Parmer ®, 15W, maximum length of 312 nm) in dorsal area to reproduce a sunburn (protocol CETEA 174/2010). P1G10 (0.1, 0.5 and 1.0% or vehicle (Natrosol®) was administered in the damage area immediately after irradiation. The mice were killed 24 h after the UVB exposure, and the full thickness of the dorsal skins were removed for further analysis. The levels of inflammatory infiltrate (N- acetyl-β-glucosaminidase –NAG and myeloperoxidade – MPO) and reduced glutathione (GSH) activity were determined by kinetic-colorimetric assay. The treatment with 0.1% of P1G10 increase neutrophil infiltration (MPO) (0.002 ± 0.000 vs 0.002 ± 0.000 D.O/mg of skin- control, p<0.05, ANOVA, Newman-Keuls post-test) and prevented the decreased of NAG (macrophages infiltration) (0.016 ± 0.001 vs 0.011 ± 0,001 D.O/mg of skin – control, p <0.05, ANOVA, Bonferroni post-test). In the treated 0.5% of P1G10 group was observed a significantly prevention of the UVB irradiation-induce depletion of GSH (1.960 ± 0.220 vs 1.260 ± 0.075 µg/mg of skin – control, p<0.05, ANOVA, Newman-Keuls post-test) .The levels of cytokines were determined by ELISA method. The VEGF levels were not significantly altered, however, the treatment with 0.5% of P1G10 decrease TNF-α levels (0.240 ± 0.030 vs 0.640 ± 0.130 ρg/mg – control, p<0.05, ANOVA, Newman-Keuls post-test). The catalase activity (spectrophotometric assay) was significantly enhanced in the presence of 0.5% of P1G10 (0.019 ± 0.003 vs 0.008 ± 0.001 ΔABS/µg of protein/min – control, p<0.05, ANOVA, Newman-Keuls post-test). Conclusion. Thus, our results suggest beneficial effectiveness of topical formulation containing P1G10 to tissue repair by neutrophils recruitment, macrophages and GSH activities maintenance and activation of antioxidant. Financial support. CNPq, FAPEMIG e CAPES.
EFFECTS OF SIARESINOLIC ACID ON THE CARRAGEENAN-INDUCED INFLAMMATION: EVIDENCE FOR THE INVOLVEMENT OF ATP-SENSITIVE POTASSIUM CHANNELS
ALMAIR FERREIRA DE ARAUJO; JAMYLLE NUNES DE SOUZA FERRO; ANDERSON MARQUES DE OLIVEIRA; LUCIA MARIA CONSERVA; EMILIANO DE OLIVEIRA BARRETO.
UNIVERSIDADE FEDERAL DE ALAGOAS, MACEIÓ - AL - BRASIL.
ALMAIR FERREIRA DE ARAÚJO(1); JAMYLLE NUNES DE SOUZA FERRO(1); ANDERSON MARQUES DE OLIVEIRA(2); LUCIA MARIA CONSERVA(2); EMILIANO BARRETO(1).
1Laboratório de Biologia Celular, 2Instituto de Química e Biotecnologia, Universidade Federal de Alagoas (UFAL), Maceió-AL, 57072-970, Brazil.
Introduction: Species of genus Sabicea are used in the Amazon region for treatment of fever and malaria, which suggests that its chemical constituents may have some effect on inflammatory diseases. Phytochemical study of the leaves of Sabicea grisea resulted in the isolation and characterization of the pentacyclic triterpene named siaresinolic acid (SA). In this study, we investigated the effects of SA on carrageenan-induced inflammation in mice. Methods and results: SA was isolated of the chloroform fraction obtained from the crude ethanol extract of dried leaves of S. grisea. The anti-inflammatory activity of SA was evaluated both in vivo using carrageenan-induced pleurisy model in mice and in vitro on production of reactive oxygen species (ROS) and TNF-α secretion in zymosan-stimulated macrophages. Pleurisy was induced by an intrathoracic injection of carrageenan in Swiss mice (20-25 g) pre-treated with SA by intraperitoneal route. Four hour after stimulus, pleural wash was used to evaluate leukocyte influx, plasma exudation and TNF-α production. The experiments were realized under approval of Committee on Ethical Use of Laboratory Animals of UFAL (license n°. 9301/2009-96). The statistically analyzed was realized by one-way ANOVA and Student–Newman–Keuls post-test (p<0.05). The pre-treatment with SA reduced the total leukocyte, neutrophil influx, plasma exudation and TNF-α production in pleurisy induced by Cg. In addition, minoxidil blocked the reduction on neutrophil influx induced by SA, indicating the involvement of ATP-sensitive potassium channels. In vitro, SA was able to inhibit both the production of ROS as the secretion of TNF-α in zymosan-stimulated macrophage without affecting cell viability. Conclusion: These results indicate that the inhibitory effects of SA on inflammatory response appear to depend of KATP pathways. These results contribute to characterization of anti-inflammatory properties of the SA suggesting that this triterpene is a potential natural product in controlling inflammatory diseases.
Financial support: CNPq.
EFFECTS OF SOY-BASED ISOFLAVONES ON NEUTROPHILS AND MACROPHAGES IN OVARIECTOMIZED RATS
FERNANDO PÍPOLE1; ANDREIA OLIVEIRA LATORRE2; GABRIELA CASTRO KELLER3; PATRICIA FRANCISCONE MENDES4; LUCIANA CASTRO DA CUNHA5; ISIS MACHADO HUEZA6.
1,3,4,5.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL; 2.2AC CAMARGO CANCER CENTER, SÃO PAULO - SP - BRASIL; 6.UNIVERSIDADE FEDERAL DE SÃO PAULO, DIADEMA - SP - BRASIL.
Introduction: The Isoflavones (ISOs) are polyphenolic compounds with chemical structures that are analogous to estradiol, the predominant female hormone. Genistein, daidzein and glycitein are the main active metabolites of ISOs. Their structure allows ISOs to fit into the binding domain of the estrogen receptor and thereby exhibit properties that are similar to the endogenous properties of estrogen. Estrogens modulate immune functions in animals and humans, altering cytokine production, receptor expression and ultimately regulating the responses of different effector cells. However, the majority of experiments assessing the effects of ISOs on the immune system have been performed using either isolated ISOs or in vitro models. Thus, the present study aimed to evaluate the possible immunomodulatory effect of increasing doses of soybean-based ISOs on innate response of ovariectomized rats.
Methods and Results: Female Wistar rats (9 weeks of age) were orally treated for 28 days, with 100 (therapeutic dose), 300 and 900 mg/kg of ISOs. To evaluate the phagocytic activity and the oxidative burst of neutrophils and macrophages, it was used 100 mL of blood samples and 10 mL of peritoneal washing, respectively. Results showed higher intensity of neutrophil phagocytosis in the group of rats treated with the intermediate dose of ISO (300 mg/kg) than in the control group. Conversely, the intensity of macrophage phagocytosis was reduced in rats treated with the higher dose of ISO.
Conclusion: It is known that ISOs metabolites, through interfering with NF-KB activity, can inhibit TNF-a expression, which is important to stimulate phagocytosis. Thus, the reduction in peritoneal macrophage-mediated phagocytosis in rats treated with 900 mg/kg of ISO could be the result of this mechanism of action. However, the possible mechanism of action of ISOs on neutrophil phagocytosis remains unclear.
Financial Support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
EFFECTS OF TERPENES ON VIABILITY OF NEURONAL SHSY-5Y CELLS AND DURING LPS INDUCED INFLAMMATION
NOELIO DE JESUS MENEZES-FILHO1; TEREZA CRISTINA SILVA COSTA2; EUDES DA SILVA VELOZO3; SONGELI MENEZES FREIRE4; SILVIA LIMA COSTA5.
1,2,5.LABORATORIO DE NEUROQUÍMICA E BIOLOGIA CELULAR-UFBA, SALVADOR - BA - BRASIL; 3.INSTITUTO DE QUÍMICA–UFBA, SALVADOR - BA - BRASIL; 4.LABORATORIO DE IMUNOLOGIA E BIOLOGIA MOLECULAR–UFBA, SALVADOR - BA - BRASIL.
Introduction: Acute and chronic inflammatory processes are associated with neurodegenerative disorders as Multiple Sclerosis (MS), a chronic disease that affects the myelin sheath with focal demyelination. This inflammation can be stimulated in vitro with lipopolysaccharide (LPS), mimicking the neuronal dysfunction and degeneration, with release of MS typical inflammatory factors. Many plant-derived drugs have been predominantly characterized as anti- inflammatory, but it is clear that effects can vary with concentration, biological activities, or magnitude of immune stimulus. There is a growing interest in natural terpenoids due to their wide spectrum of biological effects, including anti-inflammatory activity. Objectives: The present study investigated in vitro the cytotoxic effects of the terpene lupeol (triterpene-also known as phytosterols) and the ursolic acid (pentacyclic triterpene acid) in the in vitro model of SH-SY5Y neuronal cells submitted or not to the pro-inflammatory agent LPS. Methods and Results: All-trans retinoic acid (10 µM RA) differentiated SH-SY5Y cells were treated with lupeol (100-400 µg/mL) or ursolic acid (1-5 µM) after 3 h exposure to 10 ng/mL LPS. Cell viability was tested 48 h after treatments by MTT assay. LPS was not toxic to SH- SY5Y cells at the concentration used. Lupeol, at concentration of 100 µg/mL, and ursolic acid at concentrations of 1-5 µM had no effect on the viability of SH-SY5Y cells. However, the extent of MTT reduction in SH-SY5Y cells decreased with increasing lupeol concentration (400 µg/mL). Moreover, the level of MTT reduction in SH-SY5Y cells after treatment with both LPS (10 ng/mL) and Lupeol (400 µg/mL) were similar to control cultures. Conclusions: These preliminary results suggest that lupeol and ursolic acid are bioactive for neuronal cells with low cytotoxic activity and better characterization of their role during inflammatory response needs to be investigated for further elucidation of mechanisms of action and anti-inflammatory properties during CNS cells injury. Financial support: CNPq, CAPES and FAPESB.
EFFICACY OF TOCOYENA SELLOWIANA IN EXPERIMENTAL PERIODONTITIS INVOLVES PROSTAGLANDIN E2 (PGE2) DECREASE
DEBORA DA SILVA FREITAS RIBEIRO1; JORDÂNIA MARQUES DE OLIVEIRA2; SAMUEL MATEUS PEREIRA FILHO3; NATACHA CAMPOS ARRIAGA4; JÚLIO CÉSAR ARAUJO BARCELOS5; ALRIETA HENRIQUE TEIXEIRA6; GERARDO CRISTINO FILHO7; KARUZA MARIA ALVES PEREIRA8; ANTÔNIO ALFREDO RODRIGUES E SILVA9; ANDREZA MARIA LIMA PIRES10; HELLÍADA VASCONCELOS CHAVES11; MARIA ROSE JANE R. ALBUQUERQUE12; MIRNA MARQUES BEZERRA13.
1,2,3,4,5,6,7,8,9,11,13.FEDERAL UNIVERSITY OF CEARÁ, SOBRAL - CE - BRASIL; 10,12.STATE UNIVERSITY VALE ACARAÚ, SOBRAL - CE - BRASIL.
Introduction: Periodontitis is an inflammatory disease that affects the supporting structures of the tooth and is characterized by a destruction of alveolar bone, which can eventually lead to tooth loss. Prostaglandins, especially prostaglandin E2 (PGE2), are the chief mediator of inflammation contributing to the pathogenesis of several inflammatory conditions, including periodontitis. This study aimed to evaluate the effectiveness of Tocoyena sellowiana on periodontitis in rats, investigating the involvement of PGE2, and the safety of this treatment. Methods and Results: Periodontitis was induced by placing a nylon thread (3.0) in the upper molars of female Wistar rats (180 - 200 g). Rats (6 per group) were weighed and treated (per os) daily with Tocoyena sellowiana (0.1, 1 or 10 mg/kg) or vehicle (saline) for 11 days. Following the treatment course, alveolar bone resorption was measured using the ImageJ® software; the periodontal tissues were analyzed by histopathology (H&E) and by ELISA assay to determine the levels of PGE2; the gastric mucosa was examined macro and microscopically (H&E); peripheral blood was collected for biochemical analysis (alanine amino transferase - AST, aspartate amino transferase – ALT, creatinine, and total alkaline phosphatase - tAP). The study protocol was approved by the Ethics Committee of Animal Research of the Federal University of Ceará (52/12). Tocoyena sellowiana (0.1, 1 or 10 mg/kg) reduced (P < 0.05) alveolar bone resorption (4.96 ± 0.39, 3.89 ± 0.13 and 1.74 ± 0.15, respectively), compared to the vehicle group (7.09 ± 0.43). These data were confirmed by histopathology analysis of Tocoyena sellowiana (10 mg/kg) that showed reduction in both cell influx and osteoclast number, with cementum and alveolar process preserved. In addition, Tocoyena sellowiana (10 mg/kg) decreased (P < 0.05) PGE2 levels in gingival samples (2.26 ± 212.3), compared to the vehicle group (4.33 ± 355.7). Tocoyena sellowiana treatment did not change the macroscopic and histopathological analysis of gastric mucosa. Serum levels of ALT/AST, creatinine and tAP did not differ from respective controls. Conclusion: These findings suggest that Tocoyena sellowiana is safe and it reduces the alveolar bone resorption in periodontitis, at least in part, by reducing PGE2 levels. Financial Support: CAPES, CNPq, FUNCAP, and UFC.
ETHYL ACETATE EXTRACT OF MAYTENUS IMBRICATA MART. EX. REISSEK ROOTS IMPROVES INFLAMMATORY AND METABOLIC DYSFUNCTION INDUCED BY HIGH-REFINED-CARBOHYDRATE DIET IN MICE
CRISTINA DA COSTA OLIVEIRA; CLARICE DE CARVALHO VELOSO; MARINA CHAVES DE OLIVEIRA; VANESSA GREGÓRIO RODRIGUES; LUCIENIR PAINS DUARTE; MAURO MARTINS TEIXEIRA; ADALIENE VERSIANI MATOS FERREIRA; ANDREA DE CASTRO PEREZ.
UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Several studies have demonstrated that medicinal plants constitute a therapeutic approach to treat inflammatory and metabolic diseases that may arise due to obesity. Accordingly, we investigated the effects of ethyl acetate extract (EAE) of Maytenus imbricata Mart. ex. Reissek roots on the inflammatory and metabolic dysfunction induced by high-refined carbohydrate (HC) diet. Methods and Results: We observed that mice fed with EAE supplemented HC diet (n=7-9) showed improvement in glucose intolerance (n=7-9) (P< 0.001). The HC diet supplemented with EAE reduced adiposity index by 22.09% when compared to mice fed with HC diet (P<0.05). Mice fed with EAE supplemented HC diet presented fat cell area similar to animals fed with standard chow (n=7-9). EAE supplementation significantly decreased systemic leukocytes (2.73±0.29x104) and neutrophils (0.64±0.08x104) when compared to HC diet (leukocytes: 4.63±0.45x104; neutrophils: 1.67±0.25x104). EAE supplementation reduced serum resistin levels (20.67±2.68 ng/mL) when compared to HC diet group (38.92±2.44 ng/mL). TNF-α levels of HC group (417.40±21.07 pg/100 mg of liver) were significantly increased when compared to control group (242.2±38.83 pg/100 mg of liver). EAE supplementation in HC diet reduced TNF-α levels (149.10±32.24 pg/100 mg of liver) when compared to HC diet group. Conclusion: The EAE has beneficial effects on the inflammatory and metabolic dysfunction induced by HC diet. Financial support: CAPES and FAPEMIG.
EUGENIA SPP (GOMA) SHOWS PRO- AND ANTI-INFLAMMATORIES ACTIVITIES ON NEUTROPHIL MIGRATION WHITHOUT AFFECT THEIR ADHESION, DESGRANULATION AND VIABILITY
BRUNO RAFAEL LOPES; TAIS IARA JESUS; KARINA ALVES TOLEDO; CATARINA DOS SANTOS.
UNESP, ASSIS - SP - BRASIL.
INTRODUCTION: Thousands of people worldwide suffer from damage caused by chronic inflammation. Moreover, many individuals are also affected by primary immunodeficiency or secondary related to inflammatory processes. Myrtaceae family members, including the Eugenia spp, have been suggested as an alternative in the treatment of patients suffering from inflammatory disorders. OBJECTIVES: The need to enhance these effects, seeking the reduction of side effects, stimulates the study of these plant extracts. The main objective of our study was to evaluate the pro- and anti-inflammatory hydroalcoholic extract of dried leaves of Eugenia spp (Egoma) on various neutrophil functions. METHODOLOGY AND RESULTS: For this purpose, the pro- and anti-inflammatory activities Egoma were tested at different concentrations in assays (i) in vivo model of peritonitis in Swiss mice, and (ii) in vitro, using methodologies for assessing adherence, degranulation and cell viability in human peripheral blood neutrophils. The cell viability assays performed with MTT salt and Neutral Red demonstrated that different concentrations of the extract (0.01-100 µg/ml) did not exert any cytotoxic effects on neutrophils. Analysis of migration in vivo showed different results according to the protocol used. The peritoneal cavity of Swiss mice, inoculated i.p. with different concentrations of Egoma. Cell counts, after 6hs, accused increase of neutrophil migration in a dose-dependent manner, in which 0.5mg/kg showed great migration. In parallel, the peritoneal cavity of mice that were injected i.p. thioglycollate 10% after 1h having been subjected to subcutaneous (s.c.) injection of different concentrations of Egoma was evaluated. The change in the route of administration of the plant extract resulted in a drastic reduction in neutrophil migration induced by thioglycollate. Despite the results obtained with in vitro testing, none of the tested concentrations of plant extract proved inducing or inhibiting cell adhesion and degranulation induced by fMLP and PMA. CONCLUSIONS: Combined, our results demonstrate that extract of Eugenia spp owns shares pro- and anti-inflammatory activities in vivo model of peritonitis. The absences of neutrophil response in vitro studies suggest an indirect mechanism of action of this extract on neutrophil migration. The extension of these studies may help in developing new strategies biopharmacological and synthesis of more efficient products and with reduced side effects.
EUGENIA SPP. HYDRO-ETHANOLIC EXTRACT IS NOT TOXIC TO U937-DERIVED MACROPHAGES
MÍRIAN FELICIANO DA COSTA; CATARINA SANTOS; KARINA ALVES TOLEDO.
SAO PAULO STATE UNIVERSITY, ASSIS - SP - BRASIL.
Introduction: Myrtaceae family has been studied for its potential in regulating menstruation, pain, intestinal disorders, infertility, as well as antifungal, purgative and anticancer agent, and in addition to treat colds, cough and other respiratory ailments. Among its members, Eugenia spp. species appear to have pro and anti-inflammatory effect once examined in vivo and in vitro. Results from other groups state that the inflammatory activity modulation of immune cells, such as macrophages, play and important role against cancer. The main aim of this work is to evaluate the effect of co-cultivation of macrophages derived from monocyte lineage U937 with different concentrations of Eugenia spp extract. Methods: U937-derived macrophages were obtained through stimulation with PMA (phorbol-12- myristate-13 acetate) during three days. They were then co-cultivated with three different concentrations of extract (0,01, 1 e 100 µg/mL) for 24 and 48 hours. Viability cellular was evaluated by MTT assay. Results: U937-derived macrophages incubated in the presence of 1% methanol diluted into RPMI-medium were standardized as 100% of viability. Compared to these cells, any Eugenia spp extract concentration was able to reduce cell viability. These results were reproduced after 24 and 48 hours of incubation. Conclusion: Eugenia spp. hydro-ethanolic extract does not affect U937-derived macrophage cell viability. The results obtained have given us the support to keep on working with the extract and its macrophage modulation since it was not toxic in the tested concentrations (cell viability ≥90%). Further studies are in course in order to explore the profile activation of macrophage induced by this vegetable extract which may help in designing new therapeutic strategies for treating cancer.
Financial support: none.
EVALUATION IN VIVO OF ANTI-INFLAMMATORY AND ANTINOCICEPTIVE ACTIVITIES OF ETHANOLIC EXTRACT OF THE BARK OF ANADENANTHERA MACROCARPA OF FLONA
REGIANE YATSUDA; JOSELINE CEZARIO DUARTE; MAIANA FERRAZ ANDRADE; RAFAEL SANTOS DANTAS MIRANDA DÓREA; VINICIUS SABOIA MEIRELES; CASSYA MAVIONY FIUZA ANDRADE; ANDRESSA ARAÚJO OLIVEIRA; EMANUELLA GOMES MAIA; APARECIDO ALMEIDA CONCEIÇÃO; JOÃO TRINDADE SILVA; KEILA SILVA DE JESUS; ERIKA PEREIRA SOUZA; MARILUZE PEIXOTO CRUZ.
UNIVERSIDADE FEDERAL DA BAHIA-UFBA, VITÓRIA DA CONQUISTA - BA - BRASIL.
Introduction: Anadenanthera macrocarpa, popularly known as “angico, angico branco”, is used as anti-inflammatory in the folklore medicine, mainly by the population of the semi-arid region. The aim of this study was to evaluate the anti-inflammatory and antinociceptive activity in vivo of the bark ethanolic extract of the A. macrocarpa (EAM) of the National Forest Contendas do Sincorá (FLONA), semi-arid region of Bahia.
Methods and Results: The antinociceptive and anti-inflammatory activities were evaluated by the following tests: writhing evoked by acetic acid, formalin-induced hypernociception, Von Frey's test, determination of neutrophil migration to the peritoneal cavity and Evans Blue test. TNF-α and IL-10 levels from peritoneal exudates were recovered for cytokine measurement determined by ELISA. The ICAM-1 expression in mesenteric tissues induced by carrageenan injection was assessed by Western blot analysis. Ethanol 10% (v/v) was the vehicle (VH). Male Balb-C mice were used (20–25 g; n=6) and the experiments were approved by the Ethics Committee of the University of Uberaba (protocol 0107/2009). Anova one way, post tests Bonferroni or Dunnett were done, significance p<0.05. The EAM showed statistical significant results for antinociceptive activity by writhing test (VH 38.0 ± 17.25) 50 mg/kg (25.20 ± 13.07) e 100 mg/kg (34.17 ± 22.65), causes reduction the number of flinches (Control 49.83 ± 23.01) 50 mg/Kg (5.17± 4.07) and 100 mg/kg (8.67 ± 7.47) and Von Frey test (VH 9.02 ± 1.04) 100 mg/kg (4.57 ± 3.35). The EAM showed anti-inflammatory activity by inhibiting neutrophil migration (VH 2.26 ± 0.9) 100 mg/kg (2.12 ± 0.58) e 200 mg/kg (1.78 ± 0.86) (p <0.05) and vascular permeability (VH 7.0 ± 1.96) 100 mg/kg (4.25 ± 0.82) at peritonitis evoked by Cg (p <0.05). The EAM didn’t modulate TNF-α levels but increased the levels of IL-10. The ICAM-1 expression was significantly reduced compared to VH group (0.52 ± 0.99) (p<0.05).
Conclusion: A. macrocarpa showed an important antinociceptive and anti-inflammatory activity in vivo, being a promising medicinal plant to be studied to elucidated the mechanisms of these activities.
Financial support: PIBIC/CNPq, CNPq Universal 2008/2010; Decit/SCTIE/MS (CNPq/FAPESB/SESAB); CAPES.
EVALUATION IN VIVO OF ANTI-INFLAMMATORY AND ANTINOCICEPTIVE ACTIVITY OF THE BARK OF MIMOSA TENUIFLORA (WILLD.) POIR. FROM THE SEMI-ARID REGION OF BAHIA.
CASSYA MAVIONY FIUZA ANDRADE1; APARECIDO ALMEIDA CONCEIÇÃO2; MAIANA FERRAZ ANDRADE3; RAFAEL SANTOS DANTAS MIRANDA DÓREA4; KELLE OLIVEIRA SILVA5; ALINNE ANDRADE SILVA6; ANDRESSA ARAÚJO OLIVEIRA7; EMANUELLA GOMES MAIA8; VINÍCIUS SABÓIA MEIRELES9; JULIANA TRINDADE CLEMENTE – NAPIMOGA10; MARCELO HENRIQUE NAPIMOGA11; MARILUZE PEIXOTO CRUZ12; REGIANE YATSUDA13.
1,2,3,4,5,6,7,8,9,12,13.UNIVERSIDADE FEDERAL DA BAHIA-UFBA, VITÓRIA DA CONQUISTA - BA - BRASIL; 10.UNIVERSIDADE ESTADUAL DE CAMPINAS- UNICAMP- FOP-PIRACICABA, PIRACICABA - SP - BRASIL; 11.SÃO LEOPOLDO MANDIC, CAMPINAS - SP - BRASIL.
Introduction: Mimosa tenuiflora (Willd.) Poir. is used in folk medicine in northeastern Brazil for the treatment of cough and wound healing and has been described in the literature with pharmacological activity on tissue repair. This study evaluated in vivo the antinociceptive and anti-inflammatory activity of bark ethanolic extract of Mimosa tenuiflora (Willd.) Poir. collected in the semi-arid region of Bahia state.
Methods and Results: The antinociceptive and anti-inflammatory activities were evaluated by the following tests: writhing evoked by acetic acid, formalin-induced hypernociception, determination of neutrophil migration to the peritoneal cavity, Evans Blue test and Von Frey's test. The cytokines (TNF-α, IL-10 and IL-1β) were measured by ELISA from the peritoneal exudates. Male Balb-C mice were used (20–25 g; N=6) and the experiments were approved by the Ethics Committee of the University of Uberaba (protocol 0107/2009). Doses between 50 and 200 mg/kg of BEE were tested and the control group received ethanol 10%. Analysis of variance (ANOVA) followed by the Bonferroni’s or Dunnett test were done, significance p ≤0.05. The BEE showed antinociceptive activity in the writhing test (100 and 200 mg/kg), reduced the number of flinches from 49.83±23.01 to 6.67±6.44 (200 mg/kg) and edema formation (50, 100 and 200 mg/kg) in formalin-induced hypernociception test compared to the control group (p<0.05). The BEE showed also anti-inflammatory activity by inhibiting neutrophil migration at 100 and 200 mg/kg (1.0±0.50 and 1.58±0.69) caused by carrageenan (3.44±1.28). The BEE only reduced the levels of IL-1β at the dose of 100 and 200 mg/kg.
Conclusion: The bark of M. tenuiflora showed antinociceptive and anti-inflammatory activities and more studies should be done to elucidate the pharmacological mechanisms involved in these activities.
Financial support: PIBIC/CNPq, CNPq Universal 2008/2010; Decit/SCTIE/MS (CNPq/FAPESB/SESAB); CAPES.
EVALUATION OF A NEW SYNTHETIC ALKALOID IN EXPERIMENTAL MODEL OF INFLAMMATION.
LAÉRCIA PAIVA FERREIRA; RENATA COSTA VASCONCELOS; LUIZ ARAÚJO SILVA; JOÃO RODRIGUES PITA; LUÍS CÉZAR RODRIGUES; MÁRCIA REGINA PIUVEZAM.
UFPB, JOÃO PESSOA - PB - BRASIL.
Introduction:MHTP[1-(3-methoxy-4-hydroxyphenyl)-7–methoxy-1,2,3,4-tetrahydroisoquinoline] is a synthetic alkaloid, obtained through Pictet-Spengler reaction.The aim of this study was to evaluate acute toxicity and anti-inflammatory effects of MHTP in murine models. Methods and Results: Male and female Swiss mice (n=6) were pretreated (PO) with MHTP (1000 mg/kg) and acute toxicity was analyzed by measuring water and food intake, body weight, mortality, weight index of organs and hematological parameters. To assess anti-inflammatory activity, pretreated animals (n=6) received an (IP) injection of zymosan (2mg/mL) and four hs later, the peritoneum fluid was collected with 1 mL of ice- cold PBS. The peritoneum cells were counted in hemocitometer and TNF-α, IL-1, IL-6 and IL-10 levels were determined by ELISA assay. The results were expressed as mean ± SEM and analyzed by t-test and one-way ANOVA followed by Tukey’s or Bonferroni’s. It was not observed behavioral changes or death in MHTP-treated animals nor significant changes in food and water intake, index of weight gain, heart, liver, kidneys, spleen, thymus, and hematologic parameters when compared to their respective control groups. Pretreatment with MHTP (2.5 mg/kg) reduced significantly (p<0.05) cell migration to peritoneum (10.9±1.195 vs 19.99±2.113; 45%) as well as pro- inflammatory cytokine levels such as TNF-α (260±52.91 vs 1313±186.2; 80%), IL-1 (98.55±28.32 vs 178.8±28.76; 45%) and IL-6 (322.3±59.67 vs 1929±156.5; 83%). Conclusions: The MHTP presented low toxicity and reduced peritoneum cell migration and inflammatory cytokines production indicating an anti-inflammatory property.
EVALUATION OF ANTI-INFLAMMATORY ACTIVITY OF HYDROETHANOLIC EXTRACT OF DILODENDROM BIPINNATUM RALDK.
CLARISSE PINTO COELHO DE AZEVEDO NETA MAHON; RUBERLEI GODINHO DE OLIVEIRA; DOMINGOS TABAJARA DE OLIVEIRA MARTINS.
UFMT, CUIABÁ - MT - BRASIL.
Pharmacology Area, DCBS, FM, UFMT, Cuiaba, Mato Grosso, Brazil
Introduction: Dilodendron bipinnatun is popularly known as “mulher-pobre”. Its stem decoction and maceration is used in treating inflammations. Objectives: To evaluate anti-inflammatory activity of the hydroethanolic of D. bipinnatum (HEDb). Methods and Results: Wistar rats received the vehicle, HEDb (20, 100 and 500 mg/kg), 5 mg/kg indomethacin or 10 mg/kg of cyproheptadine and 1 h after, 0.1 mL of 1% carrageenan or 1.5% dextran was injected into the left posterior paw (sc), and 0.1 mL of saline 0.9% into the contralateral paw. The paw volumes were measured at 0, 30, 60, 120 and 180 for dextran or up to the 4th h for carrageenan. In peritonitis, Swiss mice received p.o. vehicle, HEDb (20, 100 and 500 mg/kg) or 0.5 mg/kg dexamethasone (dexa) and after 1 h, 250 ng LPS was given ip. Peritoneal lavage was collected for leukocyte counts (LEU). In pleurisy, rats received intrapleural injection of 0.1 ml of 2% carrageenan in sterile saline. The animals were pretreated 1 h before as in the peritonitis, and after 6 h, the pleural exudate (PL, mL) was collected for LEU. In the paw edema by carrageenan, the vehicle group showed edema peaking at the 3rd h (0.61±0.05 mL). HEDb caused edema reduction up to the 3rd h, reaching highest inhibition (36%, p<0.001) in the 2nd h at 20 mg/kg. Indomethacin reached its greatest effect (32%, p<0.01) in the 2nd. The injection of dextran in the vehicle group resulted in paw edema that peaked at 30 min (0.72 ± 0.05 mL). HEDb at the dose of 100 mg/kg reduced the edema only in the 1st h (46%, p<0.01). Cyproheptadine was active during the whole 3 h of edema, reaching the maximum effect (91.5%, p<0,001) at 30 min. In peritonitis, HEDb reduced migration of LEU at all doses, reaching maximum effect with 20 mg/kg (45%, p<0.001). With dexa, the reduction was 56.7% (p<0.001). In pleurisy the vehicle group had an PL of 1.8 ml±0.15 and an influx of 189.1±17.29 x 106 LEU. HEDb caused a greater reduction (27.7%, p<0.05) of the PL with 500 mg/kg, but fell short of impeding influx of LEU into the pleural cavity. However, dexa reduced by 51.6% (p<0.001) the PL volume and by 64.9% (p<0.001) the migration of LEU. Conclusion: HEDb possess acute anti-inflammatory activities. Financial help: CNPq, CAPES, INAU
EVALUATION OF ANTI-INFLAMMATORY EFFECTS OF NOVEL THALIDOMIDE ANALOGUES
LETÍCIA MORONI LACERDA; TÁRSILA FERREIRA GUIMARÃES GOYATÁ; MARCELA MOTTA; JULIANA MELO; SILVIA HELENA CARDOSO; MAURO VIEIRA DE ALMEIDA; GIOVANNI WILSON AMARANTE; GILSON COSTA MACEDO.
UNIVERSIDADE FEDERAL DE JUIZ DE FORA, JUIZ DE FORA - MG - BRASIL.
Introduction: In the last years, Thalidomide, a glutamic acid derivated, has received great attention from the scientific community, mainly because of its anti-inflammatory, immunomodulatory and anti-angiogenic properties. Although the specific action are not completely known, have been demonstrated that Thalidomide is able to reduce the production of several inflammatory mediators, like TNF-α, IL-1β, IL-6 and Nitric Oxide while increase IL-10 production. In addition, Thalidomide reduces NF-kB, cicloxigenase-2 and prostaglandin-E2 activity. All these characteristics give to Thalidomide the status of effective anti-inflammatory drug and enable their use to treat a variety of inflammatory diseases including erythema nodosum leprosum. However, despite of the beneficial actions, this compound presents several and severe side effects that limit its use. Thus, the goal of this study was evaluated the anti-inflammatory potential of three thalidomide analogues.
Methods and Results: Firstly, the analogues cytotoxicity was evaluated in thioglicolate elicited peritoneal macrophages, using MTT assay. After that, macrophage was treated with Thalidomide analogues at several concentrations and stimulated with Escherichia coli lypopolissacaride plus IFN-g. The Nitric Oxide or cytokine (TNF-α and IL-6) production was assessed using Griess reaction and ELISA, respectively. The results have shown that even at high concentration, the compound 20 and 24 did not exhibited relevant cytotoxicity. Only compound 23 showed significant reduction in cell viability. Concomitantly, the treatment with compounds 20 and 24 were able to induced significant reduction of Nitric Oxide, TNF-α and IL-6 production.
Conclusions: The compound analyzed showed potential applicability to control inflammatory immune response through their capacity to inhibit key molecules of this response.
Financial Support: CNPq, FAPEMIG and PROPESQ/UFJF
EVALUATION OF ANTI-INFLAMMATORY PROPERTIES OF CRUDE EXTRACT OIL FREE AND ISOLATED COMPOUND: ROSMARINIC ACID FROM ROSMARINUS OFFICINALIS L. IN IN VIVO EXPERIMENTS
JULIA SALVAN DA ROSA; BRUNO MATHEUS FACCHIN; JULIANA BASTOS; EDUARDO MONGUILHOTT DALMARCO; MOACIR GERALDO PIZZOLATTI; TÂNIA SILVIA FRÖDE.
FEDERAL UNIVERSITY OF SANTA CATARINA, FLORIANÓPOLIS - SC - BRASIL.
Introduction: Rosmarinus officinalis L. (Laminaceae) is useful to treat inflammation. The aim of this study was to evaluate the anti-inflammatory effect of Crude Extract oil free (CEOF) of R. officinalis L. and it’s isolated compound: Rosmarinic acid (RA), upon leukocytes, exudation, activities of myeloperoxidase (MPO) and adenosine deaminase (ADA), levels of nitrite/nitrate (NOx), interleukin-17A (IL-17A), interleukin-10 (IL-10), and mRNA for IL-17A and for IL- 10 in the carrageenan-induced murine model of pleurisy.
Methods and Results: The aerial parts of the R. officinalis L. were underwent extraction through the steam technique to remove the essential oils. It was macerated with ethanol at room temperature to obtain CEOF. The RA was obtained by Capillary electrophoresis and Nuclear Magnetic Ressonance methods. Swiss mice were used though the experiments (Brit.J.Pharmacol.183.811-19.1996). Ethical Committee approved (PP632/CEUA/2011). The animals (n=5/group) were treated with CEOF (10-100mg/kg, i.p.) or RA (2.5-10mg/kg, i.p.) 0.5h before Cg (1%,i.pl). The animals were previously challenged (10 min) with Evans blue dye (25mg/kg, i.v.) to evaluate the exudation. The animals were pretreated (0.5h) with CEOF (50mg/kg,i.p.) or RA (5mg/kg, i.p.) to analyse the effect of the herb upon MPO and ADA, and NOx, IL-17 and IL-10 proteins and their mRNA. The inflammation was analyzed after 4h. Statistical differences between groups were determined by ANOVA, Dunnett’s pos-Hoc test. Values of P<0.05 were considered significant. CEOF (50-100mg/kg) and RA (2.5-10mg/kg) inhibited leukocytes (CEOF: 58.3±2.4% to 61.0±2.0%, RA: 22.2±1.7% to 34.8±3.9%), neutrophils (CEOF: 60.7±2.1% to 61.0±2.2%, RA:21.1±2.2% to 35.3±3.3%) and exudation (CEOF: 31.5±3.7% to 46.0±3.3%, RA:19.4±3.4% to 14.8±2.1%) (P<0.05). CEOF(50mg/kg) and RA(5mg/kg) inhibited MPO (CEOF:61.5±6.6%, RA:37.0±4.3%), ADA (CEOF:34.4±5.6%, RA:29.5±11.7%), NOx (CEOF:50.7±4.3%, RA:60.0±9.7%), IL-17 (CEOF:60.4±7.4%, RA:58.2±4.8% and mRNA for IL-17 (CEOF: 25.9±7.5%, RA: 61.3±0.4%). CEOF and RA at the same doses increased the IL-10 (CEOF: 54.6±1.4%, RA: 67.1±3.9%) and mRNA for IL-10 (CEOF: 57.7±1.0%, RA:34.3±1.4%) (P<0.05).
Conclusion: R. officinalis L. showed anti-inflammatory activity by inhibiting leukocytes and exudation. This inhibitory effect was associated with the reduction of MPO, ADA, NOx and IL-17 and increased of IL-10, including the modulation of mRNA expression for IL-17 and IL-10.
Financial support: CAPES.
EVALUATION OF ANTINOCICEPTIVE AND ANTI-INFLAMMATORY ACTIVITY OF SCHINOPSIS BRASILIENSIS ENGLER COLLECTED IN SEMI-ARID REGION OF BAHIA
RAFAEL SANTOS DANTAS MIRANDA DÓREA; VINÍCIUS SABÓIA MEIRELHES; MAIANA FERRAZ ANDRADE; ERIKA PEREIRA DE SOUZA; JOSELINE CEZARIO DUARTE; KELLE OLIVEIRA SILVA; EMANUELLA GOMES MAIA; ANDRESSA ARAÚJO OLIVEIRA; CASSIA MAVIONY FIUZA ANDRADE; JOÃO PAULO DA LUZ; LUCAS MIRANDA MARQUES; MARILUZE PEREIRA CRUZ; REGIANE YATSUDA.
FEDERAL UNIVERSITY OF BAHIA, VITÓRIA DA CONQUISTA - BA - BRASIL.
Introduction: Schinopsis brasiliensis Engler, Anacardiaceae family, popularly known as "Braúna", is widely used in folk medicine to treat headache, hypertension, diarrhea and intestinal infection. This study aims to evaluate the antinociceptive and anti-inflammatory activity of the ethanolic extract of leaf of S. brasiliensis Engler (EESB), collected in the National Forest Contendas Sincorá, semiarid region of Bahia. Methods and Results: The antinociceptive and anti-inflammatory activity of EESB were analyzed by the methods of writhing induced by acetic acid, hypernociception formalin-induced, migration of neutrophils and Evans blue extravasation into the peritoneal cavity. The cytokines (TNF-α, IL-10 and IL-1β) were measured by ELISA from the peritoneal exudates. Male Balb/c mice were used at all tests with 6 mice per experimental group. Ethanol 10% (v/v) was the vehicle (VH). The statistical comparisons between groups were made using analysis of variance (ANOVA) followed by the Bonferroni’s or Dunnett test. Values were considered significance when p < 0.05. Antinociceptive activity was observed in the test of writhing induced by acetic acid (100 mg/kg - 14.83±13.15; VH - 37.29±14.37), in the test of hypernociception induced by formalin (100 mg/kg – 17.80±3.96; VH - 30.40±6.95). The EESB also inhibited the increase of vascular permeability (50 mg/kg – 0.71±0.39; VH - 3.03±2.14) and the migration of neutrophils (50 mg/kg - 1.90±0.67; VH 6.64±1.82) on carrageenan- induced peritonitis. EESB also showed reduction in the levels of IL-1β (43.21±17.77) and higher level of IL-10 (177±70.29). Conclusion: The ethanolic extract of leaf of S. brasiliensis demonstrated antinociceptive and anti- inflammatory, being a promising medicinal plant, and more studies should be conducted to understand the pharmacological mechanism of these extract.
Financial Support: PIBIC/CNPq, CNPq Universal 2008/2010; Decit/SCTIE/MS (CNPq/FAPESB/SESAB); CAPES.
EVALUATION OF CYTOTOXICITY OF AMPHOTERICIN B IN THE PRESENCE AND ABSENCE OF RUTIN
FRANCINE ALESSANDRA MANENTE1; ANA CAROLINA URBACZEK2; LÍVIA CAROLINE RIBEIRO3; ANA FLÁVIA TOSTES SILVA4; JOSIANE APARECIDA SCHEMBERGER5; JOSÉ CARLOS REBUGLIO VELLOSA6; IRACILDA ZEPPONE CARLOS7.
1,2,3,7.FACULDADE DE CIÊNCIAS FARMACÊUTICAS - UNESP ARARAQUARA, ARARAQUARA - SP - BRASIL; 4,5,6.UNIVERSIDADE ESTADUAL DE PONTA GROSSA - UEPG, PONTA GROSSA - PR - BRASIL.
Introduction: Amphotericin B (AmB) is the gold standard drug for the treatment of invasive fungal infections since 1960. However, amphotericin B has high toxicity, which manifests itself most commonly in the kidneys (nephrotoxicity) and less frequently in the liver (liver toxicity). It is known since 1985 that self-oxidation of amphotericin B gives different forms of reactive oxidative species and these are responsible in part by toxicity. An antioxidant, such as rutin, could contribute to protect against cell injury. Methods: Different concentrations of rutin alone or in association to amphotericin were evaluated through: i) erythrocytes (RBC) suspension in the presence of 50mM 2,2′-Azobis(2- methylpropionamidine) dihydrochloride radical (AAPH) (150min, 370C, sodium phosphate buffer 10mM, NaCl
150mM). Hemolytic effects were revealed by haemoglobin releasing (A414nm); ii) cell culture using a method based on the ability of viable cells to cleave the tetrazole ring MTT producing formazan crystals. It was performed in a microtiter plate of 96-well flat bottom containing HUVEC 5x105 (adherent cells) / ml RPMI Medium 1640, per well. Results:
Expressed as means and standard deviation. Hemolysis: RBC+AAPH: A414nm= 0.840 ±0.0474; RBC + AmB
(0.002mg/mL) + AAPH: A414nm = 1.2765±0.0092; RBC + AmB (0.002mg/mL) + AAPH + rutin (0.02, 0.01 and
0.005mg/mL): A414nm= 0.1285 ±0.0035, 0.1623 ±0.0169 and 0.3543 ±0.0297, respectively. The viability (%) of HUVEC cells in the presence of LPS (stimulus) and: i) Amb (0.3125mg/mL, 0.0391mg/mL, 0.0195mg/mL, 0.0098mg/mL) were 33.26±4.12, 88.93±4.99, 98.68±1.40 and 99.27±0.89, respectively; ii) Rutin (0.3125mg/mL, 0.0391mg/mL, 0.0195mg/mL, 0.0098mg/mL) was 71.64±2.56, 88.76±4.62, 92.92±4.07, 96.64±3.89, respectively. When it was associated different concentrations of AmB with 0.0781mg/mL rutin, the cell viability has decreased compared to the most concentrations of AmB alone (0.3125mg/mL: 19.29±0.25; 0.0391mg/mL: 72.78±1.52; 0.0195mg/mL: 78.94±3.74; 0.0098mg/mL: 100±0). Conclusion: When the antioxidant was added to the complete hemolitic system (RBC+AAPH+AmB) the hemolysis has descreased indicating protetive effect of rutin against AmB exacervated hemolysis. However, in stimulated HUVEC cells, the antioxidant association to AmB has increased the citotoxic effect demonstrating that in different conditions and front of different cellular types, an antioxidant may act as a lesive agent.
Financial support: CAPES
EVALUATION OF THE ANTI-INFLAMATORY ACTIVITY OF NEW ISOXAZOLINE-ACYLHYDRAZONE DERIVATIVE
ELIZABETH OLIVEIRA BORBA; FERNANDA BARRETO MOTA; SANDRA CABRAL SILVA; ALINE GATIS CARRAZZONI; LARISSA CORRÊA ARAÚJO; VALDERES MORAES ALMEIDA; ANTÔNIO RODOLFO FARIA; TERESINA GONÇALVES SILVA.
UFPE, RECIFE - PE - BRASIL.
Introduction: Preliminary studies have shown that acylhydrazone and isoxazoline derivatives have several pharmacological activities, among them anti-inflammatory activity. This study aimed to evaluate the anti-inflammatory activity of the isoxazoline-acylhydrazone derivative [4-METOXIBENZILIDENO)-HIDRAZIDA DO ÁCIDO-6-(4-NITRO- BENZOIL)-4,5,6,6a-TETRAHIDRO-3aH-PIRROLO[3,2-d]ISOXAZOL-3-CARBOXÍLICO (R-123)] using experimental model of paw edema in mice induced by different phlogistic agents. Methods and results: The derivative R-123 was provided by the Laboratório de Síntese Orgânica Aplicada a Fármacos from UFPE. Were used albino swiss mice (Mus musculus) (n=6) (Committee for Ethics in Animal Research n° 23076.018987/2012-82). The paw edema was induced by injecting of 50 µL of carrageenan (1%), dextran (0.2%), histamine (100 µg) or 5-HT (100 µg) into the right hind paw. The animals received the R-123 orally (15 mg/kg) one hour before induction of edema. Indomethacin and cyproheptadine were used as standard reference (10 mg/kg, p.o) and the control group received saline (NaCl 0.9%, p.o). The paw volume was measurement immediately after induction of edema and in different times, according to the flogistic agent, with a pletismometer. Statistical analysis was performed by ANOVA Two-way followed by Bonferroni’s test using the software GraphPad prism v.5.0. The compound R-123 inhibited the edema, with percentages of 41% (0.020 ± 0.0 mL), 29% (0.064 ± 0.005 mL) and 49% (0.0406 ± 0.0 mL) of the paw edema induced by dextran (4th hour), histamine (15 min) and 5-HT (15 min), respectively. However, there was no inhibition of the edema induced by carrageenan. Conclusion: The derivative R-123 inhibits the edema induced by dextran, histamine and 5-HT, but not by carrageenan, showing that the compound acts probably inhibiting the action/release of the vasoactive amines, histamine and serotonin.
Financial support: FACEPE and CNPq.
EVALUATION OF THE ANTI-INFLAMMATORY EFFECTS OF MYRTENOL, A PLANT-DERIVED MONOTERPENE ALCOHOL, IN MICE.
CAMILA DE FÁTIMA CARVALHO BRITO1; RENAN OLIVEIRA SILVA2; MAISA DE SOUSA DOS SANTOS3; FRANCISCA BEATRIZ DE MELO SOUSA4; IRISMARA SOUSA SILVA5; SAMARA RODRIGUES BONFIM DAMASCENO6; NATHALIA SANTOS CARVALHO7; OSKAR ALMEIDA SILVA8; KAROLINE SABÓIA ARAGÃO9; ANDRÉ LUIZ DOS REIS BARBOSA10; RIVELILSON MENDES DE FREITAS11; JAND-VENES ROLIM MEDEIROS12.
1,2,3,4,5,6,7,10,11,12.BIOTECHNOLOGY AND BIODIVERSITY CENTER RESEARCH (BIOTEC), FEDERAL UNIVERSITY OF PIAUÍ, PARNAÍBA - PI - BRASIL; 8.POST-GRADUATION PROGRAM IN PHARMACEUTICAL SCIENCES, FEDERAL UNIVERSITY OF PIAUÍ, TERESINA - PI - BRASIL; 9.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: There is a continuous search for new compounds as therapeutic alternative because many anti- inflammatory drugs present considerable side effects (Biol. Pharm. Bull. 30:1217-1220, 2007). So, it has been natural products sources study to find more effective therapeutic agents. The aim of this study was evaluate the anti- inflammatory effects of myrtenol in mice. Methods and Results: The present study was approved by the local ethics committee (protocol no. 0066/10). Myrtenol was used in classical models of inflammation in mice: paw edema induced by different agents, carrageenan-induced peritonitis, MPO levels and cytokine measurement. In models of paw edema measured by plesthismometer, the results show that pre-treatment of mice with myrtenol (n = 6) presented a dose- dependent inhibitory effect, at all doses tested, with maximal effect at dose of 75 mg/kg i.p. (0.020±0.008 ml) inhibiting edema, as compared to carrageenan group (0.107±0.016 ml), well as significantly reduced the paw edema induced by compound 48/80 (0.108 ± 0.011 ml) to 0.064 ± 0,007 ml, serotonin (0.084 ± 0.008 ml) to 0.030 ± 0.005 ml, histamine (0.082 ± 0.003 ml) to 0.028 ± 0.003 ml and PGE2 (0.071 ± 0.002 ml) to 0.018 ± 0.008 ml. For evaluation of neutrophil migration (n = 6), myrtenol (75 mg/kg, i.p.), injected thirty minutes before of carrageenan, produced a decrease in leukocyte count (1.26 ± 0.22 leukocytes × 103/ml) and neutrophils (0.67 ± 0.22 neutrophils × 103/ml) in the peritoneal cavity, as compared to carrageenan group (13.22 ± 2.610 leukocytes × 103/ml and 11.41 ± 2.392 neutrophils × 103/ml). In the evaluation of myeloperoxidase activity by spectrophotometry (n = 6), myrtenol (75 mg/kg, i.p.) significantly reduced the MPO activity to 1.8 ± 0.1 U/mg of exudate, as compared to carrageenan group (7.7 ± 0.8 U/ml of exudate). The levels of IL-1β and TNF-α were evaluated by ELISA (n = 6) and pretreatment with myrtenol (75 mg/kg, i.p.) significantly reduced IL-1β (1044.0 ± 221.5 pg/ml) levels, but not TNF-α (194.0 ± 64.09 pg/ml), as compared to the carrageenan control group (1995.0 ± 13.19 pg/ml, 191.6 ± 18.7 pg/ml, respectively). Conclusions: Thus, myrtenol reduces the inflammatory response due to the inhibition of the release of inflammatory mediators and cell migration.
Financial support: CNPq and FAPEPI.
EVALUATION OF THE ANTIEDEMATOGENIC ACTIVITY OF NEW ISOXAZOLINE-ACYLHYDRAZONE DERIVATIVE
FERNANDA VIRGINIA BARRETO MOTA; SANDRA CABRAL DA SILVA; ALINE STAMFORD HENRIQUE DA SILVA GUERRA GATIS CARRAZZONI; LARISSA CARDOSO CORRÊA DE ARAÚJO; VALDERES MORAES DE ALMEIDA; ANTÔNIO RODOLFO DE FARIAS; TERESINHA GONÇALVES DA SILVA.
UFPE, RECIFE - PE - BRASIL.
Introduction: The compounds containing isoxazolinic ring and acylhydrazone function have been reported as anti- inflammatory and analgesic agents. This study aimed to evaluate the anti-inflammatory activity of the new isoxazoline- acylhydrazone derivative (R-99) using an experimental model of paw edema in mice induced by different phlogistic agents.
Methods and results: The derivative R-99 was provided by the Laboratório de Síntese Orgânica Aplicada a Fármacos from UFPE. Were used albino swiss mice (Mus musculus) (n=6) (Committee for Ethics in Animal Research n° 23076.018987/2012-82). The paw edema was induced by injecting of 50 µL of carrageenan (1%), 0.2% dextran, histamine (100 µg) or 5-HT (100 µg) into the right hind paw. The animals received the compound orally (15 mg/kg) one hour before induction of edema. Indomethacin and cyproheptadine were used as standard reference (10 mg/kg, p.o) and the control group received saline (NaCl 0.9%, p.o). The paw volume was measurement immediately after induction of edema and in different times, according to the flogistic agent, with a pletismometer. Statistical analysis was performed by ANOVA Two-way followed by Bonferroni’s test using the software GraphPad prism v.5.0. The compound R-99 inhibited paw edema induced by carragenan in 58% (0.036 ± 0.009 mL) (4th hour); induced by dextran (3rd hour) 74% (0.016 ± 0.009 mL); or induced by histamine (15 minutes) in 49% (0.046 ± 0.005 mL). However, showed no significant inhibition of the edema induced by 5-HT.
Conclusion: The derivative R-99 inhibits the edema induced by carrageenan, dextran and histamine, but not in 5-HT- induced edema. The results showed that the compound acts inhibiting the action/release of histamine and possibly prostaglandins.
Financial support: FACEPE and CNPq.
EVALUATION OF THE ANTINOCICEPTIVE EFFECTS OF MYRTENOL, A PLANT-DERIVED MONOTERPENE ALCOHOL, IN MICE
MAISA DE SOUSA DOS SANTOS1; RENAN OLIVEIRA SILVA2; OSKAR ALMEIDA SILVA3; FRANCISCA BEATRIZ DE MELO SOUSA4; SAMARA RODRIGUES BONFIM DAMASCENO5; CAMILA DE FÁTIMA CARVALHO BRITO6; IRISMARA SOUSA SILVA7; NATHALIA SANTOS CARVALHO8; KAROLINE SABÓIA ARAGÃO9; ANDRÉ LUIZ DOS REIS BARBOSA10; RIVELILSON MENDES DE FREITAS11; JAND-VENES ROLIM MEDEIROS12.
1,2,4,5,6,7,8,10,11,12.BIOTECHNOLOGY AND BIODIVERSITY CENTER RESEARCH (BIOTEC), FEDERAL UNIVERSITY OF PIAUÍ, PARNAÍBA - PI - BRASIL; 3.POST-GRADUATION PROGRAM IN PHARMACEUTICAL SCIENCES, FEDERAL UNIVERSITY OF PIAUÍ, TERESINA - PI - BRASIL; 9.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: There large number of analgesics currently available for clinical use, however the analgesic drugs exert a wide range of side effects (Rev Bras Anestesiol. 48:137-58, 1998). In this context, natural products have been one of the most successful sources for the discovery of new therapeutic agents to benefit those afflicted by inflammatory diseases (Drug Discov Today. 5:294-300, 2000). The aim of this study was evaluate the antinociceptive effects of myrtenol, a plant-derived monoterpene alcohol, in mice and its possible mechanisms. Methods and Results: Experimental protocols were approved by the Ethics Committee in Research of the Federal University of Piauí (protocol no. 0066/10). The analgesic effects of the myrtenol were evaluated in classic models: writhing test, hot plate test, formalin-, glutamate- and capsaicin-induced paw licking and rota-rod test. Writhing test models of abdominal pain was induced by the intraperitoneal injection of acetic acid (1%) (n=6), myrtenol (75 mg/kg, i.p.) produced a significant (p < 0.05) inhibition (69.3%) on the writhing responses. In hot plate test (55 ± 1 °C) (n=6), myrtenol (75 mg/kg, i.p.) did not significantly (p < 0.05) prolong the latency time in the hot plate test in time analyzed, as compared to time zero. Formalin test (n=6) occurs in a biphasic pattern, nociceptive behavior was induced by injecting 2.5% formalin (20 μl, i.p.) in right hind paw, myrtenol (75 mg/kg, i.p.) showed a significant antinociceptive effect, reducing the formalin induced paw licking time, though it did not reduce significantly (p < 0.05) only the second phase (inflammatory) of the test with a reduction of 97.6%, as compared to the control group. Myrtenol (75 mg/kg, i.p.) (20.90 ± 2.65 s; 36.8%) presented a significant reduction (p < 0.05) of the capsaicin-induced paw licking (n=6) (33.08 ± 1.27 s), the myrtenol (30.90 ± 6.78 s; 44.9%) also significantly reduced (p < 0.05) glutamate-induced paw licking (56.05± 11.21 s). In the rota-rod test (n=10) the animals pretreated with myrtenol (25, 50 or 75 mg/kg) showed no significant motor performance alterations in the rota-rod test. Conclusion: Myrtenol reduces the nociception in mice due to the inhibition of the release of inflammatory mediators, cell migration and also to the signaling pathway of receptors involved in the transmission of pain.
Financial Support: CNPq, FAPEPI, UFPI.
EVALUATION OF THE ANTITUMOR EFFECT OF THE EXTRACT AND THE DICLOROMETHANE FRACTION OF EUPHORBIA TIRUCALLI
MAYARA DE AZEREDO REZENDE1; KATIA COSTA DE CARVALHO SABINO2; IVANA CORREA RAMOS LEAL3; MARINA LETÍCIA DO CARMO CAXITO4.
1.INSTITUTO NACIONAL DO CÂNCER, FACULDADE BEZERRA DE ARAÚJO, RIO DE JANEIRO - RJ - BRASIL; 2,4.DEPARTAMENTO BIOQUIMICA/IBRAG/UERJ, RIO DE JANEIRO - RJ - BRASIL; 3.CENTRO DE TECNOLOGIA - CENTRAL ANALÍTICA - CT-UFRJ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Leukemias are disorders characterized by the accumulation of malignant leukocytes in bone marrow and peripheral blood. The current drugs to treat this pathology can cause cytotoxic effects or may not produce the expected effect. Euphorbia tirucalli popularly known as Aveloz is considered an important plant in Brazilian folk medicine for treatment of cancer. The aim of this study was to characterize in vitro its antitumor effect and the relationship of this effect with the induction of tumor cell death by pure extract and dichloromethane fraction of the plant E. tirucalli. Methods: The leaves were extracted by maceration in ethanol 70%, filtered and dried by evaporation at 60ºC. The extracts were fractionated by liquid-liquid partition. The dichloromethane fraction was analyzed by gas chromatography coupled with mass detector. K562 and Lucena tumor cell lines were characterized by flow cytometer. The antitumor effects of Aveloz were evaluated by MTT, Cell Cycle and Cell Death Assays. Results: Analysis of CG/MS showed stearic acid, palmitic acid and malic acid as major compounds. The treatment of the K562 cell line with both pure extract and dichloromethane fraction revealed a decrease in mitochondrial activity (55.2 ± 0.2 N=3 and 64.13 ± 0.9 N=3, respectively). Also, it was observed an important modulation of cell cycle, as well as a significant increase in early phase of cell death (47.02 ± 5.01% N=3). Concerning Lucena cell line, reduction of mitochondrial activity (56.3 ± 2.3% N=3), changes in the cell cycle and increased in early phase of cell death (43.55 ± 7.7% N=3) were observed only after treatment with the dichloromethane fraction of E. tirucalli. The pure extract of this plant did not demonstrated any effect in all tests used, suggesting that the antitumor chemical constituents are mainly present in the dichloromethane fraction. Conclusion: These data suggest that the dichloromethane fraction of E. tirucalli may represent a promising prototype drug with antitumor activity. Further studies are necessary to elucidate the pharmacological mechanism involved in this effect.
Financial support: CNPq, FAPERJ.
EVALUATION OF THE EFFECTS OF HEXANE/ETHYL ETHER (1:1) EXTRACT OF MAYTENUS IMBRICATA MART. EX. REISSEK SUPPLEMENTATION ON THE TREATMENT OF INFLAMMATORY AND METABOLIC DYSFUNCTION INDUCED BY HIGH-REFINED CARBOHYDRATE DIET
CLARICE DE CARVALHO VELOSO; MARINA CHAVES DE OLIVEIRA; VANESSA GREGÓRIO RODRIGUES; CRISTINA DA COSTA OLIVEIRA; LUCIENIR PAINS DUARTE; MAURO MARTINS TEIXEIRA; ADALIENE VERSIANI MATOS FERREIRA; ANDREA DE CASTRO PEREZ.
UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Several studies have demonstrated that medicinal plants constitute a therapeutic approach for the treatment of metabolic diseases. We investigated the effects of hexane/ethyl ether (1:1) extract of Maytenus imbricata Mart. ex. Reissek (HEE) roots supplementation for the treatment of inflammatory and metabolic dysfunction induced by high-refined-carbohydrate (HC) diet. Methods and Results: The HC diet supplemented with HEE (n=7-9) did not change adiposity index when compared with mice fed with HC diet (7-9). However, we observed that mice fed with HC diet supplemented with HEE decreased adipocyte area (P<0.001), improved the glucose intolerance (P<0.001), inhibited the systemic increase of total leukocytes (2.83±0.42x104) and mononuclears (1.39±0.23x104) induced by HC diet (leukocytes: 4.63±0.45x104; mononuclears: 2.85±0.27x104). The levels of resistin of HC (38.92±2.44 ng/mL) were increased when compared to control group (n=7-9) (27.16±3.15 ng/mL). The supplementation with HEE in HC diet reduced resistin levels (25.96±3.50 ng/mL) when compared to HC diet. The levels of TNF-α of HC (417.40±21.07 pg/100 mg of liver) were increased when compared to control group (242.2±38.83 pg/100 mg of liver). HEE supplementation in HC diet reduced TNF-α levels (166.80±27.87 pg/100 mg of liver). Conclusion: The results showed that HEE may treat inflammatory and metabolic dysfunction induced by HC diet. Financial support: CAPES and FAPEMIG.
EVALUTION OF ANTINOCICEPTIVE EFFECT OF RIPARIN II: ROLE OF TRANSIENT POTENTIAL AND ASICS RECEPTORS
ALYNE MARA RODRIGUES CARVALHO1; NAYRTON FLAVIO MOURA ROCHA2; LEONARDO FREIRE VASCONCELOS3; EMILIANO RICARDO VASCONCELOS RIOS4; MARILIA LEITE DIAS5; LAURA MARIA TEODORIO VIDAL6; STANLEY JUAN CHAVEZ GUTIERREZ7; JOSE MARIA BARBOSA FILHO8; FRANCISCA CLEA FLORENÇO SOUSA9.
1,2,3,4,5,6,9.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FACULTY OF MEDICINE UFC, FORTALEZA - CE - BRASIL; 7.DEPARTMENT OF BIOCHEMISTRY AND PHARMACOLOGY UFPI, TERESINA - PI - BRASIL; 8.LABORATORY OF PHARMACEUTICS TECHNOLOGY UFPB, JOAO PESSOA - PB - BRASIL.
Introduction: Riparin II (RipII) is an alkamide compound isolated from Aniba riparia, collected from the Amazonas’s forest. This substance presents anxiolytic, antidepressant-like and antiinflamatory effects in animal models. The aim of this work was verify the involvement of TRPV1, TRPA1, TRPM8 and ASIC receptors in the antinociceptive mechanism of this substance. Methods: We used Swiss male mice weighing 25-32g (n= 7-9 mice/group). Data were analyzed using One-Way ANOVA and Student Newman-Keuls test as post hoc. To verify the participation of this receptors, the animals were pretreated with RipII (25 and 50mg/kg; p.o.) or vehicle and 60min after the animals received 20µL of capsaicin (2.2µg/paw, TRPV1 agonist), cinnamaldehyde (10nmol/paw, TRPA1 agonist) menthol (1.2µmol/paw, TRPM8 agonist) or acidified saline (2% acetic acid in 0.9% saline, pH 1.98, 20µL /paw, activation of ASIC) in the right hind paw. Ruthenium red (3mg/kg, i.p., non-selective TRP antagonist) or camphor (7.6mg/kg, s.c., TRPA1 antagonist) were used as reference drugs. In another set of experiments, RipII (25 and 50µg/paw) was co-administered to the hind paw together with capsaicin, cinnamaldehyde, menthol or acidified saline in the same doses cited above, and the behavioral responses were recorded as described above. Results: The orally administration of RipII (25 and 50mg/kg) produced inhibition of the licking induced by capsaicin (36.8% and 50%), cinnamaldehyde (72.1% and 72.1%), menthol (70.5% and 88.7%) and acidified saline (49.6% and 61.4%), respectively, when compared with vehicle group. Ruthenium red and camphor were effective in reduced nociceptive response in 98.5% and 65.1%, respectively. Intraplantar co-administration of RipII (25 and 50µg/paw) decreased the responses induced by capsaicin (56.1% and 42.2%, respectively) or acidified saline (62.2% and 57.0%, respectively). Whereas, did not alter the responses induced by cinnamaldehyde or menthol. Conclusion: Based on the obtained results it is suggested that RipII presents antinociceptive activity probably due by binding in the TRPV1 and mediating the TRPM8 and ASIC. Support Financial: CNPq/CAPES/FUNCAP
EVIDENCE FOR THE ANTIPRURITIC AND ANTIINFLAMATORY ACTIVITIES OF THE SLOW-RELEASING HYDROGEN SULFIDE DONOR, GYY4137
LEANDRO RODRIGUES1; JULIANA FLORENZANO2; BRUNA CAETANO DOS SANTOS3; KAREN TIAGO DOS SANTOS4; KELLI CARDOSO LOPES5; FLÁVIA BATISTA CHAVES DE LIRA6; FILIPHE DE PAULA NUNES MESQUITA7; SIMONE APARECIDA TEIXEIRA8; MARK WOOD9; MATTHEW WHITEMAN10; MARCELO NICOLÁS MUSCARÁ11; SORAIA KÁTIA PEREIRA COSTA12.
1,2,3,4,6,7,8,11,12.DEPT. OF PHARMACOLOGY, INSTITUTE OF BIOMEDICAL SCIENCES, UNIVERSITY OF SAO PAULO, SÃO PAULO - SP - BRASIL; 5.DEPT. OF MICROBIOLOGY, INSTITUTE OF BIOMEDICAL SCIENCES, UNIVERSITY OF SAO PAULO, SÃO PAULO - SP - BRASIL; 9,10.UNIVERSITY OF EXETER MEDICAL SCHOOL, ST. LUKE'S CAMPUS, EXETER - REINO UNIDO.
Introduction and aims: Poor diabetes control is commonly associated with pruritus, a sensory modality that similar to pain acts as a protective mechanism (Nat Rev Neurosci. 7:535 – 47, 2006). Recently, we showed that either histamine or Compound 48/80 (C48/80)-induced pruritus and associated skin inflammation is reduced by treatment with fast hydrogen sulphide (H2S)-releasing donors Lawesson’s reagent and Na2S. The aims of this study are: i) to determine the anti-inflammatory and anti-pruritic effectiveness of the novel slow H₂S-releasing donor GYY4137 and possibly involved mechanisms, and ii) to measure cutaneous mRNA expression for H2S-producing enzymes, cystathionine-β- synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST).
Methods and Results: Experiments were approved by the ICB-USP Ethics Committee (no. 33, book 2/2010). In order to produce itching behaviour or skin inflammation, male BALB/c mice were anaesthetised with isoflurane and i.d. injected with histamine alone or in the presence of test agents GYY4137 (0.3-30 nmol site-1; 0.05 ml) and glibenclamide (ATP-sensitive potassium channels blocker - KATP, i.p., – 40 min). Over a period of 40 min, itching behaviour was assessed by measuring the number of scratching bouts whereas skin oedema was quantified via extravascular accumulation of 125I-labelled serum albumin. The mRNA was detected by qualitative RT-PCR. Data were analyzed by ANOVA plus Dunnett test. P<0.05 values were considered significant. Histamine i.d. injected significantly increased the itching frequency (220±12%, n=5) as compared to its vehicle Tyrode (n=5). Co-injection of increasing doses of GYY4137 (1-10 nmol site-1; n=5) inhibited, in a dose-dependent manner, histamine-induced pruritus (76±8%, 54±10% and 54±7%, respectively; n=5-6) but failed to affect the related plasma extravasation. The suppression of histamine-induced pruritus by GYY4137 was not reversed by glibenclamide (n=5-7), thus suggesting that KATP channels are not involved. Either CBS or 3-MST, but not CSE, are highly (mRNA, n=3) expressed in mice dorsal naive skin.
Conclusion: GYY4137 inhibits, in a dose-dependent fashion, histamine-induced pruritus without affecting the associated local inflammation via a mechanism independently of KATP channels activation.
Financial support: FAPESP, CAPES and CNPq.
Acknowledgement: Gouvea IM and Barreto MA for their technical assistances.
EVIDENCE OF ANTI-INFLAMMATORY PROPERTIES OF AGERATUM CONYZOIDES L. IN THE MURINE MODEL OF PLEURISY INDUCED BY CARRAGEENAN
SILVANA VIGIL DE MELLO; BRUNO MATHEUS FACCHIN; ANA BEATRIZ GOBBO LUZ; CRISTIANE FRACARI BOSI; DANIELA WEINGÄRTNER ROSA; MAIQUE WEBER BIAVATTI; TÂNIA SILVIA FRÖDE.
FEDERAL UNIVERSITY OF SANTA CATARINA, FLORIANÓPOLIS - SC - BRASIL.
Introduction: A.conyzoides L. has analgesic and anti-inflammatory actions. The aim of this study was to evaluate the anti-inflammatory effect of the Crude Extract(CE), and fractions: Ethanol(EtOH) and Hexane(HEX), and compound: Methoxy Nobiletin(MeONOB) upon leukocytes, exudation and myeloperoxidase(MPO) and adenosine- deaminase(ADA) activities, and nitrate/nitrate(NOx) levels, using a murine carrageenan-induced pleurisy.
Methods and Results: The aerial parts of herb were air-dried at 50ºC, crushed and stored at 8ºC. The CE (3g) was placing in 150mL of boiling water. The aqueous extract was lyophilized and analyzed by Nuclear Magnetic Resonance and High Performance Liquid Chromatography-High Resolution Mass Spectrometry. The MeONOB was isolated from EtOH by silica gel column chromatography, using the n-hexane-ethyl acetate-methanol. Swiss mice were used though the experiments (Br.J.Pharmacol.183.811-19.1996). Ethical Committee approved (PP00757/CEUA/2012). Different groups of animals (n=5) were treated with CE(10-200mg/kg,i.p.), EtOH(5-25mg/kg,i.p.), HEX(25-50mg/kg,i.p.) or MeONOB(2.5-10mg/kg,i.p.) 0.5h before Cg (1%,i.pl.). The inflammation was analyzed after 4h. The animals were previously challenged with Evans blue dye (25mg/kg,i.v.) to evaluate the exudation. The doses of CE(50mg/kg), EtOH(10mg/kg), HEX(50mg/kg) or MeONOB(5mg/kg) administered 0.5 h before Cg injection were selected to evaluate the effect of the herb upon MPO and ADA and NOx. Statistical differences were determined by ANOVA and Student-Newman-Keuls post-hoc analysis. Values of p<0.05 were considered significant. CE(50-200mg/kg), EtOH(10- 25mg/kg), HEX(50mg/kg) and MeONOB(5-10mg/kg) inhibited leukocytes (CE:35.0±6.8 to 79,3±1.8%, EtOH:57.0±6.6 to 75.6±0.3%, HEX:56.7±7.5%, MeONOB: 58.5±2.2 to 60.0±6.2%), neutrophils (CE:37.4±7.1 to 80.1±1.7%, EtOH:68.3±2.2 to 77.0±0.2%, HEX:54.3±8.1%, MeONOB:63.1±2.0 to 63.8±5.5%), exudation (CE:26.5±2.8 to 68.2±2.9%, EtOH:58.5±3.9 to 63.3±2.9%, HEX:33.3±2.4%, MeONOB:51.3±7.2 to 54.3±2.7%). The herb inhibited MPO (CE:34.7±5.6%, EtOH:31.6±5.3%, HEX:29.6±4.2%, MeONOB:28.6±2.3%), ADA (CE:70.5±6.3%,
EtOH:71.0±4.9%, HEX:72.1±4.8%, MeONOB:67.3±2.1%) and NOx (CE:79.1±7.2%, EtOH:63.2±12.9%, HEX:71.2±9.4%, MeONOB:55.5±13.9%) (p<0.05).
Conclusion: A.conyzoides L. presented important anti-inflammatory activity not only by inhibiting leukocytes migration but activated neutrophils. This effect was also associated with exudation and NOx decrease.
Financial support: CAPES
EXPERIMENTAL ACUTE PANCREATITIS PRESENTS INCREASED OF THE CYTOKINES PLUS IMPORTANT PULMONAR DYSFUNCTION
CECÍLIA MENDES MORAIS1; PRISCILLA FERNANDA CAMPOS JUSTINO2; CAIO LUCIO ANDRADE3; JOSÉ ATHAYDE VASCONCELOS MORAIS4; LUARA MANUELA NEVES SILVA5; WALBER OLIVEIRA MENDES6; MARINA RODRIGUES COSTA7; ALVARO XAVIER FRANCO8; MARCELLUS HENRIQUE LOIOLA PONTE SOUZA9; DAVID NEIL CRIDDLE10; PEDRO MARCOS GOMES SOARES11.
1,2,3,4,5,6,7,8,9,11.UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA - CE - BRASIL; 10.UNIVERSITY OF LIVERPOOL, LIVERPOOL - REINO UNIDO.
Introduction: Acute pancreatitis is an acute clinical disease, characterized by rapid progression, systemic inflammatory response (SIR) and high mortality. Lung injury is a severe complication of acute pancreatitis that increases the mortality rate. The literature has demonstrated that cytokines are involved in organ damage distance as observed in acute pancreatitis. Objectives: Evaluate the involvement of inflammatory cytokines and spirometric parameters in the course of experimental acute pancreatitis. Methods: Male Wistar rats (100-150g) were treated four times with one hour interval, intraperitoneally with 20μg/kg caerulein (C) or saline (S). Twenty-four hours after the first injection of cerulein, the animals were sacrificed and plasmatic amylase and lipase was measured. Besides, the concentrations of the cytokines (TNF-α, IL-1β, IL-6, IL-18, EPO, IL-2, IL-10, GRO-KC, and VEGF) were measured by ELISA. Another groups was performed the same treatment, after animals were anesthetized, tracheostomized and connected to a spirometer specific for small animals. The following parameters were evaluated: Flow Rate (FR), Tidal Volume (TV) and Minute Volume (MV). All animal procedures were approved by the local ethics committee (protocol 88/11). Values were considered significant with p<0.05, using ANOVA and Bonferroni tests. Results: Caerulein induced a raise plasma levels of amylase (UI/dL) (C=5863.2 ± 73.2; S=2897.4 ± 29.3) and lipase (UI/dL) (C=113.9 ± 19.1; S=57.3 ± 15.7). Caerulein also was cable of the induce important increased of concentrations of cytokines (pg/mL) TNF- α (S=5.5 ± 2.8; C=7.2 ± 1.2); IL-18 (C=674.7 ± 114.8; S=373.2 ± 59.3), IL-6 (C=17.4 ± 3.5; S=11.6 ± 2.1); IL-1β (C=6.6 ±0.7; S=3.9 ± 1.2), EPO (S=50.8 ± 10.9; C=103.5 ± 16.7); IL-2 (S=68.8 ± 4.8; C=88.8 ± 1.8); IL-10 (S=26.1 ± 3.7; C=39.4 ± 3.6) and VEGF (S=4.4 ± 0.6; C=6.8 ± 1.9). Acute pancreatitis induced by caerulein showed significant changes in lung function represented by values of the FR(mL/s) (C=14.4 ± 0.6; S=20.3±1.6), TV(mL) (C=0.7 ± 0.2; S=0.8 ± 0.3) and MV (mL/min) (C=87.1 ± 3.9; S=111.3 ± 5.6). Conclusion: Acute pancreatitis induced by caerulein shows involvement of the cytokines, which correlate with important functional pulmonary alterations.
Financial Support: CNPq, CAPES and FUNCAP.
EXPOSURE TO OCCUPACIONAL POLLUTANT DURING INTRAUTERINE PHASE INCREASES ALLERGIC LUNG INFLAMMATION IN OFFSPRING
MARILIA MAIELLARO; LUANA BEATRIZ VITORETTI; JOÃO ANTÔNIO GIMENES; HELORY VANNI DOMINGUES; WOTHAN TAVARES-DE-LIMA; SANDRA POLISELLI FARSKY; ADRIANA LINO-DOS-SANTOS-FRANCO.
UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL.
EXPOSURE TO OCCUPACIONAL POLLUTANT DURING INTRAUTERINE PHASE INCREASES ALLERGIC LUNG INFLAMMATION IN OFFSPRING
MARÍLIA MAIELLARO (IC)(1); LUANA BEATRIZ VITORETTI (PG)(2); JOÃO ANTONIO GIMENES (PG)(2); HELORY
VANNI DOMINGUES (2); WOTHAN TAVARES DE LIMA(2); SANDRA HELENA POLISELI FARSKY(1), ADRIANA
LINO DOS SANTOS FRANCO(1).
(1)Department of Clinical and Toxicological Analyses, University of São Paulo, São Paulo, Brazil; (2)Department of Pharmacology, University of São Paulo, São Paulo, Brazil.
Introduction: Formaldehyde (FA) is an occupational pollutant widely used in many industries, laboratories and, is expelled in the smoke of the cigarette. Data of literature has shown correlation between exposure to pollutants during the pregnancy and increased risk for development of allergic lung diseases in young or adult phase. The objective of this study was evaluated the effects of FA exposure in uterus and it is repercussion on the development of allergic lung inflammation in offspring.
Methods: Female 3-month-old Wistar rats were divided in 2 experimental groups: C (control group) and P (group exposed to FA 5 ppm for 1h during 21 days of pregnancy. After 30 days of birth, male and female rats were sensitized with ovalbumin (OVA)-alum by the subcutaneous route. Two weeks later the rats were challenged with aerosolized OVA (1%, 15 min) for three consecutive days. After 24 h the last OVA challenge the inflammatory parameters were evaluated in terms of the cells recruited into the lung, blood and bone marrow, cytokines released, IgE synthesis and the pulmonary function to cholinergic agonist.
Results: Our data showed that offspring of rats exposed to FA (P group) increased the number of cells recruited in bronchoalveolar lavage in relation to Control and naive groups. The number of cells in peripheral blood and bone marrow was reduced in C and P groups when compared to the naïve group. We showed that IL-6 and IL-17 were increased in P group when compared to the C and naive groups.The IgE synthesis and pulmonary function were not modified by exposition of FA.
Conclusion: Our study revealed that FA exposure in uterus increases the risk for development of allergic lung inflammation and these effects are mediated by cytokines such as IL-6 and IL-17. Thus, the data obtained in this study may contribute to the understanding the effects of pollutants as risk factor to development or deterioration of asthma symptoms.
Financial support: FAPESP (2013/08796-9; 2011/51711-9).
GABAPENTIN IMPROVES THE INFLAMMATORY RESPONSE BY INHIBIT THE CARRAGEENAN AND DEXTRA- INDUCED PAW EDEMA AND MPO CONCENTRATION.
DIVA DE AGUIAR MAGALHÃES; PAMMELA WERYKA DA SILVA SANTOS; TARCISIO VIEIRA BRITO; RAFAEL SILVA PRUDÊNCIO; JORDANA MAIA DIAS; JAND-VENES ROLIM MEDEIROS; ANDRÉ LUIZ REIS BARBOSA.
UNIVERSIDADE FEDERAL DO PIAUÍ, PARNAÍBA - PI - BRASIL.
GABAPENTIN IMPROVES THE INFLAMMATORY RESPONSE BY INHIBIT THE CARRAGEENAN AND DEXTRA- INDUCED PAW EDEMA AND MPO CONCENTRATION.
DIVA DE AGUIAR MAGALHÃES1; PAMMELA WERYKA DA SILVA SANTOS1; TARCISIO VIEIRA BRITO1; RAFAEL SILVA PRUDÊNCIO1; JORDANA MAIA DIAS1; JAND-VENES ROLIM MEDEIROS1; ANDRÉ LUIZ REIS BARBOSA1.
1LAFFEX – Laboratory of Experimental Physiopharmacology, Biotechnology and Biodiversity Center Research (BIOTEC), Federal University of Piauí.
Introduction: Gabapentin (GBP) is used in anticonvulsant clinical behavior. Recently data were demonstrated that GBP diminished the inflammatory response in models of inflammation. However, there are no studies that demonstrated by which this phenomenon occur. Methods and Results: To investigate the possible mechanisms of gabapentin reduces the inflammatory response mice were pre-treated with GBP (0.1, 0.5 or 1 mg.kg-1, i.p). One hour later, carrageenan (Cg,500 μg/paw; 50 μl) or dextran (Dxt, 500 μg/paw; 50 μl) were administered by subplantar injection into the right paw. Paw volume was measured with a plethysmometer (Ugo-Basile 7140) immediately before injections and then 1, 2, 3, and 4 h later for carrageenan or 30 min, 1, 2, 3, and 4 h later for dextran injections. To evaluated the neutrophil infiltration into the right hind paw, was performed the MPO assay. This experimental protocol was made immediately after carrageenan-induced paw edema. GBP (1 mg.kg-1) significantly reduces the paw edema- induced by Cg (3 h: 0.225 ± 0.01109 ml; 4 h:0.025 ± 0,007638 ml) and Dxt (30 min: 006667± 0.004944; 1h: 0.01333 ± 0.006146) when compared those groups with Cg group (3h: 0.1017 ± 0,01046; 4h: 0.08167 ± 0.001667) and Dxt (30 min: 0.0340± 0.002449; 1h: 0.0420 ± 0.004899) group respectively. In the MPO assay the GBP decrease the MPO concentration (1.100 ± 0, 3340 UMPO/mg plantar tissue) when compared this group with the animals that receive only carrageenan into the right paw (17.73 ± 3,850 UMPO/mg plantar tissue).Conclusion: The current study demonstrated that GBP decreases several parameters of acute inflammation by diminished carrageenan and dextran-induced paw edema and neutrophil infiltration into the site of inflammatory process.
GABAPENTIN INHIBIT THE INFLAMMATORY RESPONSE BY MODULATING THE NEUTROPHIL MIGRATION AND THE RELEASING OF CYTOKINES IN THE PERITONEAL FLUID.
JORDANA MAIA DIAS; PAMMELA WERYKA DA SILVA SANTOS; DIVA DE AGUIAR MAGALHÃES; JALLES ARRUDA BATISTA; TARCISIO VIEIRA BRITO; RAFAEL SILVA PRUDÊNCIO; SAMARA RODRIGUES BONFIM DAMASCENO; JAND-VENES ROLIM MEDEIROS; ANDRÉ LUIZ REIS BARBOSA.
UNIVERSIDADE FEDERAL DO PIAUÍ, PARNAÍBA - PI - BRASIL.
GABAPENTIN INHIBIT THE INFLAMMATORY RESPONSE BY MODULATING THE NEUTROPHIL MIGRATION AND THE RELEASING OF CYTOKINES IN THE PERITONEAL FLUID.
JORDANA MAIA DIAS1; PAMMELA WERYKA DA SILVA SANTOS1; DIVA DE AGUIAR MAGALHÃES1; JALLES ARRUDA BATISTA1; TARCISIO VIEIRA BRITO1; RAFAEL SILVA PRUDÊNCIO1; SAMARA RODRIGUES BONFIM DASMACENO1; JAND-VENES ROLIM MEDEIROS1; ANDRÉ LUIZ REIS BARBOSA1.
1LAFFEX – Laboratory of Experimental Physiopharmacology, Biotechnology and Biodiversity Center Research (BIOTEC), Federal University of Piauí.
Introducion: The neutrophil migration into the peritoneal cavity induced by the flogisitic agent carrageenan is dependent on macrophage activation and release of pro-inflammatory cytokines such as IL-1β and TNF-α resulting in protein extravasation and cellular infiltration at the site of inflammation. Recently data has been demonstrated that Gabapentin (GBP) decreased inflammatory response in several models of inflammation. However, no studies have shown the action of GBP in the neutrophil infiltration during inflammatory response induced by carrageenan. Knowing this, the main goal of study to verify the effect of GBP on the peritonitis model. Methods and Results: Mice were pre- treated with GBP (1mg.kg-1) and one hour later, the animals were injected intraperitoneally with 250 µl carrageenan (Cg, 500 µg) into the peritoneal cavity; 4 h later total cell counts were performed in a Neubauer chamber, and differential cell counts (100 cells total) were carried out on cytocentrifuge slides stained with haematoxylin and eosin. From the peritoneal exudates which was induced peritonitis IL-1β and TNF-α were measured. In this study, the group GBP showed a significant reduction in peritoneal leukocyte count (1760 ± 123.9 x 103 cells/ml), neutrophils count (0.7842 ± 0.05502 x 103 cells/ml) and peritoneal concentration of IL-1β (599.3 ± 113.1 pg/ml) and TNF-α (90.12± 14.22) compared to the Cg (peritoneal leukocyte count: 16675± 2252 x 103 cells/ml; neutrophils count :11.41 ± 2.392 x 103 cells/ml; IL-1β :1125 ± 37.40 pg/ml; and TNF-α: 564.9± 52.32). Conclusion: This study demonstrated that GBP reduced inflammation by decreasing neutrophil migration into the site of inflammatory process and release of pro- inflammatory cytokines.
GABAPENTIN REDUCES THE INFLAMMATORY PROCESS BY MODULATING THE ACTION OF THE HISTAMINE, SEROTONIN, PGE2 AND 48/80-INDUCED PAW EDEMA.
PAMMELA WERYKA DA SILVA SANTOS; DIVA DE AGUIAR MAGALHÃES; JALLES ARRUDA BATISTA; TARCISIO VIEIRA BRITO; RAFAEL SILVA PRUDÊNCIO; RENAN OLIVEIRA SILVA; JORDANA MAIA DIAS; JAND-VENES ROLIM MEDEIROS; ANDRÉ LUIZ REIS BARBOSA.
UNIVERSIDADE FEDERAL DO PIAUÍ, PARNAÍBA - PI - BRASIL.
GABAPENTIN REDUCES THE INFLAMMATORY PROCESS BY MODULATING THE ACTION OF THE
HISTAMINE, SEROTONIN, PGE2 AND 48/80-INDUCED PAW EDEMA.
PAMMELA WERYKA DA SILVA SANTOS1; DIVA DE AGUIAR MAGALHÃES1; JALLES ARRUDA BATISTA1; TARCISIO VIEIRA BRITO1; RAFAEL SILVA PRUDÊNCIO1; RENAN OLIVEIRA SILVA1; JORDANA MAIA DIAS1; JAND-VENES ROLIM MEDEIROS1; ANDRÉ LUIZ REIS BARBOSA1.
1LAFFEX – Laboratory of Experimental Physiopharmacology, Biotechnology and Biodiversity Center Research (BIOTEC), Federal University of Piauí.
Introduction: Studies demonstrated that carrageenan injection into the mouse paw induces a biphasic edema. The early phase is mainly mediated by histamine, serotonin, and by an increasing synthesis of prostaglandins such as prostaglandin E2 (PGE2). These mediators are involved in induction of paw edema. Recently data were demonstrated that Gabapentin (GBP) diminished the inflammatory response in models of inflammation. However, there are no studies that demonstrated by which this phenomenon occur. Methods and Results: To investigate the anti- inflammatory response of GBP, mice are pre-treated with this drug (1mg.kg-1). One hour later, Histamine (1% w/v) or Serotonin (1% w/v) or PGE2 (5µg/paw) or 48/80 (2µg/paw) were administered by subplantar injection into the right paw. Paw volume was measured by pletismometry. immediately before injections and then 30,60,90 and 120 min later for Serotonin, PGE2 and 48/80 and 30 min, 1, 2, 3 and 4 h later for Histamine injections. GBP (1 mg.kg-1) significantly reduces the paw edema-induced by Histamine (30 min: 0.0380 ± 0.003742; 1h: 0.03167± 0.007032), Serotonin (30 min: 0.0440 ± 0.004000; 60 min: 0.0300 ± 0.004082), PGE2 (30 min: 0.0250±0.0050) and 48/80 (30 min: 0.0700±0.01581; 60 min: 0.0525±0.01109) and when compared those groups with Histamin (30 min: 0.1320 ± 0.009165; 1h: 0.1880± 0.01428), Serotonin (30 min: 0.0900±0.008165; 60 min: 0.0525±0.004787), PGE 2 (30 min: 0.0750±0.002887 ) and 48/80 (30 min: 0.1175 ± 0.008539; 60 min: 0.0800±0.009129) groups respectively. Conclusion: The study revealed that GBP decrease the action of several mediators involved in vascular events of systemic acute inflammation.
GASTROINTESTINAL DYSMOTILITY ASSOCIATED WITH A SEVERE ACUTE PANCREATITIS IS DEPENDENT OF NEUTROPHIL INFILTRATION.
ANA CARLA DA SILVA CARVALHO DIAS1; RHAMON BARROSO SOUSA2; RONALDO ALBUQUERQUE RIBEIRO3; ROBERT SUTTON4; DAVID NEIL CRIDDLE5; PEDRO MARCOS GOMES SOARES6; MARCELLUS HENRIQUE LOIOLA PONTE SOUZA7.
1,2,3,7.INSTITUTE OF BIOMEDICINE OF BRAZILIAN SEMI-ARID, DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FORTALEZA - CE - BRASIL; 4,5.NIHR PANCREAS BIOMEDICAL RESEARCH UNIT, UNIVERSITY OF LIVERPOOL, LIVERPOOL - REINO UNIDO; 6.INSTITUTE OF BIOMEDICINE OF BRAZILIAN SEMI-ARID, DEPARTMENT OF MORPHOLOGY, FORTALEZA - CE - BRASIL.
Introduction: Despite the gastrointestinal dysmotility is commonly encountered in patients with severe acute pancreatitis, the pathogenesis of this condition is not totally defined. We demonstrated that fucoidan reduced the severity of acute pancreatitis in mice by decreasing neutrophil infiltration. The present study has evaluated the effect of the neutrophil infiltration blocker fucoidan in the gastrointestinal dysmotility associated with severe acute pancreatitis. Methods: Acute pancreatitis was induced in mice (n=6/group) by the retrograde infusion of taurolithocholic acid (TLC-S) into the pancreatic duct. Experimental groups received fucoidan (25 mg/kg, i.v.) before pancreatitis induction, control groups received only saline. After 24 hours, serum amylase and lipase, myeloperoxidase (MPO) activity (lung, pancreas, stomach and jejunum), histological assessment (pancreas), gastric emptying (GE) and gastrointestinal transit (GIT) were determined. Gastric fundus and jejunum contractility in vitro was evaluated using carbachol (0,01-30 µM), KCl 60mM and electrical field stimulation (0.5-8.0 Hz; 1ms; 40 V). Results: TLC-S infusion increased serum level of amylase (6154.0±417.6 U/l), lipase (865.0±75.9 UI), pancreatic (7.3±0.9 UMPO/mg) and lung MPO (7.7±0.9 UMPO/mg) compared with Saline (amylase= 3435.0±170.7 U/l; lipase= 449.2±37.9 UI; pancreatic MPO = 2.4±0.3 UMPO/mg; lung MPO= 4.9±0.5 UMPO/mg). Fucoidan decreased the augmented levels of amylase (3878.0±519.5 U/l), lipase (488.6±32.7 UI), pancreatic (3.4±1.7 UMPO/mg) and lung MPO (5.1±0.6 UMPO/mg). Pancreas histological changes were significantly (P<0,05) attenuated by fucoidan. Acute pancreatitis induced by TLC-S caused delayed GE (31.8±3.0%) and GIT (1.8±0.1), increased gastric (0.9±0.02 UMPO/mg) and jejunum MPO (1.4±0.03 UMPO/mg) compared with control group (saline: GE= 25.5±0.8%; GIT= 2.2±0.02; gastric MPO= 0.6±0.1 UMPO/mg; jejunum MPO= 0.7±0.02 UMPO/mg) and jejunum hypercontractility in vitro. Fucoidan reversed the gastrointestinal disorders in vivo (GE= 23.2±1.7%; GIT= 2.3±0.1), in vitro and the neutrophil infiltration in the stomach (0.6±0,1 UMPO/mg) and in the jejunum (0.9±0,1 UMPO/mg) Conclusion: The reduction of the neutrophil infiltration induced by fucoidan reversed the gastrointestinal dysmotility associated with a severe acute pancreatitis in mice. Financial support: CAPES, CNPq.
GEDUNIN IMPAIRS LPS-INDUCED TOLL-LIKE RECEPTOR 4 SIGNALING IN MACROPHAGES
PERLA VILLANI BORGES1; KATELIM HOTTS MORET2; LEONARDO SEITO3; THADEU MOREIRA COSTA4; CLARISSA MAYA MONTEIRO5; PAULO RICARDO BATISTA6; ERNESTO CAFFARENA7; PATRÍCIA PACHECO8; MARIA DAS GRAÇAS HENRIQUES9; CARMEN PENIDO10.
1,2,3,4,8,9.LABORATÓRIO DE FARMACOLOGIA APLICADA, FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL; 5.LABORATÓRIO DE IMUNOFARMACOLOGIA, FUNDAÇÃO OSWALDO CRUZ, INSTITUTO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL; 6,7.PROGRAMA DE COMPUTAÇÃO CIENTÍFICA, GRUPO DE BIOFÍSICA COMPUTACIONAL E MODELAGEM MOLECULAR, FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL; 10.CENTRO DE DESENVOLVIMENTO TECNOLÓGICO EM SAÚDE, FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by CD14/TLR4/MD-2 complex. Additional receptors, such as heat shock proteins (Hsp), have been suggested to be part of this activation cluster (Trends of immunol. 23:301-304, 2002). Previous data from our group and others have demonstrated that gedunin, a tetranortriterpenoid obtained from the seeds of Carapa guianensis, present marked antiinflammatory properties via the modulation of Hsp90 (Bioorganic & Medicinal Chemistry 19:684–692, 2011). In the present study, we have investigated the role of gedunin in LPS-induced inflammatory response in macrophages.
Methods and Results: Our results demonstrate that the incubation of immortalized murine macrophages with gedunin (0.01-10 mM) did not induced citotoxicity (100% viability), as assessed by resazurin reduction method. The pretreatment of macrophages with gedunin (10 µM) and dexamethasone (50 nM) significantly inhibited LPS (50 ng/ml)-induced cytokine production, as assessed by ELISA: IL-6 (Medium: 0.02±0.01; LPS: 53.4±1.6; DEXA: 0.03±0.01; GED: 3.6±0.01 ng/ml, n=5, p<0.05) and TNF-α (Medium: 5.3±0.23; LPS: 16.0±0.28; DEXA: 1.6±0.1; GED: 7.3±0.3 ng/ml, n=5, p<0.05). Pretreatment of macrophages with Hsp90 inhibitor 17AAG (1 µM) also impaired the production of IL-6 (17AAG: 0.6±0.02 ng/ml, n=5, p<0.05) and TNF-α (17AAG: 6.0±0.2 ng/ml; n=5, p<0.05). The production of nitric oxide (NO) by macrophages stimulated with LPS (50 ng/ml) plus interferon-γ (IFN-γ: 25 U/ml) was also inhibited by dexamethasone (100 nM) and gedunin (10 µM) (Medium: 10.86±0.22; LPS+IFN: 35.3±1.3; DEXA: 21.83±0.65; GED: 11.5±0.3 µM of nitrite, n=5, p<0.05), as determined by Griess method. Pretreatment of macrophages with gedunin (1 µM) also impaired LPS-induced NFκB translocation into the nucleus, as revealed by western blot. We performed computational modeling studies and proposed through in silico docking calculations that gedunin could directly bind to MD-2 protein. Gedunin efficiently docked into the MD-2 LPS binding site, suggesting that it might act as a competitive inhibitor of LPS, blocking the formation of TLR4/MD-2/LPS complex.
Conclusion: Gedunin modulates LPS-induced macrophage activation, impairing TLR4 signaling, probably blocking the complex TLR4/MD-2/LPS.
Financial support: FIOCRUZ/ CNPQ/FAPERJ.
GERANIOL ACTS STIMULATING THE PROTECTIVE FACTORS OF GASTRIC AND DUODENAL MUCOSA IN MODELS OF EXPERIMENTAL PEPTIC ULCER DISEASE
KATHARINNE INGRID MORAES DE CARVALHO1; FLAVIA BONAMIN2; LAÍSA PINHEIRO DA SILVA3; DAMIÃO PERGENTINO DE SOUSA4; CLÉLIA AKIKO HIRUMA-LIMA5.
1.UFRJ, RIO DE JANEIRO - RJ - BRASIL; 2,3,5.UNESP, BOTUCATU - SP - BRASIL; 4.UFPB, JOÃO PESSOA - PB - BRASIL.
Introduction: The global expansion in the consumption of alcohol and NSAIDs has contributed to an increased incidence of peptic ulcers in the population. One of the biggest problems relative on disease is recurrence of it after the treatment, justifying the search for new treatments more effectives that strengthen the mucosal defense. Geraniol, an acyclic monoterpene alcohol has been recognized by to exhibit important biological activities, such as antioxidant, antiinflammatory, antimicrobial and anti-apoptotic effects (Mol and Cell Biochem. 369:17-25, 2012; Exp Mol Pathol. 94:419-429, 2013). Thus, this study aims evaluate if the antioxidant and anti-inflammatory actions of geraniol could be effective in preventing the damage caused by ulcerogenic agents. Methods and Results: Male Wistar rats (n = 6) were treated whit tween 80 ® 8% (vehicle), geraniol 7.5 mg/kg or carbenoxolone 100 mg/kg (positive control) one hour before of ulcerogenic agent. After 1 h of induction of lesions, the animals were sacrificed and their stomachs were removed to evaluate the number of lesions and strips of the stomach were removed, weighed for determination of total glutathione content (GSH), activity of myeloperoxidase (MPO) and mucus quantification. The results showed the treatment with geraniol significantly inhibited gastric lesions induced by ethanol (24.40 ± 4.53 mm 2) when compared the vehicle (82.93 ± 5.75 mm2), decreased the number of ulcerative lesions induced by ischemia/reperfusion (30.60 ± 5.28 mm2 vs vehicle 106.70 ± 17.73 mm2) and decreased the duodenal ulcers induced by cysteamine (30.46 ± 10.85 mm2 vs vehicle 96.70 ± 12.70 mm2). Analysis of the gastric tissue of rats treated with geraniol revealed GSH levels increased (1708 ± 79.21 nmol/g tissue vs vehicle 1319 ± 60.05 nmol/g tissue), levels of MPO in the gastric mucosa decreased (266.5 ± 31.31 u/g tissue vs vehicle 441.2 ± 58.61 u/g tissue) and increased mucus production (1509 ± 64.85 vs vehicle 854.80 ± 29.26 μg Alcian Blue/mL/g tissue). The use of specific antagonists showed that action of geraniol was mediated by the activation of defensive mucosa-protective factors such as the NO pathway, endogenous prostaglandins, increased sulfhydryl compounds and the stimulation of CGRP release through the activation of TRPV. Conclusion: The multifaceted mechanisms of gastroprotective geraniol represent a promising option for the treatment of gastric and duodenal mucosal injury.
Financial support: CNPq, FAPESP.
GLUCAGON INHIBITS AIRWAY SMOOTH MUSCLE CONTRACTION AND HYPERREACTIVITY IN VIVO THROUGH NITRIC OXIDE GENERATION
DANIELLA BIANCHI REIS INSUELA1; JULIO BELTRAME DALEPRANE2; RAFELE RODRIGUES ALMEIDA3; ANA CAROLINA SANTOS ARANTES4; RENATO SERGIO BALÃO CORDEIRO5; PATRICIA MACHADO RODRIGUES SILVA6; MARCO AURELIO MARTINS7; VINICIUS FRIAS CARVALHO8.
1,3,4,5,6,7,8.FUNDAÇÃO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL; 2.UNIVERSIDADE DO ESTADO DO RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL.
Introduction: In our previous work, we demonstrated that glucagon presents an antispasmodic effect on airway smooth muscle contractility in vitro and this action occurs in an indirectly way, by activation of its receptor expressed only in airway epithelium with consequent release of NO. In this study, we investigated the effect of glucagon on airway smooth muscle contractility and hyperreactivity in vivo and possible mechanism involved. Methods and Results: All the procedures used in this study were in accordance with the guidelines of the Ethic Committee on Use of Laboratory Animals of the Oswaldo Cruz Foundation, License LW – 23/10. The action of glucagon (0.1 – 10 μg/Kg, i.n) on methacholine-provoked airway obstruction in mice was evaluated in noninvasive and invasive plethysmography. In some experiments, the mice received i.p. L-NAME (20 mg/Kg) 30 min before glucagon. The effect of glucagon on NOS3 expression in lungs was verified by Western blot. To assessed the airway hyperreactivity, the mice were sensitized with ovalbumin (OVA) plus aluminum hydroxide in days 0 and 7 and challenged with OVA (1 μg/μL, i.n.) for 2 consecutive days in days 21 and 22 after sensitization. Treatment with glucagon was administrated 1 h after each provocation, and all analysis were realized 24h after the last challenge. Glucagon (1 μg/Kg, i.n.) inhibited methacholine-induced bronchoconstriction 1 h (saline i.n.: 67.03 ± 6.60 AUC of Penh; glucagon i.n.: 49.09 ± 3.05 AUC of Penh; mean ± SEM; n = 7) and 3 h (saline i.n.: 165.50 ± 21.51 AUC of Penh; glucagon i.n.: 82.14 ± 9.44 AUC of Penh; mean ± SEM; n = 6) after treatment. All the doses of glucagon (0.1 - 10 µg/kg) prevented the decrease in lung compliance promoted by methacholine, but only the dose of 1 µg/Kg inhibited the increase in airways resistance. L- NAME abrogated the antispasmodic action of glucagon. Moreover, glucagon induced an up-regulation of NOS3 expression in lungs. Finally, treatment with glucagon inhibited airway hyperreactivity and accumulation of eosinophils and neutrophils but did not change mononuclear leukocytes in BAL. Conclusion: Our results show that glucagon presents an anti-spasmodic effect on airway smooth muscle contraction in vivo and inhibits airway hyperreactivity in a murine model of asthma. This anti-spasmodic action is mediated by inducing an increased in the expression of NOS3 in the lungs with concomitant elevation of NO production. Financial support: CNPq, FAPERJ and FIOCRUZ.
HYDROGEN SULFIDE (H2S) AND NITRIC OXIDE (NO) ARE MUTUALLY DEPENDENT IN THE GASTRIC MOTILITY CONTROL.
LARISSE TAVARES LUCETTI1; ANA PAULA MACEDO SANTANA2; BRUNO DE MELO TAVARES3; RONALDO ALBUQUERQUE RIBEIRO4; PEDRO MARCUS GOMES SOARES5; JAND-VENES ROLIN MEDEIROS6; MARCELLUS HENRIQUE LOIOLA PONTE SOUZA7.
1,2,3,4,5,7.FEDERAL UNIVERSITY OF CEARA, FORTALEZA - CE - BRASIL; 6.FEDERAL UNIVERSITY OF PIAUI, PARNAIBA - PI - BRASIL.
INTRODUCTION: H2S has regulatory effects in the control of muscles physiology and inflammatory process. It was described that the actions of H2S could be at least in part dependent of NO. Recently, we demonstrated that H2S donors had effects in gastric motility (Eur J Pharmacol. 2012, 15;693(1-3):57-63). In this study, we evaluated the interrelation between H2S and NO in gastric emptying and in gastric muscle relaxation in mice. METHODS: Swiss mice (20- 25g) were divided in experimental groups:Saline; NaHS (H2S donor, 150µmol/kg p.o.); Sodium niproprusside (NPS, NO donor, 10 mg/kg p.o.); NaHS + L-NAME (NOS inhibitor,3 mg/kg i.p.); NPS + PAG (CSE inhibitor, 50 mg/kg); L-NAME and PAG. To evaluate the effects of these treatments in the gastric emptying, the animals were gavage-fed (0,3 ml) with the test meal (5% glucose solution with 0.05g/ml phenol red). Twenty minutes later, gastric dye recovery was measured by spectrophotometry. In addition, gastric fundus or pyloric sphincter muscle layers were mounted in an organ bath system. After 1 h of equilibration, L-Name (300µM), PAG (1mM) or Tyrode´s solution (control) were added. After 30 min, NaHS (10-1000µM) or NPS (0,3- 100µM) by a cumulative curve concentration-response. Data were expressed as percentages of the maximum relaxation obtain by papaverin (1- 1000μM). The project was approved by the local ethics committee (protocol No 63/07).RESULTS: NaHS decreased the gastric retention (18.9±0.9%,N=6) as compared to saline (25.9±1.5,N= 6) and L-Name + NaHS (31.5±2.1%,N=6) reversed the NaHS effect. We also observed that, in the pyloric sphincter, NaHS (37.3±1.7%) (100 µM), caused relaxation and the pre incubation with L-Name(26.50±1.41) reversed the NaHS effect. This effect was not observed in gastric fundus (NaHS=13.80±3.0% (100µM) and pre incubation with L-Name(10.7± 3.1%). NPS increased the retention (72.8±3.6%,N=6) as compared to saline (21.9±2.9%, N=6) and PAG + NPS (34.0±10.1%, N=6) reversed the NPS effect. In the pyloric sphincter (63.5±9.7%)(10µM) and in gastric fundus (65.4±7.0%)(10µM), NPS caused relaxation and pre incubation with PAG in the pyloric sphincter (37.4±6.7%) and in gastric fundus (35.4±8.2%) reversed the NPS effect . CONLUSION: In the gastric motility, the effect of H2S-donor was dependent of NOS activity and the effect of NO- donor was dependent of CSE activity. Then, we can infer that H2S and NO are mutually dependent in the gastric motility control. FINANCIAL SUPPORT: Capes and CNPq.
HYDROGEN SULFIDE INDUCES NEUROGENIC VASODILATATION IN THE MOUSE EAR
ERIKA PINTER; ZSOFIA HAJNA; GABOR POZSGAI; ZSUZSANNA HELYES.
DEPARTMENT OF PHARMACOLOGY, UNIVERSITY OF PECS, PECS - HUNGRIA.
Introduction: Capsaicin-sensitive sensory neurons express several ion channels of the TRP family, such as transient receptor potential ankyrin 1 (TRPA1) receptors. TRPA1 can be activated by several chemical stimuli, such as allylisothiocyanate (AITC) or cinnamaldehyde resulting in the release of inflammatory neuropeptides, e.g. Substance P
(SP) and calcitonin gene-related peptide (CGRP) [1]. Recently, the gaseous mediator hydrogen sulfide (H2S) was suggested to have effect on capsaicin-sensitive sensory neurons acting via TRPA1 receptors. Microcirculatory changes in the mouse ear have been investigated in this study.
Methods and Results: Balb/c, C57BL/6, TRPA1 KO, TRPV1 KO and NK1 KO mice (20-30 g) were treated with H2S donor sodium hydrogen sulfide (NaHS) or AITC. Ipsilateral ears of the animals were treated with 2% AITC or 5% NaHS. Contralateral ears received respective vehicles. Balb/c mice received selective TRPA1 receptor antagonist HC- 030031 (i.p. 30 or 100 mg/kg), NK1 receptor antagonist CP-99994 (i.p. 10-50 mg/kg), non-peptide CGRP antagonist BIBN4096 (i.p. 0.1-10 mg/kg). A separate group of mice were pretreated with resiniferatoxin (RTX) Ear blood flow was detected by laser Doppler imaging.
AITC treatment of mouse ears led to 29.8±2.8% increase in blood flow. NaHS elevated cutaneous blood flow of the mouse ears by 61±4.5%. Effect of NaHS on microcirculation was ameliorated by HC-030031, BIBN4096 as well as CP-99994. Blood flow of TRPA1 KO and NK1 KO mice showed significantly smaller increase in response to NaHS compared to the wild-type counterparts. Microcirculatory responses to NaHS were reduced in RTX-pretreated animals.
Conclusion: We conclude that CGRP and SP-mediated neurogenic components via activation of TRPA1 channels on the capsaicin-sensitive sensory nerves play pivotal role in the NaHS-induced vasodilatation in the mouse ear.
Financial support: This work was supported by research grants OTKA K-81984 and TÁMOP 422A-11/1/KONV-2012- 0024.
HYPOXIA ALTERED ALVEOLAR NA/+K/+ATPASE AND ENAC EXPRESSION IS SYNCHRONIZED VIA OXIDATIVE STRESS INFLAMMATORY RESPONSE: MODULATION BY CURCUMIN TREATMENT
TITTO MATHEW; SARADA SURYA KUMARI S.
DEFENCE INSTITUTE OF PHYSIOLOGY AND ALLIED SCIENCE, DELHI - ÍNDIA.
Introduction: In cultured alveolar epithelial cells, hypoxia induces down regulation of the two main proteins, the epithelial Na channel (ENaC) and the Na/+K/+ATPase. However, the time dependant in vitro effects of hypoxia on alveolar epithelial transport and pathway(s) regulating the same have not been well studied. Therefore, the objective of this study was to investigate in vitro time dependant model of hypoxia induced reduction in vectorial Na and fluid transport across the alveolar epithelium.
Materials and Results: Human lung adenocarcinoma cell line (A549) was exposed to 3% O2, for different time periods (1 h, 3h, 6h, 12h, 24h and 48 h). The cells were treated with curcumin (10μM) and exposed for 6h similarly male SD rats were exposed to hypobaric hypoxia (7620m) for 6 h with and without curcumin (orally, 50mg/KgBW).The hypoxic exposure increased reactive oxygen species (ROS), lipid peroxidation (MDA) with concomitant reduction in antioxidants levels (GSH, GPx and SOD) in A549 cells, (P<0.001).The hypoxic exposure also significantly up regulated NFkB thereby triggering a pro-inflammatory milieu in A549 cells. Hypoxia exposure induced progressive decrease in Na/+K/+ATPase and ENaC reaching negligible levels after 6 and 12 h respectively in A549 cells. The hypoxia exposure also resulted in decrease in Na+K+ATPase activity (0.134±0. 04 μM/Pi/μg protein /h, P<0.001). The curcumin supplementation at 10µM enabled the cells in maintaining a balanced oxidative state thereby attenuating the NFκB activation and enhanced Na/+K/+ATPase activity and protein expression. The in-vivo hypoxia exposure studies also revealed reduced Na/+K/+ATPase activity (2.5 μM/Pi/μg protein /h, P<0.00,n=6) and α ENaC expression levels. However, curcumin administration has enhanced Na+K+ATPase activity (12.2 μM/Pi/μg protein /h, P<0.001, n=6) and α ENaC expression levels by attenuating NFκB activation.
Conclusion: This study showed that curcumin treatment is capable of alleviating hypoxia induced down regulation of key ion channels of alveolar epithelium (Na/+K/+ATPase and ENaC) which is synchronised via NFkB.
Funding Source: The study was funded by Ministry of Defence; Govt. of India
IB4+ SUBPOPULATION OF C- NOCICEPTORS CONTRIBUTES TO INFLAMMATORY PAIN IN MICE
LARISSA GARCIA PINTO; GUILHERME RABELO SOUZA; ALEXANDRE HASHIMOTO PEREIRA LOPES; JHIMMY TALBOT; FERNANDO DE QUEIROZ CUNHA; THIAGO MATTAR CUNHA; SERGIO HENRIQUE FERREIRA.
FMRP-USP, RIBEIRAO PRETO - SP - BRASIL.
Introduction: The small-diameter nociceptive fibers (C fibers) have an important role in detecting noxious stimuli, initiating the transmission of painful information. They are classified as nonpeptidergic (IB4 positive) and peptidergic (IB4 negative) C fibers. However, a possible functional difference between these two classes of C fibers in the genesis of acute nociception as well as inflammatory pain is still unclear. This study aims to clarify the role of nonpeptidergic C fibers in acute nociception induced by mechanical, thermal and chemical stimuli as well as in inflammatory hypernociception. Methods and Results: In order to elucidate differences between these two classes of C fibers, a neurotoxin was used to selectively eliminate the nonpeptidergic C fibers: a saporin conjugated to isolectin B4 (IB4). Nociceptive threshold was evaluated through thermal (Hargreaves) and mechanical (filaments and electronic von Frey) tests in C57BL/6 mice. Nociception models were induced by intraplantar (i.pl.) injection of capsaicin and formalin (acute nociception) or by i.pl. administration of prostaglandin E2 (PGE2), epinephrine, endothelin, NGF, GDNF and carrageenan
(inflammatory hypernociception). P2X3 and TRPV1 expression were analyzed by Western blot of dorsal root ganglion
(DRG). The expression of IB4-labeled in spinal cord was determined by immunofluorescense using confocal microcopy. Firstly, it was observed that the intrathecal administration of IB4-saporin did not change baseline thermal and mechanical nociceptive threshold of the mice paw when compared to saline and saporin-control groups. The intrathecal administration of IB4-saporin reduced mechanical inflammatory hypernociception induced by carrageenan, epinephrine, endothelin, PGE2 or GDNF, but not NGF, in mice. Similarly, the treatment with IB4-saporin inhibited the nociception caused by intraplantar injection of the capsaicin. By contrast, the acute nociception induced by formalin did not change by administration of IB4-saporin. In addition, the expression of TRPV1 and P2X3 in DRG were reduced after treatment with IB4-saporin. Consistent with these findings, we found that IB4-saporin injection decreased the expression of IB4-labeled in spinal cord.
Conclusion: Our results indicate that the nonpeptidergic C fibers are important for the development of nociception in the paw of mice induced by inflammatory stimuli but not in basal nociceptive threshold.
Financial support: This work was supported by grants from FAPESP and CNPq (Brazil).
IL-6 IS INVOLVED IN THE DEVELOPMENT OF THE SEPSIS-INDUCED IMMUNOSUPPRESSION
VINÍCIUS LOPES ANDRADE; DANIELE CARVALHO NASCIMENTO; JOSÉ CARLOS FARIAS ALVES-FILHO.
FMRP-USP, RIBEIRÃO PRETO - SP - BRASIL.
IL-6 IS INVOLVED IN THE DEVELOPMENT OF THE SEPSIS-INDUCED IMMUNOSUPPRESSION
VINICIUS LOPES ANDRADE1; DANIELE BERNARDO CARVALHO NASCIMENTO1; JOSÉ CARLOS FARIAS ALVES-FILHO1
1Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo – FMRP-USP
Introduction: Sepsis is a systemic inflammatory response due to infection that remains the main cause of morbidity in intensive care units (Hotchkiss & Karl, 2003). A compensatory anti-inflammatory response develops concomitantly and/or subsequent course of systemic pro-inflammatory response induced during the sepsis. The imbalance of the immune response in favor of the anti-inflammatory response can result in immunosuppression. Interleukin-6 (IL-6) is a pleiotropic cytokine implicated in the regulation of immune response. Thus, we investigated the involvement of IL-6 in the development of immunosuppression during sepsis. Methods and Results: The WT and IL-6-/- mice were induced to sepsis by cecal ligation and puncture (CLP) model followed by a basic support (hydration and antibiotics). We found that survival was approximately 40% for both groups. Sepsis surviving mice were then infected with Legionella pneumophila nasally 15 days after CLP. Interestingly, IL-6-/- mice were more resistant to secondary infection (40% survival) than WT mice (100% mortality). In a new set of experiments, bacterial load was measured in the spleen 3 days after nasal infection with L. pneumophila in CLP surviving mice. Reduced number of bacterial counts was found in IL-6-/- mice compared to WT mice. Finally, we found an increase number of Tregs (CD4+Foxp3+ cells) in the spleen from WT sepsis surviving mice, which was not observed in IL-6-/- mice. Conclusion: These preliminary studies revealed a important role of IL-6 in the development of sepsis-induced immunosuppression, and the regulation of Tregs may be one of the mechanisms. Financial Support: CNPq and FAPESP
IMMUNOMODULATORS EFFECTS OF GLUCANS FROM CARIPIA MONTAGNEI MUSHROOM ON TNBS- INDUCED COLITIS
MARILIA NASCIMENTO SANTOS; JOEDYSON E.M. MAGALHÃES; THUANE S. PINHEIRO; LUIZA S.E.P.W. CASTRO; HUGO W.B. ALMEIDA; KAHENA Q. FLORENTIN; CELINA M.P.G. DORE; IURI G. BASEIA; EDDA L. LEITE.
UFRN, NATAL - RN - BRASIL.
Introduction: The mushrooms have been aim of intense research about its potential of application in different sectors of the pharmacology and food industry. Caripia montagnei is a basidiomycete species which contains polysaccharides with immunomodulatory properties. This study was conducted to avaluate some chemical characteristics of polysaccharides from Caripia montagnei mushroom and the effect of different doses of these polysaccharides at different intervals of treatment on anti-inflammatory responses in colonic injury at model of colitis induced by 2,4,6- trinitrobenzene sulfonic acid (TNBS).
Methods and Results: The induction of colitis was conducted by intracolonic administration of 30 mg by 2,4,6- trinitrobenzene sulfonic acid (TNBS) in 0.25 mL of 40% ethanol (v/v) via a polyethylene catheter inserted into the lumen of the colon of Wistar rats (n=10) (Int Immun. 8:1481-92, 2008). Treatments were administered intraperitoneally and started 24 h after induction of experimental colitis and were performed in two ways: from 12 to 12 h or 24 to 24 h, both for 60 h. The tests were approved (nº 014/2010) by the Ethics Committee of the Federal University of Rio Grande do Norte (UFRN). The FT-IR analysis and NMR showed that this species of mushroom polysaccharides are composed of α- and β-glucans. The dose of 75 mg/kg glucans reduced the MPO activity by about 3.7 and 3.8 times (p < 0.001) when administered every 12 h and 24 h, respectively. The activity of the colonic enzyme alkaline phosphatase (AP) was significantly reduced in the groups treated with glucans at 75 mg/kg (0.75 ± 0.028 U/mg; p < 0.01) at intervals of 24 h. Futhermore, also verified was the effect of the glucans from C. montagnei on the release of IL-6 and a dose-dependent decrease even reaching 64.8 ± 4.11% and 57.2 ± 6.45% of the cytokine levels in colonic tissue of the group treated with 75mg/kg in intervals of 12h and 24h, respectively. In all groups treated with the glucans, a moderate increase was observed in CAT levels in a dose-dependent manner up to 1300.61 ± 151 (p<0.05) and 1577.28 ± 170 (p<0.05) in the groups treated with 75 mg/kg at intervals of 12 and 24 h, respectively.
Conclusions: These findings confirms the anti-inflammatory potential of the polysaccharides from Caripia montagnei mushroom and shows some possible immunomodulatory mechanisms of action of these polysaccharides.
Financial support: The authors would like to thank CNPq and the CAPES for the financial support.
IMMUNOMODULATORY ACTIVITY OF ROSMARINIC ACID ON LPS-INDUCED TLR4 ACTIVATION, IN VITRO.
NORMA VILANY QUEIROZ CARNEIRO1; RYAN SANTOS COSTA2; TAMIRES CANA BRASIL CARNEIRO3; PEDRO AUGUSTO SILVA DOS SANTOS4; KEINA MACIELY CAMPOS DOURADO5; ANA TEREZA CERQUEIRA LIMA6; LAIN CARLOS PONTES DE CARVALHO7; NEUZA MARIA ALCANTARA NEVES8; CAMILA ALEXANDRINA FIGUEIREDO9.
1,2,3,4,5,6,8,9.INSTITUTO DE CIENCIAS DA SAUDE-UFBA, SALVADOR - BA - BRASIL; 7.CENTRO DE PESQUISA GONÇALO MONIZ- FIOCRUZ, SALVADOR - BA - BRASIL.
Introduction: Rosmarinic acid is a polyphenol found on Ocimum gratissimum and has been shown to have immunomodulatory activity by suppressing T cell receptor (TCR) signaling. Lipopolysaccharide (LPS) is a component from gram-negative bacteria responsible to induce inflammation by activating Toll-like receptor 4 (TLR4) signal pathway, producing mediators such as pro-inflammatory cytokines and Nitric Oxide (NO). The aim of the present study was to evaluate the anti-inflammatory effect of Rosmarinic acid in stimulated culture with LPS.
Methods and Results: Spleen cells from BALB/c mice were stimulated with 20µg/mL of LPS with or without Rosmarinic acid (RA) at a concentration of 50, 75 or 100µg/mL. After 72h of culture, the supernatants were collected and IFN-γ and IL-10 levels were determined by ELISA. Cell proliferation was estimated based on MTT-tetrazolium method. In order to evaluate nitric oxid production, BALB/c mice received 20µg/animal of LPS,i.p. After 72h of injection, peritoneal lavage was performed to obtain macrophages. NO production in macrophage culture was induced by LPS (5µg/ml) stimulation in vitro, with 25, 50 or 100µg/mL of RA. After 24h incubation, NO production was determined by Griess’ reaction. Our results have shown that LPS increased spleen cells proliferation (p<0.001) as well as IFN-γ in culture (p<0.05) and NO production by macrophages (p<0.01). RA decreased LPS-induced cells proliferation (p<0,001) and IFN-γ production (p<0,05) at concentration 100µg/mL. RA increased levels of IL-10 at concentrations of 100µg/mL (p<0,05), 50µg/mL and 25µg/mL (p<0,001). RA at concentrations 100µg/mL (p<0,01) and 50µg/mL (p<0,05) reduced the NO production as well.
Conclusion: RA attenuates inflammation probably by increasing IL-10 levels in LPS-induced TLR4 activation model.
Financial support: CNPq and FAPESB
IMMUNOMODULATORY EFFECT OF HISTAMINE ON CHEMOKINES SECRETION INDUCED BY TOLL-LIKE RECEPTOR ACTIVATION IN NEONATAL CELLS
ANNA CLÁUDIA CALVIELLI CASTELO BRANCO; ADRIELLI MENDES COSTA; NÁTALLI ZANETE PEREIRA; LUANDA MARA DA SILVA OLIVEIRA; ALBERTO JOSÉ DA SILVA DUARTE; MARIA NOTOMI SATO.
FACULDADE DE MEDICINA DA USP, SÃO PAULO - SP - BRASIL.
Introduction: Histamine is a biological amine, interacting through the histamine receptors (HR) 1, 2, 3 and 4 on dependency of the cell type, leading for a broad action on immune response, as inflammatory response and type I hypersensitivity reactions. To evaluate the effect of histamine and its receptors on mononuclear cells from umbilical cord blood and adult individuals on the chemokines secretion induced by Toll-like receptors (TLR) 4 and TLR7/TLR8 activation.
Methods and Results: Mononuclear cells (MNC) from umbilical cord (CB, n=9) obtained from cesarean delivery and healthy adult individuals (n=13, female=9, 4=male, median age=26 years) were incubated with histamine concentrations (10-8-10µM) in presence of ligands for TLR 4 (LPS) or TLR7/8 (CL097) and antagonists of HR1 (Pyrilamine), HR2 (Cimetidine), HR3 (Thioperamide) or HR4 (JNJ7777120) for 24h. Cultures supernatants were determined for CXCL10, CXCL9, CCL2, CCL5 and CXCL8 by Cytometry Bead Array. The findings showed that in baseline condition, only MNC from adult secrets CXCL10 and CXCL9, while CBs secretes higher levels of CCL2. Histamine exerts an inhibitory effect only in adult group, in a dose-independent manner. Activation via TLR4 and TLR7/8 induced increased CCL5, CXCL10 and CXCL9 secretion levels and decreased CCL2 secretions in adults compared to CB and similar CXCL8 levels in both groups. Histamine exerted both inhibitory and stimulatory effects, whereas to evaluate the influence of HRs we choose the inhibitory histamine effect on CCL5 and CCL2 secretion. Upon LPS stimulation, the inhibitory histamine effect was detected in 46% of adults through H2R and H4R while in CB reached 33%, without HR predominance. Histamine inhibitory effect on CCL2 secretion occurred in 61% of adults, via H1R, and 89% of CBs, without HR predominance. Upon TLR7/TLR8 stimulation, histamine inhibits CCL5 secretion in 60% of adults and in 20% of newborns, without preferential HRs engagement. Again, CCL2 secretion-induced by CL097 was higher in newborns than in adults, in which histamine was inhibitory in 54% of adults, using H2R and H4R and 56% of CBs without preferential HR involvement.
Conclusion: The use of HR antagonists as therapeutic approach to prevent exacerbation of the innate immune response during the neonatal period, suggest a partial involvement of the all receptors. It is a possible compensatory mechanism for the immunological immaturity in early period of life.
Financial support: FAPESP/LIM-56/HCFMUSP
IMMUNOMODULATORY EFFECTS OF CAFFEIC ACID ON BLOMIA TROPICALIS MURINE MODEL OF RESPIRATORY ALLERGY
TAMIRES CANA BRASIL CARNEIRO1; RYAN DOS SANTOS COSTA2; ANA TEREZA CERQUEIRA LIMA3; KEINA MACIELE CAMPOS DOURADO4; TATIANE OLIVEIRA5; LEONARDO NASCIMENTO SANTOS6; DARÍZY FLÁVIA SILVA AMORIM DE VASCONCELOS7; LAIN CARLOS PONTE DE CARVALHO8; NEUZA MARIA ALCANTARA NEVES9; CAMILA ALEXANDRINA VIANA DE FIGUEIREDO10.
1,2,3,4,5,6,7,9,10.UNIVERSIDADE FEDERAL DA BAHIA, SALVADOR - BA - BRASIL; 8.CENTRO DE PESQUISAS GONÇALO MONIZ (FIOCRUZ), SALVADOR - BA - BRASIL.
Introduction: Allergic asthma is a chronic airway inflammatory disorder characterized by reversible airway obstruction and hyperresponsiveness and production of Th2 cytokines such as IL-4, IL-13 and IL-5. The drugs used to treat respiratory diseases can control the illness symptoms but, on the other hand, can also induce long-term side effects. Therefore, new treatments are needed to optimize disease control having fewer side effects. Caffeic acid (CA), a phenolic acid, is likely to have various biological properties, such as anti-oxidant and anti-inflammatory. The present study aimed at studying the antiallergic effects of caffeic acid in a model of experimental murine asthma induced by the Blomia tropicalis mite.
Methods and Results: Groups of 6 mice AJ were sensitized (100 µg/ animal s.c) and challenged (10μg/ animal i.n) with Bt mite extract. Sensitized animals were treated or not with CA (10, 100 or 200mg/Kg) or with 3 mg/kg of Dexametazone (Dex) and the following parameters were analyzed: number of leukocytes/eosinophils in bronchoalveolar lavage (BAL); eosinophil peroxidase activity (EPO) in BAL; histopathological changes in the lung and levels of Th2 cytokines on spleen. Oral treatment with CA led to a decrease on cellular infiltrate, EPO and mucus production in the lungs, also reduces levels of IL-13, IL-5 and IL-4 in spleen cells culture.
Conclusion: These results suggest that the treatment with CA could be a promising treatment for allergic asthma. Further studies are needed in order to elucidate the CA’s mechanism of action.
Financial support: CAPES/ FAPESB
IMMUNOSTAINING FOR IBA-1 (MICROGLIA MARKER) IN THE PREFRONTAL CORTEX OF RATS SUBMITTED TO THE NEURODEVELOPMENTAL MODEL OF SCHIZOPHRENIA INDUCED BY NEONATAL IMMUNE CHALLENGE.
ALINE SANTOS MONTE; BRUNA MARA MACHADO RIBEIRO; MARIANA LIMA VALE; ANTÔNIO TELES DE MENEZES; MARTA REGINA DOS SANTOS DO CARMO; DANIELLE SILVEIRA MACÊDO.
FEDERAL UNIVERSITY OF CEARA (UFC), FORTALEZA - CE - BRASIL.
Introduction: Recently, the involvement of neuroinflammation in schizophrenia has been a subject of many studies. Indeed, evidences point towards a microglial activation in key brain areas related to the pathophysiology of schizophrenia. Currently, the use of animal models of neonatal immune challenge is becoming an essential tool to prove the association between postnatal infections as risk factor for schizophrenia development, these are known as neurodevelopmental models of schizophrenia and reproduce wide spectrum of symptoms of schizophrenia in the adulthood. Thus, the aim of this study is determine the immunostaining for IBA-1 in the prefrontal cortex of rats submitted to the model of schizophrenia induced by neonatal immune challenge. Methods and Results: Wistar rats of both sexes (n=6/group), were treated from the 5th to 7th postnatal (PN) days with Poly I: C at the dose of 2mg/kg,i.p. The animals were evaluated at distinct neurodevelopmental periods corresponding to early adolescence (PN 35), adult (PN 60) and adulthood (PN 74) in humans. The antipsychotic drug, clozapine (CZL) 25 mg/kg, i.p. was administered from PN60-74. Control group received saline under the same protocols. In the last day of administration the animals had their brains removed, processed, fixed and mounted on silanized slides. Histological sections, in turn, passed through antigen recovery process, and subsequently immunofluorescence according to the standard laboratory protocol where brain sections were incubated with rabbit polyclonal antibody anti-Iba1 (MNK 4428, Wako Pure Chemicals, Neuss, Germany). The images were analyzed by Image J. ANOVA with Student-Newman Keuls as post hoc test were used to determine variations among the groups tested considering p< 0.05. Results showed that branches and processes of microglial cells derived from adult rats exposed to Poly I:C had a strong decrease (8.89%), while the microglial cell body was enlarged as compared to control adult animals (18.69%), corresponding to a microglial activation. Young animals treated with Poly I: C this activation appeared as mild to moderate (13.45%). Clozapine reversed the changes caused by the immune challenge in adult animals (14.95%). Conclusion: The results of this work showed that microglial activation is progressive during the neurodevelopmental course of schizophrenia and that the antipsychotic CLZ is able to reverse this alteration.
Financial support: CNPq.
IMPORTANT ROLE OF PAF RECEPTOR IN POSTISCHEMIC HINDLIMB REVASCULARIZATION, INFLAMMATION AND BONE MARROW STEM CELL MOBILIZATION
BRÍGIDA GOMES DE ALMEIDA SCHIRMER (PG)1; JOUSIE MICHEL PEREIRA (PG)2; ALAN SALES BARBOSA (IC)3; MARIA CECILIA CAMPOS CANESSO (IC)4; ROBSON AUGUSTO SOUZA DOS SANTOS5; BRUNO MELO MENDES (PG)6; ADRIANA MÁRCIA GUIMARÃES ROCHA (PG)7; CARLOS MALAMUT8; MAURO MARTINS TEIXEIRA9; LUCÍOLA DA SILVA BARCELOS10.
1,2,3,4,10.IMMUNOPHARMACOLOGY GROUP, DEPARTMENTS OF PHYSIOLOGY AND BIOPHYSICS, ICB- UFMG, BELO HORIZONTE - MG - BRASIL; 5.DEPARTMENTS OF PHYSIOLOGY AND BIOPHYSICS, ICB-UFMG, BELO HORIZONTE - MG - BRASIL; 6,7,8.RADIOPHARMACEUTICALS RESEARCH AND PRODUCTION UNIT, UPRR-CDTN, BELO HORIZONTE - MG - BRASIL; 9.IMMUNOPHARMACOLOGY GROUP, DEPARTMENTS OF BIOCHEMISTRY AND IMMUNOLOGY, ICB-UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Peripheral arterial disease is characterized by obstruction of arterial blood flow in lower limbs and may evolve to amputation in severe cases. Valuation of mechanisms involved in the revascularization of ischemic tissues is of great importance in order to uncover potential therapeutic targets. Platelet Activating Factor (PAF) is an endogenous regulator of angiogenesis in the inflammatory and tumor microenvironments, but its role during limb ischemia is unknown. Here, we evaluate the role of PAF receptor (PAFR) in the spontaneous revascularization of ischemic hindlimbs.
Methods and results: Hindlimb ischemia was induced by permanent left femoral artery occlusion (FAO) of C57Bl/6 wild-type (WT) and PAFR knockout (KO), n=5-8 mice/group. By laser Doppler perfusion imaging, we observed a marked reduction in perfusion recovery of ischemic hindlimb in KO mice (KO 0.5±0.05 vs WT 0.8±0.04, p<0.001, at day 5 post-FAO). In accordance, when evaluating neovascularization response to ischemia, we observe that capillary/myocyte ratio (p<0.01) and collateral vessels remodeling (p<0.001) at day 7 post-FAO and arterioles density (p<0.05) at day 14 post-FAO were significantly reduced in the KO group. By ELISA assay, we observed reduction in VEGF (p<0.01) and CXCL12 (p<0.05) in the ischemic muscles of KO animals and an increase of TNF-α levels (p<0.05) at day 3 post-FAO. At this time-point, total leukocyte count in peripheral blood was reduced in the KO group (p<0.05); such reduction was primarily due to reduced monocyte count (p<0.01). Interestingly, macrophages content (assessed by N-acetil-β-D-glucosaminidase activity) in the ischemic muscle was significant reduced in KO mice (p<0.05). Intriguingly, we observed an increased number of putative angiogenic stem cells such as Lin-/Sca-1+/cKit+, Lin-/cKit+/CD31+ and Lin-/cKit+/VEGFR2+ cells (p<0.01) in the bone marrow (BM) of KO animals. However, we found a reduced number of Sca-1+/cKit+, cKit+/CD31+ and cKit+/VEGFR2+ cells (p<0.05) in the blood of KO animals. In micro PET (positron emission tomography) studies, after i.v. infusion of 2-deoxy-2-(18F)fluoro-D-glucose (18FDG), we observed a more intense activity of the radiopharmaceutical in BM of KO mice compared with WT (p<0.05).
Conclusions: Our results disclosure an important role for PAFR in orchestrating post-ischemic hindlimb revascularization, inflammation and stem cell mobilization from BM to bloodstream.
Support: FAPEMIG, INCT-Nanobiofar, CNPq, CAPES.
INFLAMMATORY MECHANISMS INVOLVED IN ACUTE MUSCLE PAIN
BRUNA DE MELO1; DIOGO FRANCISCO DOS SANTOS2; CAROLINA OCANHA JORGE3; JALILE GARCIA4; CARLOS AMILCAR PARADA5; MARIA CLÁUDIA GONÇALVES OLIVEIRA-FUSARO6.
1,2,3,4,6.FCA UNICAMP, LIMEIRA - SP - BRASIL; 5.IB UNICAMP, LIMEIRA - SP - BRASIL.
Introduction: Muscle pain induced by sustained isometric contraction has important socioeconomic impact. However, the inflammatory mechanisms underlying this kind of pain are unknown. The aim of this study was to investigate the inflammatory mechanisms involved in mechanical hyperalgesia induced by sustained isometric contraction of gastrocnemius muscle of rats. Methods and results: The sustained isometric contraction was performed by electrical stimulation directly to the belly of gastrocnemius muscle of rats. The muscle hyperalgesia was assayed by Randall- Selitto test applied directly on the muscle belly. Intramuscular administration of the selective bradykinin B1 or B2 receptor antagonist DALBK and bradyzide, respectively, the cyclooxygenase inhibitor Indomethacin, selective β1 or β2 receptor antagonist Atenolol and ICI 118,551, respectively, selective P2X3 and P2X2/3 receptor antagonist
A317491 and inhibitor of neutrophil migration Fucoidan were used to investigate the role of bradykinin, PGE2, sympathetic amines, ATP and neutrophils on mechanical muscle hyperalgesia induced by sustained isometric contraction. Male Wistar rats (200-250g) were used in this study and all procedures were approved by the Ethics Committee in Animal Research at the UNICAMP (protocol number 2448-1). Electrical stimulation with 19-ms duration, at a frequency of 50Hz and intensity of 2V for 1h induced mechanical hyperalgesia ½ (32,5±3,1) and 1h (37,5±1,2) post sustained isometric contraction significantly greater than that induced by sham (1,9±0,8, p<0.05, Tukey test, n=5). Pretreatment with Fucoidan (25mg/Kg,1,1±1,5) or administration of DALBK (30µg, 16,5±4,7), Bradyzide (15µg, 10,3±3,0), Indomethacin (100µg, 3,8±2,1), Atenolol (6.0µg, 9,2±1,3), ICI 118,551 (1.5µg, 5,1±1,7) or A317491 (60µg, 1,1±0,5) in the ipsilateral but not in the contralateral gastrocnemius muscle prevented the mechanical hyperalgesia
(p<0.05, Tukey test, n=5). Conclusion: These data demonstrated the involvement of bradykinin, PGE2, sympathetic amines, ATP and neutrophils in acute muscle pain induced by sustained isometric contraction. Support: FAPESP (2011/11064-4; 2012/10402-6).
INFLUENCE OF THE GENETIC BACKGROUND ON INTRAPERITONEAL ADHESION FORMATION IN MICE:RESPONSE TO DIPYRIDAMOLE
SUZANE MOTA MARQUES COSTA; POLLYANA RIBEIRO CASTRO; CELSO TARSO VIANA; SILVIA PASSOS ANDRADE.
UNIVERSITY FEDERAL OF MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.
Introduction: Individual differences in the induction of fibroproliferative tissue may influence the rate of progression and severity of the disease processes and the sensitivity/resistance of the individuals to drugs. In this project, we evaluated the influence of genetic heterogeneity in intraperitoneal adhesion formation and the response to dipyridamole (DP) in different strains of mice. Methods and Results: Polyether-polyurethane sponge discs were surgically implanted in peritoneal cavity of anesthetized DBA, Swiss, C57BL/6 and BALB/C mice to induce adhesions. Dipyridamole (100/mg/Kg) was administered by gavage for 6 days in the treated groups and water in the control animals. At day 7 post implantation, the animals were killed and the implant-induced fibroproliferative tissue was removed to determine inflammation, angiogenesis and fibrogenesis. Neutrophil recruitment/activation, as assessed by myeloperoxidase (MPO) activity (OD/mg) and macrophage accumulation/activation as NAG (n-acetyl- β-D- glucosaminidase) activity (OD/mg) differed among the strains. Dipyridamole significantly reduced the levels of MPO in BALB/ implants from 3.4±0.7 to 2.1 ±0.6 (n=7) and in Swiss implants from 2.4 to 1.9±0.2 (n=9).The treatment reduced the levels NAG in C57BL/6 implants from 12.4 to 7.6±1.8 (n=8). The levels of the pro-inflammatory cytokines (TNF-α and CCL2/JE) also varied among the strains and responded differently to the treatment. Angiogenesis (hemoglobin; µg Hb/mg wet tissue) and vascular endothelial growth factor levels -VEGF) differed among the strains and after the treatment. Dipyridamole reduced Hb content in Swiss implants from 3.5 to 2.1±0.6 (n=7) but increased VEGF levels 0.1 to 0.3±0.1 (n=7). In DBA implants, the treatment increased both Hb content (1.9 to 2.9±0.2; n=7) and VEGF levels (0.1 to 0.2±0.1; n=6). The treatment reduced this parameter in C57BL/6 from 0.4 to 0.2±0.1 (n=7). Fibrosis also differed among the strains. Collagen deposition (µg/mg wet tissue) was more intense in C57BL/6 implants compared with the implants of the other strains. Dipyridamole treatment decreased collagen levels in C57BL/6 implants only (2.0±0.1; n=8 to 1.5±0.07; n=8). Conclusion: These findings emphasize the contribution of the genetic background in the induction of peritoneal fibrosis and in the pharmacological sensitivity to dipyridamole.
Financial support: CNPq, CAPES, FAPEMIG.
INFLUENCE OF THE RENIN-ANGIOTENSIN SYSTEM IN THE INFLAMMATORY PROFILE IN OSTEOARTHRITIS
GRAZIELLE CORDEIRO AGUIAR1; FLÁVIO ALMEIDA AMARAL2; MAURO MARTINS TEIXEIRA3; ANDERSON JOSÉ FERREIRA4.
1,4.DEPARTMENT OF MORPHOLOGY, FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 2,3.DEPARTMENT OF BIOCHEMISTRY AND IMMUNOLOGY, FEDERAL UNIVERSITY OF MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.
Introduction: Osteoarthritis (OA) is the most common rheumatic disease characterized by chronic joint degeneration. It is associated with increases in pro-inflammatory mediators and reductions in anti-inflammatory factors. The renin- angiotensin system (RAS) can modulate the inflammatory process and the hyaline cartilage contains many components of this system. Here, we evaluated if the RAS can affect the inflammatory profile in OA.
Methods and Results: Wistar rats (n=7 per group) were submitted to intra-articular injection of 2mg (~50µL) of monosodium iodoacetate in the knee joint in order to induce OA. Control animals were injected with saline (~50µL). Following 28 days of OA induction, the rats were orally treated for 8 weeks with one of the following treatments: saline; captopril [inhibitor of angiotensin converting enzyme (ACE), 30mg/kg/day]; losartan (antagonist of AT1 receptors, 30mg/kg/day); or diminazene aceturate (DIZE) (activator of ACE2, 1mg/kg/day). Paw hyperalgesia, swelling of the knee, mean arterial pressure (MAP), total leukocytes counting in the joint and levels of TNF-α, IL-1ß and IL-10 were evaluated before and after of the treatments. To evaluate the role of nitric oxide (NO) in the effect of captopril, rats were treated with NO synthase inhibitor L-NAME (15mg/kg/day). Data are expressed as mean ± SEM and analyzed by one-way ANOVA/Newman-Keuls post-test (p<0.05). This study was approved by the local ethics committee. OA animals presented more leukocytes in the knee cavity than control rats (0.9±0.09 vs. 0.2±0.03 leukocytes x 105/cavity). The treatment with captopril and losartan, but not with DIZE, significantly reduced the number of leukocytes in the joint cavity (captopril:0.4±0.04; losartan:0.4±0.06; DIZE:0.8±0.1). L-NAME did not change the effect of captopril in the number of leukocytes. The levels of IL-10 were reduced in OA animals (15.9±15.9 vs. 372.9±84.8 in control rats) and administration of captopril or DIZE did not alter this effect. Also, OA rats presented lower nociceptive threshold for paw withdrawal, which was not reversed by any of the treatments. No significant differences in the levels of IL-1ß and TNF-α, as well as in the swelling in the knee and in the MAP were observed among any of the groups.
Conclusion: Our data indicate that ACE inhibition and AT1 antagonism reduce the inflammatory process in the knee in OA, thereby suggesting that the RAS might be related to the pathophysiology of OA.
Support: FAPEMIG and CNPq.
INHIBITION OF CELL MIGRATION PROMOTED BY PLANTAGO BERROI IN THE PLEURISY MODEL IN RATS
JOSÉ C. DA SILVA1; DRIELLY S. FLORENTINO2; LUIZ C. VIEIRA3; FABRÍCIA C. PETRONILHO4; ANNA PAULA PIOVEZAN5.
1.GRADUATION IN MEDICINE, TUBARÃO - SC - BRASIL; 2.GRADUATION IN PHARMACY, TUBARÃO - SC - BRASIL; 3.GRADUATION IN BIOLOGY, TUBARÃO - SC - BRASIL; 4,5.MASTER’S DEGREE IN HEALTH SCIENCE PROGRAMM, UNIVERSITY OF SOUTHERN CATARINA (UNISUL), TUBARÃO, - SC - BRASIL.
Introduction: Several species of plants from Plantago genus have demonstrated anti-inflammatory effect in different animal models in the literature. Besides this, the specie Plantago berroi remains poorly investigated for such property. The present study investigated the anti-inflammatory activity for the hydroalcoholic crude extract (HCE) from leaves of this plant in the pleurisy model in rats.
Methods and Results: The procedure for induction of the pleurisy was completed in male Wistar rats (around 300 g). Animals were anesthetized and received intrathoracic administration of λ-carrageenan (2% in 0.2 mL) into the pleural cavity, via the intercostals space. Animals were sacrificed 4 h later, their thorax were opened and the pleural cavity were washed with sterile saline in order to obtain aliquots of this bronchoalveolar liquid, containing the pleural exudates. These samples were analyzed in a Neubauer chamber by adding Türk solution for erythrocyte hemolysis and the total number of leukocytes/mL was recorded. The possible influence of the plant on leukocytes migration was assessed by treatment of different groups (n= 9-12) with HCE of Plantago berroi (doses from 10 to 100 mg/kg, orally, 1 h after pleurisy induction). Control groups received treatment, in the same conditions, with saline (0.1 mL/100g) or acetylsalicylicacid (ASA, 100 mg/kg). All protocols were approved by the Ethics Committee for Animal Use (CEUA)- UNISUL under number 12.017.4.01 III. The doses of 30 mg/kg or 100 mg/kg of HCE from plant leaves have reduced the number of leukocytes present in the pleural exudates (3079.0 ± 790.7 or 2908.0 ± 446.8 leukocytes/mL, respectively) when compared to the carrageenan group (7339.0 ± 2017 leukocytes/mL); this effect was also similar to that observed in the ASA group (1680 ± 412.3 leukocytes/mL).
Conclusion: Anti-inflammatory effect of HCE from leaves of the plant Plantago berroi seems to be, at least in part, mediated by its action on inhibition of cell migration.
Financial support: UNISUL
INHIBITION OF THE HUMAN NEUTROPHIL OXIDATIVE METABOLISM BY 3-PHENYLCOUMARIN DERIVATIVES AND BACCHARIS DRACUNCULIFOLIA EXTRACT: INVESTIGATION OF THE UNDERLYING ANTIOXIDANT MECHANISMS
MICÁSSIO FERNANDES DE ANDRADE1; ANDRÉA SILVA GARCIA FIGUEIREDO-RINHEL2; LUCIANA MARIKO KABEYA3; ANA ELISA CALEIROS SEIXAS AZZOLINI4; FLÁVIO DA SILVA EMERY5; MÔNICA TALLARICO PUPO6; JAIRO KENUPP BASTOS7; YARA MARIA LUCISANO-VALIM8.
1.FMRP-USP, RIBEIRÃO PRETO - SP - BRASIL; 2,3,4,5,6,7,8.FCFRP-USP, RIBEIRÃO PRETO - SP - BRASIL.
Introduction: Production of reactive oxygen species (ROS) by neutrophils is an important mechanism of microbial killing, modulation of intra- and extracellular redox balance and regulation of inflammatory processes. The NADPH •- - oxidase complex, which reduces O2 to O2 , and myeloperoxidase (MPO), which converts H2O2 and Cl into the strong oxidant HOCl, are the main enzymes that generate ROS during the neutrophil oxidative metabolism activation. However, increased ROS generation can damage the surrounding tissues and needs to be controlled. The present study aims to investigate the antioxidant mechanisms underlying the modulatory effect of 3-phenylcoumarin derivatives (3-PD) and Baccharis dracunculifolia D.C. (Asteraceae) leaf extracts (BdE) on the ROS generation by immune complex-stimulated human neutrophils. Methods and Results: The inhibitory effect of the samples on the •- O2 and total ROS generation by neutrophils was evaluated by the lucigenin (CL-luc)- and luminol (CL-lum)-enhanced chemiluminescence assays. These cells were stimulated by insoluble IgG/ovalbumin immune complexes, prepared at equivalence, or serum-opsonized zymosan. All the 3-PD and BdE samples suppressed the neutrophil CL-luc and CL- lum in a concentration-dependent manner. Such effect was not due to cytotoxicity of the samples, as evaluated by trypan blue exclusion and measurement of lactate dehydrogenase release. BdE and 3-PD slightly inhibited the neutrophil O2 uptake due to NADPH oxidase activity, as measured with the Clark electrode. The 3-PD but not BdE inhibited MPO activity, assessed by guaiacol oxidation in the presence of H2O2. Both 3-PD and BdE effectively inhibited the HOCl-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine; thus, they were efficient HOCl scavengers. Conclusion: The BdE and 3-PD samples does not inhibit the neutrophil CL-luc by suppressing the NADPH oxidase •- activity but probably by scavenging O2 . Furthermore, the neutrophil CL-lum inhibition by the 3-PD is mediated by their ability to suppress MPO activity and scavenge HOCl, whereas CL-lum inhibition by the BdE is strongly related to their HOCl scavenging activity. Therefore, the 3-PD and BdE samples tested herein modulate the human neutrophil ROS generation through a combination of different antioxidant mechanisms.
INHIBITORY ACTIVITIES OF ETHANOL EXTRACT FROM AERIAL PARTS OF ALTERNANTHERA MARITIMA AT INFLAMMATORY PAIN, LEUKOCYTE MIGRATION AND OEDEMA IN MICE.
DIANA FIGUEIREDO DE SANTANA AQUINO1; ISABELLA CRISTINA DIAS2; RENAN DONOMAE IWAMOTO3; ANA CLAÚDIA PICCINELLI4; MARCOS JOSÉ SALVADOR5; CANDIDA APARECIDA LEITE KASSUYA6.
1,2,3,4,6.UNIVERSIDADE FEDERAL DA GRANDE DOURADOS, DOURADOS - MS - BRASIL; 5.UNIVERSIDADE ESTADUAL DE CAMPINAS, CAMPINAS - SP - BRASIL.
INTRODUCTION: The Alternanthera maritima belong to the genus Alternanthera Forkssal comprising 80 species and approximately 30 of them occurring in Brazil. (Z. Naturforch. C 62:339-347; Braz. J. Microbiol. 35:131-136) Many species of Aternanthera are used in the treatment of infections and some properties such as analgesic, anti- nociceptive, anti-viral, anti-inflammatory and immunomodulatory were documented in scientific works (Biochem. Syst. Ecol. 32:107-110; Braz. J. Med. Biol. Res.. 36:1215-1219; Z. Naturforch. C. 59: 499-505). Based on this context, the aim of this study was to verify if ethanol extract from aerial parts of A. maritima (EAM) induced inhibitory activities at inflammatory pain, leukocyte migration and oedema induced by carrageenan in mice. METHODOLOGY AND RESULTS: Experiments were performed after approval of the protocol 12/2013 by the Institutional Ethics Committee and using Swiss mice (25-35 g). After the identification, the aerial parts of A.maritima were collected and the ethanol extract was made (Z. Naturforch. C. 59: 499-505). The evaluation of anti-inflammatory activity was carried out through experimental models of carrageenan-induced paw oedema, alldynia, and pleurisy, at doses of 100, 300 and 500mg/kg of extract. Oral administration of EAM at a dose of 300 and 500mg/kg, significantly inhibited the oedema induced by carrageenan, with inhibition de 79±8% and 77±4%, four hours after carrageenan injection, respectively. In the pleurisy, at dose of de 100 and 300 mg/Kg, promoted the reduction of leukocyte migration, with inhibitions were 68±5% and 65±5%, respectively. The same form, orally administration of EAM significantly prevented the reduction in the sensitivity threshold at 72.5% and 90%, in the third and fourth hour after the administration of carrageenan, respectively, at a dose of 500mg/kg, unlike of the dose 100mg/kg. CONCLUSION: The oral administration of the EAM in mice at doses of 100 and 300mg/kg significantly inhibited paw oedema and leukocyte migration into the pleural cavity. The evaluation of inflammatory pain activity, the EAM significantly prevented the reduction in the sensitivity threshold, demonstrating a significant anti-nociceptive effect. However, more studies are necessary to identify the possible compounds responsible for these activities, as well as to elucidate its mechanism of action.
Acknowledgments: CAPES, CNPq, FAPESP e FAEPEX-UNICAMP.
INHIBITORY EFFECT OF FRIEDELIN, A PENTACYCLIC TRITERPENE, ON LPS-INDUCED INFLAMMATION.
JAMYLLE NUNES DE SOUZA FERRO1; JOÃO PAULO NOÉ DA SILVA2; MARVIM PAULO LINS3; ALMAIR FERREIRA DE ARAÚJO4; FERNANDA LIMA DE AQUINO5; SAMÁRIO LINO6; NATHALIE OBADIAS7; VANESSA ESTATO8; MARCO ADRIANO LESSA9; LÚCIA MARIA CONSERVA10; EMILIANO BARRETO11.
1,2,3,4,5,6,10,11.UNIVERSIDADE FEDERAL DE ALAGOAS, MACEIÓ - AL - BRASIL; 7,8,9.INSTITUTO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Pentacyclic triterpenes are secondary metabolites produced in plants belonging to the genus Clusia. Friedelin appear in leaves and barks from Clusia nemorosa, and has been associated as anti-proliferative and antioxidant responses. However, the anti-inflammatory effect of friedelin on acute LPS-induced inflammation is poorly understood. Here, we evaluated anti-inflammatory effects of friedelin against LPS-mediated inflammation. Methods and results: Swiss male mice (25-30 g, n=7) were treated with friedelin (FD, 1, 10 and 50 mg/kg, i.p.) 1h prior LPS- induced pleurisy, and 4h later the exudates were collected to determinate total leukocyte and neutrophil. In order to better establish the effect of friedelin on leukocyte mobilization we performed the intravital microscopy assay on cerebral microcirculation after topical application of LPS. Data were expressed as mean±SEM and were statistically analyzed using Student’s t test or one-way ANOVA. LPS injection (250 ng/cavity) increased the number of total leukocytes (from 31.8±0.4 to 12.6±1.5 x106/mL) mainly characterized by neutrophils (from 1.1±0.4 to 8.8±1.2 x106/mL) as compared to the vehicle-stimulated mice. FD (1, 10 and 50 mg/kg) inhibited the total leukocyte count (to 6.0±0.8, 4.9±1.0 and 5.8±0.6 x106 cells/mL, respectively) and neutrophil (to 2.5±0.3, 2.7±0.8 and 2.6±0.4 x106 cells/mL, respectively). After LPS injection into the pleural cavity was observed a blood neutrophilia (from 6.0±2.5 to 13.6±2.3 x106/mm3) and release of neutrophils from bone marrow (from 4.9±0.6 to 1.7±0.2 x106/femur), events that were inhibited by FD treatment at dose 1 mg/kg (9.3±1.5 x106/mm3 and 3.4±0.6 x106/femur, respectively). Intravital microscopy studies reveled that in FD-treated (10 mg/kg) mice there was suppression in the rolling (from 8.5±1.9 to 4.2±0.5 cell/mim) and adhesion (from 11.1±2.3 to 4.8±1.3 cell/mim/100µm) of leukocyte without interfere with functional capillary density (from 625.0±86.8 to 588.0±45.1 capillaries/mm2) after LPS stimulation. Conclusion: Taken together, our data show that friedelin prevents the LPS-induced neutrophil migration to the inflammatory site, by affecting the adhesion molecule expression in neutrophils. Thus, these results provide new insights about the new candidate of anti-inflammatory therapies.
Financial support: CNPq, CAPES.
INSULIN ATTENUATES CLP-INDUCED LIVER DYSFUNCTION IN THE DIABETIC RATS
EDUARDO LIMA NOLASCO; FERNANDO LUIZ ZANONI; FERNANDA PEIXOTO BARBOSA NUNES; MARIANA CRISTINA FERREIRA SILVA; SABRINA SOUZA FERREIRA; LORRAINE ISTÉFANE ANDRADE COSTA; JOILSON OLIVEIRA MARTINS.
UNIVERSITY OF SAO PAULO, SAO PAULO - SP - BRASIL.
Introduction: Diabetes mellitus is associated with higher rates of recurrent infections and about 20% of all septic patients are diabetic. The objective of present study was to investigate the influence of insulin on the systemic inflammation caused by cecal ligation and puncture (CLP) of two punctures.
Methods and Results: Diabetic male Wistar rats (n=24, alloxan, 42 mg/kg i.v., 10 days) and control rats (n=25) were submitted to two puncture-CLP procedure. On the 6th hour, the following analyses were performed: (a) total and differential cell counts in bronchoalveolar lavage (BAL) fluid; (b) quantification of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-10, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2 in the BAL by enzyme-linked immunosorbent assay (ELISA); (c) hemogram by veterinary haematology analyser (ABV VET HORIBA®) and leukocyte differentiation by Leishman stain (d) biochemical parameters (urea, creatinine, alanine aminotransferase - ALT, aspartate aminotransferase - AST and alkaline phosphatase - ALP) by colorimetric analyses (e) lung, kidney and liver morphological analyses (Hematoxylin and eosin staining) and activity of myeloperoxidase (MPO). Regarding sham animal, CLP rats exhibited a reduction in the number of leukocyte counts (50%) in both diabetic and non-diabetic groups. The CLP control group, CLP-diabetic rats exhibited an increased in the concentration of ALT (2.7-fold), AST (2.3-fold), FAL (2.7-fold) and urea (2.7-fold). Sham diabetic animals exhibited 2.3-fold higher FAL level when compared to control group. Treatment of diabetic rats with neutral protamine Hagedorn insulin (NPH, 4 IU, s.c.), 2 h before the CLP procedure, completely restored ALT, AST and FAL levels. In addition, total and differential cell counts and concentration of cytokines in the BAL fluid, red blood cells, hemoglobin, platelets and creatinine levels did not differ between groups as well as the morphological analyses and MPO activity from lung, kidney and liver samples. Treatment of non-diabetic rats with insulin (NPH, 1IU, s.c.), 2 h before CLP procedure, did not change any of these parameters.
Conclusion: Data presented suggest that insulin only attenuates CLP-induced liver dysfunction in the diabetic rats.
The authors would like to thank the founding agencies FAPESP, CNPq and PRP/USP - PROJETO 1.
INTERFERENCE OF ESTRADIOL ON THE LEUKOCYTE MOBILIZATION AND EXPRESSION OF ICAM-1 IN THE LUNG OF RAT SUBJECTED TO INTESTINAL ISCHEMIA/REPERFUSION
EVELYN THAIS FANTOZZI1; ANA CRISTINA BREITHAUPTH-FALOPPA2; MARIA BEATRIZ BERNARDEZ AMORIM3; FERNANDA YAMAMOTO RICARDO SILVA4; RICARDO MARTINS OLIVEIRA-FILHO5; BORIS BERNARDES VARGAFTIG6; WOTHAN TAVARES-DE-LIMA7.
1,3,4,5,6,7.PHARMACOLOGY DEPT. ICB/USP, SÃO PAULO - SP - BRASIL; 2.LIM 11 - HC - FMUSP, SÃO PAULO - SP - BRASIL.
Introduction: Experimental evidence indicates that female sex hormones may exert protective effect on organ injury caused by trauma hemorrhagic shock model. In addition, intestinal ischemia reperfusion (I/R) causes neutrophil- dependent acute lung injury. In this context female are more resistant than the male to develop adverse effects in the lung after ischemic event. Here, in order to assess the role of estradiol on the traffic of leukocytes into the lung, we have quantified the number of circulating blood leukocytes and the expression of adhesion molecule ICAM-1 in the lung of ovariectomized rats subjected to intestinal I/R. Methods and Results: Intestinal I/R was induced by 45 min occlusion of the superior mesenteric artery, followed by 2 h reperfusion in female Wistar (60 days old). Ovaries removal (OVx) was carried 7 day before I/R induction. OVx rats received one single dose (280 µg/kg, s.c.) of estradiol, 24 h before induction of I/R. OVx rats not submitted to I/R (Sham) and non-manipulated rats were used as controls. The expression of ICAM-1 in the lung was performed by imunohistochemistry. Leukocytes in the lung were measured by optical microscopy after extraction in the lung digested by collagenase method whereas circulating leukocytes were quantified in blood samples. Estradiol treatment (E) of OVx I/R rats prevented the increased of lung neutrophils induced by non-treated I/R rats (OVx I/R= 40.3 ± 5.1; vs OVx I/R+E= 25.2 ± 2.2 % cells/mm2, n=9). Estradiol treatment also reduced the number of neutrophils and monocytes in the blood of OVx I/R rats (Neutrophils: OVx I/R= 17,873 ± 3,289 vs OVx I/R+E= 7,765±1,693/mm3 ; n=8; Monocytes: OVx I/R= 15,439 ± 1,464 vs OVx I/R+E= 4,714 ± 798/mm3; n=8). Estradiol treatment caused a significant reduction of ICAM-1 expression (OVx I/R= 27.3 ± 3.0 vs OVx I/R+E= 11.4 ± 2.7 objects x103/mm2; n=11). Conclusion: Our results suggest the estradiol exerts a direct control of the lung inflammatory response caused by gut injury. Moreover, since blood leukocytes did not increase after estradiol treatment, our results could suggest that this steroid could exert some inhibitory effect on the bone marrow functional activity. Financial support: CNPq and FAPESP.
INTRAUTERINE MALNUTRITION DOWREGULATES LEPTIN RECEPTOR EXPRESSION AND MODULATE LIPID MEDIATORS PRODUCTION, IN PRIMARY CULTURE OF LUNG ENDOTHELIAL CELLS STIMULATED BY LPS.
LEILA APARECIDA SANTOS1; ALEKSANDRO MARTINS BALBINO2; REBÉCA MANTUAN GASPARIN3; RENAIDE FERREIRA4; LILIAM FERNANDES5; MARISTELLA ALMEIDA LANDGRAF6; RICHARDT GAMA LANDGRAF7.
1,2,3,5,7.LABORATÓRIO DE INFLAMAÇÃO E FARMACOLOGIA VASCULAR - UNIVERSIDADE FEDERAL DE SÃO PAULO, DIADEMA - SP - BRASIL; 4.BIOTÉRIO CENTRAL ICB - UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL; 6.LABORATÓRIO DE HIPERTENSÃO - UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL.
Introduction: Intrauterine undernourishment can induce a range of fetal adaptations, which can lead to permanent alterations in adult life, such as reduced inflammatory response. The vascular endothelium is closely related with the circulatory control, and plays an important role in cellular and molecular events which occur during immune system reactions and tissue injuries. Leptin, a hormone mainly synthesized by adipose tissue, is involved in various biological systems, acting in the food intake control and energetic metabolism; in addition, it modulates immune response, hematopoiesis and lymphopoiesis.
Objectives: To evaluate the expression of long-form leptin receptor in rat pulmonary endothelial cells intrauterine malnutrition and your role in the production of lipid mediators.
Methods and Results: Pulmonary endothelial cells were obtained from intrauterine malnourished rats or nourished rats, at 12 weeks age. These cells were stimulated with leptin (10ng/mL) or LPS (1µg/mL) or leptin plus LPS; additionally, cells were stimulated with saline, as a control. Two hours after the stimulation, the production of inflammatory mediators (PGE2 and LTB4) and western blots analysis (leptin receptor) were performed. All the procedures used in this study were approved and are in accordance with the rules established by Ethics Committee of UNIFESP. Western blot assay showed that expression of long-form leptin receptor is decreased (63%) in the primary cultures of endothelial cells derived from intrauterine malnourished rats. Leptin alone did not induce any alteration on the levels of the inflammatory mediator evaluated, whereas LPS increased the PGE2 (250%) and LTB4 (29%) levels.
Only in endothelial cells from nourished rats, leptin enhanced lipid mediators production induced by LPS (PGE2 - 28% and LTB4 - 18%). Interestingly, the same was not observed in endothelial cells from intrauterine malnourished rats.
Conclusion: Our preliminary results suggest that intrauterine malnutrition dowregulates leptin receptor expression and modulate lipid mediators production in primary culture of pulmonary endothelial cells stimulated by LPS.
Financial support: FAPESP (2010/01404-0, 2012/51104-8, CNPq and CAPES/REUNI.
INVESTIGATION OF THE ANTINOCICEPTIVE MECHANISMS OF CITRONELLYL ACETATE
EMILIANO RICARDO VASCONCELOS RIOS1; NAYRTON FLÁVIO MOURA ROCHA2; ALYNE MARA RODRIGUES CARVALHO3; LEONARDO FREIRE VASCONCELOS4; MARILIA LEITE DIAS5; LAURA MARIA TEODORIO VIDAL6; DAMIÃO PERGENTINO DE SOUSA7; FRANCISCA CLEA FLORENÇO DE SOUSA8; MARTA MARIA DE FRANÇA FONTELES9.
1,2,3,4,5,6,8,9.UFC, FORTALEZA - CE - BRASIL; 7.UFS, SÃO CRISTOVÃO - SE - BRASIL.
Introduction: Citronellyl acetate (CAT) has demonstrated to possess various biological effects, and many works suggest the antinociceptive activity of other essential oils. So, we aimed to study the antinociceptive action of the CAT and to verify the antinociceptive mechanisms involved in this action. This work was approved in local Ethics Committee on Animal Research by protocol number: 31/12. Methods and Results: Male Swiss mice with 25-32g were used in all experiments, and the tests were conducted with 7-8 animals per group. CAT (200 mg/kg, p.o.) was tested at the model of visceral nociception induced by acetic acid 0.6%. The animals were pretreated with L-arginine (NO donor, 150mg/kg, i.p.), yohimbine (α2-antagonist, 2.5 mg/kg, p.o.), haloperidol (non-selective dopamine antagonist, 0.2 mg/kg, i.p.) or atropine (non-selective muscarinic antagonist, 1 mg/kg, i.p.) 15 minutes before receiving the CAT, and after 30 minutes submitted to the writhing test induced by acetic acid, and observed for 30 minutes. Finally, the glutamate-induced paw linking test was conducted 30 or 60 minutes after the pre-treatment with CAT (100 or 200 mg/kg, p.o.) or MK-801 (NMDA antagonist, 0.5 mg/kg, i.p.), injected 20μL of glutamate (20 μg/paw) into the plantar surface of the right hind paw and observed for 15 minutes. For statistical analysis, we used ANOVA and Student-Newman-Keuls as post hoc and p<0.05 was accepted. The systemic increasing of NO promoted by L- arginine or the pre-treatment with yohimbine were not capable of changing the antinociceptive effect of CAT. Moreover, the pre-treatment with haloperidol or atropine were capable of decreasing the antinociceptive effect of CAT by 48.02% and 46.68%, respectively, when compared with the group of animals that received CAT only. However, it did not abolish the antinociceptive effect completely. Finally, CAT was capable of decreasing the pain behavior of the animals by 43.33% (100 mg/kg) and 51.22% (200 mg/kg) in glutamate-induced paw-linking model, and a similar result was found with MK-801 group (93.18% of reduction). Conclusion: The results indicate that the modulation of pain caused by CAT might be mediated, at least in part, by dopaminergic and muscarinic receptors, but there is still necessary to conduct more experiments in order to identify a specific receptor, as well as the glutamate pathway, probably peripheric nociceptors glutamatergic.
Financial support: CNPq, CAPES and FUNCAP.
INVESTIGATION OF THE IMMUNOPOTENTIATING EFFECT OF THE ATOMIZED EXTRACT OBTAINED FROM THE LIANA SARACURA-MIRÁ (AMPELOZIZYPHUS AMAZONICUS).
MARINA VIEIRA AGOSTINHO PEREIRA1; FERNANDA FERREIRA BARBOZA2; TATIANA JOTHA-MATTOS3; DANILO RIBEIRO DE OLIVEIRA4; PRISCILA FINOTELLI5; SUZANA GUIMARÃES LEITÃO6; LIGIA MARIA TORRES PEÇANHA7.
1,2,7.DEPARTAMENTO DE IMUNOLOGIA, INSTITUTO DE MICROBIOLOGIA PAULO DE GÓES, CCS, UFRJ, RIO DE JANEIRO - RJ - BRASIL; 3,4,5,6.DEPARTAMENTO DE PRODUTOS NATURAIS E ALIMENTOS, FACULDADE DE FARMÁCIA; CCS, UFRJ, RIO DE JANEIRO - RJ - BRASIL.
INTRODUCTION: An infusion obtained from the bark of the liana Ampelozizyphus amazonicus, popularly known as Saracura-mirá (SAR), is traditionally used by indigenous communities in the North and Northeast of Brazil for the treatment of malarial infection. Previous studies have shown that SAR does not have a direct effect upon the protozoan Plasmodium in vitro. In the present study we evaluated whether the antimalarial effect of this infusion would be due to an immunomodulatory effect.
METHODS AND RESULTS: A spray-dryer atomized extract obtained from the barks of SAR and a saline solution of this extract was administered daily at the dose of 10mg/Kg; this dose was determined based on the popularly employed malaria treatment dose. BALB/c mice were infected by the intraperitoneal injection of 106 Plasmodium chabaudi parasitized red blood cells and the effect of SAR administration on the progression of infection induced by the protozoan was investigated. We measured survival rate, parasitemia, and total immunoglobulin levels (evaluated by ELISA). To investigate the immunomodulatory effect of SAR, the effect of the treatment on antigen-specific immunoglobulin production induced by immunization with either TNP-Ficoll (a T-independent type 2 antigen) or ovalbumin (a T-dependent antigen) was also evaluated. Besides that, the basal circulating levels to the natural anti- dextran antibody were examined on SAR-treated animals. Antigen-specific immunoglobulin levels were measured by ELISA. It was observed that SAR oral treatment increased survival rate of mice infected with P. chabaudi and also significatively augmented total circulating IgG and IgM antibody levels. However, this treatment did not have any effect on parasitemia levels. The treatment with SAR increased significatively the production of antibodies against the polysaccharide TNP-Ficoll as measured 21 days after immunization but did not modify the specific anti-ovalbumin response in immunized mice. Despite having no effect on anti-ovalbumin-specific antibodies in immunized animals, SAR treatment induced an increase in basal anti-ovalbumin immunoglobulin levels in unimmunized SAR-treated animals. The basal levels of natural antibodies to the polysaccharide dextran were also significatively increase by treatment with SAR 1 and 2 weeks after continuous daily SAR treatment.
CONCLUSIONS: Taken together, our data suggest that SAR acts as an immune response modulator by increasing the basal levels of antibodies and possibly by improving the individual's ability to respond to antigens. These data support the hypothesis that SAR would have an overall immunopotentiating effect.
FINATIAL SUPPORT: FAPERJ
INVOLVEMENT OF CC-CHEMOKINE RECEPTOR 2 (CCR2) IN SEPSIS: FOCUS IN COGNITIVE IMPAIRMENT
MARIANA GISELY AMARANTE TEIXEIRA-DA-CUNHA1; FERNANDO BOZZA2; SILVIO CAETANO ALVES JUNIOR3; ROSALIA MENDEZ OTERO4; PATRICIA BOZZA5; RACHEL NOVAES GOMES6; HUGO CAIRE CASTRO-FARIA- NETO7.
1,5,7.LAB. IMUNOFARMACOLOGIA-FIOCRUZ-IOC, RIO DE JANEIRO - RJ - BRASIL; 2.FIOCRUZ-IPEC, RIO DE JANEIRO - RJ - BRASIL; 3.LAB. IMUNOFARMACOLOGIA- FIOCRUZ-IOC, RIO DE JANEIRO - RJ - BRASIL; 4.LAB. NEUROBIOLOGIA CELULAR E MOLECULAR-UFRJ - CCS, RIO DE JANEIRO - RJ - BRASIL; 6.FIOCRUZ - IOC/IPEC, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Sepsis is a major disease entity with important clinical implications. Critical illness survivors present long-term cognitive impairment, including problems with memory and learning. Chemokines are important to the recruitment of leukocytes to infectious tissue, but a little bit of studies described the role of the CCL2 in the cognitive process. In this study, we analyze the involvement of CCR2 in physiopathology of sepsis, especially in development of cognitive dysfunction.
Methods and Results: The CCR2 deficient mice (CCR2-/-) were submitted to CLP model and we analyze the survival rate, the severity score of the animals during 144 hours and 15 days after the CLP, we analyzed the memory of the animals. To analyze the contextual memory, the mice were submitted to open field and the water maze procedure. For evaluate the aversive memory, the passive avoidance test was performed. First, we observed that the CLP group had a cognitive impairment, but the CCR2-/- group submitted to CLP had a more severe cognitive impairment in comparison with the WT-CLP group. Interesting, the CCR2-/- SHAM group presented cognitive impairment, suggesting that CCR2 is important to physiological process of cognition. Then, we submitted CCR2-/- naive mice to water maze and passive avoidance test. We found that the CCR2-/- naive mice have an impairment of aversive and contextual memory. The cognitive impairment was associated with a decrease of BDNF expression in hippocampus. When we analyze the expression of β-amyloid protein in brain of CCR2-/- naive mice, we observed the increased in β-amyloid protein expression in cortex and hippocampus of this animals, accompanied by increased cell proliferation in dentate girus, increased of caspase-3 and caspase-12 expression in hippocampus and cortex. We didn't observed difference in the numbers of neurons in brain from CCR2-/- naïve mice, as well the numbers of microglial cells. But, surprisingly, there was an increase of astrocytes in hippocampus of CCR2-/- mice.
Conclusion: The CCR2 is involved with the physiology of the cognition, with the important role in arising of the amyloid accumulation in brain and induction of caspase-3 pathway.
Financial support: FIOCRUZ, FAPERJ, CNPq
INVOLVEMENT OF GCS/KATP PATHWAY IN NITROSYL - RUTHENIUM (CIS[RU(BPY)2SO3NO]+ (PF6) PROTECTIVE EFFECT IN EXPERIMENTAL GASTRIC DAMAGE MODELS.
ANA PAULA SANTANA1; BRUNO DE MELO TAVARES2; ANA CARLA CARVALHO3; LARISSE TAVARES LUCETTI4; KAROLINE SABÓIA ARAGÃO5; FRANCISCO ORDELEI DA SILVA6; LUIZ GONZAGA LOPES7; RONALDO DE ALBUQUERQUE RIBEIRO8; PEDRO MARCOS SOARES9; JAND-VENES ROLIM MEDEIROS10; MARCELLUS HENRIQUE DE SOUZA11.
1,2,3,4,5,6,7,8,9,11.FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 10.FEDERAL UNIVERSITY OF PIAUÍ, PARNAÍBA - PI - BRASIL.
INTRODUCTION: Nitric Oxide (NO) plays an important regulatory role in maintaining gastric mucosal integrity. Recently, new NO donors with ruthenium metal in its composition were developed. AIM: To evaluate the effect of NO donor nitrosyl-ruthenium (Rut-NO) in experimental models of gastric damage induced by ethanol or naproxen (NPX), and the involvement of soluble guanylate cyclase (sGC) and KATP channels in this event. METHODS: Swiss mice (25-
30 g) were pre-treated with saline, ODQ (sGC inhibitor, 10mg/kg,p.o) or glibenclamide (GLIB, KATP inhibitor, 10mg/kg, i.p). After 30 min or 1 h respectively, Rut-NO (3mg/Kg,p.o) was administrated. At the end of 30 min, the animals received 50% ethanol (0.5 ml/25g) by gavage. After 1h, the animals were sacrificed and gastric damage (macroscopic), glutathione (GSH) and malondialdehyde (MDA) gastric levels were determined. Other group, were pre- treated with saline, ODQ or GLIB. After 30 min or 1 h respectively, Rut-NO (3mg/Kg,p.o) was administrated. At the end of 30 min, the animals received naproxen (300 mg/kg) by gavage. After 6h, the animals were sacrificed and gastric damage (macroscopic), MPO activity, TNF-α and IL-1β gastric concentration was evaluated. In addition, it was determined the effect of Rut-NO (3mg/Kg,p.o) on naproxen-induced mesenteric leukocytes adherence by intravital microscopy. Local ethics committee protocol 33/10. RESULTS: Rut-NO prevented the macroscopic (EtOH: 136±10; Rut-NO: 7.6±5.0) gastric damage, decrease GSH (EtOH: 176±25; Rut-NO: 299±35) and increase MDA (EtOH: 58±4.0; Rut-NO: 34±4.0) induced by ethanol. It was also observed, that Rut-NO prevented naproxen-induced gastric damage (NPX: 11.3±1.0; Rut-NO: 5.1±1.0), increase in MPO activity (NPX: 124±11; Rut-NO: 67±9.7), and TNF-α (NPX: 5.3±0.3; Rut-NO: 3.2±0.2), IL-1β (NPX: 2.0±0.1; Rut-NO: 1.3±0.1) concentrations. Rut-NO (0.3±0.1) was also able to decrease the leukocytes adherence induced by naproxen (0.6±0.1). ODQ or GLIB completely reversed the Rut-NO protective effect against ethanol (ODQ/GLIB: macroscopic 191±40/186±29; GSH 128±41/106±13; MDA 67±9.0/69±40) or naproxen (ODQ/GLIB: macroscopic 11±1.0/11±2.0; MPO 124±15/144±16) induced gastric damage.
CONCLUSION: We can infer that Rut-NO prevented the gastric damage through an activation of the sGC and KATP channels, with a decrease in the free radical and cytokines production by the blocking neutrophil adhesion and infiltration.
FINANCIAL SUPPORT: CNPq, CAPES, FUNCAP.
INVOLVEMENT OF MAST CELLS AND HISTAMINE IN INFLAMMATORY EVENTS INDUCED BY SCOLOPENDRA VIRIDICORNIS CENTIPEDE VENOM
BIANCA DE CARVALHO LINS FERNANDES TÁVORA1; LOUISE FAGGIONATO KIMURA2; ELIANA FAQUIM DE LIMA MAURO3; NICOLE ASSIS PEREIRA4; IRENE KNYSAK5; SAMUEL GIOIA GUIZZE6; KATIA CRISTINA BARBARO7.
1,2,3,4,7.LABORATORY OF IMMUNOPATHOLOGY - BUTANTAN INSTITUTE, SÃO PAULO - SP - BRASIL; 5,6.LABORATORY OF ARTHROPODS - BUTANTAN INSTITUTE, SÃO PAULO - SP - BRASIL.
Introduction: The Scolopendra genus is commonly found throughout Brazil. Envenomation caused by centipede bites is generally mild, and human victims usually manifest burning pain, paresthesia and edema. Since previous report have shown that Scolopendra viridicornis venom (Sv) induces a rapid and persistent edematogenic activity followed by leukocyte influx in mice, the aim of this work was to evaluate the involvement of mast cells and histamine on these inflammatory events induced by Sv centipede venom in vivo and in vitro.
Methods and Results: Groups of Swiss mice were pretreated with cromoglycate (inhibitor of mast cell degranulation) or the histamine-receptor antagonists promethazine (H1R antagonist), cimetidine (H2R antagonist), or thioperamide
(H3/H4R antagonist), and injected i.pl. with Sv (15 µg/paw) to evaluate edema forming and leukocyte recruitment modulation. Edema was measured by plethysmometer (15 min, 30 min, 1, 4, 6, 24 and 48 h) and the maximum peak was observed at 15 min after Sv injection, returning to baseline within 48 h. Cromoglycate significantly reduced paw edema (40-90%) induced by Sv in all periods evaluated. The pretreatment of mice with promethazine (all periods varying between 50-100%) and thioperamide (4 h, 30%) decreased paw edema induced by Sv. On the other hand, cimetidine did not reduce edema in any of the evaluated time periods. Moreover, Sv injection evoked a complex inflammatory reaction with leukocyte recruitment to the footpad, which is observed at 15 min after venom injection in comparison with PBS-injected mice; however, none of these drugs inhibited cell migration in mice. In addition, Sv (7.5- 60 µg/well) induced mast cell degranulation in both PT-18 (mouse mast cell line) and RBL-2H3 (rat basophilic leukemia) cell lineages, with no effect on cell viability.
Conclusion: Mast cells and histamine release are involved in local reaction induced by Sv. However, other mechanisms may be involved in the inflammatory process caused by Sv, since none of the treatments inhibited the cell migration.
Financial support: INCTTox (FAPESP grant #2008/57898-0 and CNPq grant #573790/2008-6).
INVOLVEMENT OF NFAT TRANSCRIPTION FACTORS IN THE REGULATION OF GENES RELATED TO LIPID METABOLISM, PRODUCTION OF INFLAMMATORY MEDIATORS AND LIPD BODY FORMATION IN COLON ADENOCARCINOMA
MARINA AUGUSTIN1; ANDRÉ CRUZ2; JOÃO P.B VIOLA3; PATRÍCIA BOZZA4; MIRIAM WERNECK5.
1,4.OSWALDO CRUZ INSTITUTE INSTITUTE AND OSWALDO CRUZ INSTITUTE, RIO DE JANEIRA - RJ - BRASIL; 2,3,5.NATIONAL CANCER INSTITUTE AND OSWALDO CRUZ INSTITUTE, RIO DE JANEIRA - RJ - BRASIL.
Introduction: The increase in lipogenesis is a common phenotype of various tumors and is associated with a poor prognosis in some types of cancer, including colon carcinoma. Changes in lipid metabolism in cancer cells include modulation of lipogenic enzymes and accumulation of lipid bodies. It was believed that these organelles had their role restricted to lipid stock and trafficking. However, we now know that lipid bodies also contain PI3K, cyclooxygenase-2 (COX-2) and other enzymes, suggesting a role in cell signaling. COX-2 is involved in the synthesis of prostaglandin
E2, an inflammatory mediator that plays a major role in tumor development and progression. The transcription factor nuclear factor of activated T cells (NFAT) regulates the expression of COX-2, genes involved in cellular metabolism and in the process of adipocyte differentiation. However, the involvement of NFAT in the regulation of lipid metabolism in tumor cells has not been evaluated. The aim of this study was to evaluate the involvement of NFAT in the regulation of genes related to lipid metabolism, production of inflammatory mediators and lipid body biogenesis in colon adenocarcinoma. Methods and Results: Our results indicate that the colon cancer cell line HT-29 upregulates genes encoding (i) the enzyme fatty acid synthase (FAS), which main function is to catalyze the synthesis of palmitate and is involved in colon cancer tumorigenesis, (ii) ADRP, a major protein in lipid bodies and (iii) COX-2, when activated with ionomycin or PMA and ionomycin, as measured by real-time PCR. Increased expression of COX-2 was accompanied by an increase in PGE2 synthesis, dosed by enzyme immunoassay, and formation of lipid bodies, analyzed by staining of lipid bodies with lipophilic dyes. Treatment with cyclosporin A (CsA) reversed the increase in gene expression of
FASN, ADRP and COX-2, as well as PGE2 synthesis, but did not alter lipid body biogenesis in these cells. Conclusion: These data indicate that NFAT may have an important role in regulating genes involved in lipid metabolism and the production of PGE2, but is not essential for the formation of lipid bodies in colon cancer cells.
Financial Support: CAPES, CNPq, FAPERJ, INCT-Cancer, MS/INCA.
INVOLVEMENT OF SYSTEM CHOLINERGIC, ADRENERGIC AND DOPAMINERGIC IN THE ANTINOCICEPTIVE ACTIVITY OF RIPARIN III
LEONARDO FREIRE VASCONCELOS1; ALYNE MARA RODRIGUES CARVALHO2; NAYRTON FLAVIO MOURA ROCHA3; EMILIANO RICARDO VASCONCELOS RIOS4; MARILIA LEITE DIAS5; LAURA MARIA TEODORIO VIDAL6; STANLEY JUAN CHAVEZ GUTIERREZ7; JOSE MARIA BARBOSA FILHO8; FRANCISCA CLEA FLORENÇO SOUSA9.
1,2,3,4,5,6,9.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FACULTY OF MEDICINE UFC, FORTALEZA - CE - BRASIL; 7.DEPARTMENT OF BIOCHEMISTRY AND PHARMACOLOGY UFPI, TERESINA - PI - BRASIL; 8.LABORATORY OF PHARMACEUTICS TECHNOLOGY UFPB, JOAO PESSOA - PB - BRASIL.
Introduction: Riparin III (RipIII) is an alkamid compound that was firstly isolated from unripe fruit of Aniba riparia, but now it can be synthesized. This substance presents antimicrobial, anxiolytic and antidepressant-like effects in different animal models. The objective of this work was to investigate the antinociceptive effect of RipIII and the involvement of cholinergic, α2-adrenergic and dopaminergic systens in this action. This work was submitted to local Ethics Committee on Animal Research (protocol 41/10). Methods and Results: Male Swiss mice, weighing 25-32g, were used and all experiments were conducted with 7-9 animals per group. Data were analyzed using One-Way ANOVA and Newman Keuls test as post hoc. Firstly, RipIII was tested in the acetic acid-induced abdominal writhing and Time Course test. Indomethacin 5mg/kg (p.o.) was used as standard drug. In order to investigate the involvement of adrenergic, cholinergic and dopaminergic system, different groups received yohimbine (2.5mg/kg, i.p; selective α2-adrenergic antagonist), atropine (1mg/kg, i.p; non-selective cholinergic antagonist) or haloperidol (0.2mg/kg, i.p.; non-selective dopamine antagonist). Subsequently, animals received RipIII-50 mg/kg (p.o.) or vehicle (2% Tween 80 in distilled water) and were submitted to the test of abdominal writhing induced by acetic acid. RipIII administration (p.o.) produced an antinociceptive effect at 25 and 50mg/kg doses (reduction of 60.2 and 59.57%, respectively), whereas at 6.125, 12.5, 100 and 200mg/kg doses, a weaker and non-significant antinociceptive action was observed when compared to vehicle. In relation to the onset and duration of RipIII effect, the results showed that the response started after 30 min (reduction of 28.83%) after its administration and lasted for about 6h (reduction of 42.35%) after injection of acetic acid. The results show that yohimbine was not able to significantly reverse the antinociceptive effect caused by RipIII. Atropine and haloperidol were able to reverse the effect generated by RipIII. Conclusion: RipIII has a great antinociceptive activity that may be, at least in part, related to cholinergic and dopaminergic system. Support Financial: CNPq/CAPES/FUNCAP
INVOLVEMENT OF TOLL-LIKE RECEPTOR 2 AND MYD88 IN IRINOTECAN-INDUCED INTESTINAL MUCOSITIS PATHOGENESIS
DEYSI VIVIANA TENAZOA WONG1; VANESSA DE FÁTIMA BORGES (PG)2; AMANDA XIMENES COUTO BEM (IC)3; EDWYN LOWISI FERNANDES COSTA (IC)4; CARLOS WAGNER DE SOUZA WANDERLEY (PG)5; CAIO ABNER VITORINO GONÇALVES LEITE (PG)6; FABRICIO OLIVEIRA SOUTO7; GERLY ANNE DE CASTRO BRITO8; ROBERTO CÉSAR PEREIRA LIMA-JÚNIOR9; FERNANDO DE QUEIROZ CUNHA10; RONALDO ALBUQUERQUE RIBEIRO11.
1,3,4,5,6,9,11.DEPARTMENT OF PHISIOLOGY AND PHARMACOLOGY. FEDERAL UNIVERSITY OF CEARA, FORTALEZA - CE - BRASIL; 2,8,10.FACULTY OF MEDICINE OF RIBEIRÃO PRETO. UNIVERSITY OF SÃO PAULO, RIBEIRÃO PRETO - SP - BRASIL; 7.FACULTY OF MEDICINE. UNIVERSITY OF SÃO PAULO, RIBEIRÃO PRETO - SP - BRASIL.
Introduction: Severe diarrhea and the associated intestinal mucositis (IM) are common side effects (15-25%) of colorectal anticancer therapy with Irinotecan (IRI). Gut injury induced by chemotherapeutic agents may result in bacterial/endotoxin translocation from the intestine to the systemic circulation. Pathogens recognition is partially due to the activation of toll-like receptors (TLR) by the pathogens-associated molecular patterns (PAMPs). Furthermore, necrotic cells and extracellular matrix, which release damage-associated molecular patterns (DAMPs), activate the synthesis of pro-inflammatory mediators through TLRs, causing intestinal injury. Then, we aimed to evaluate the involvement of TLR2 and MyD88, which is an adaptor protein to TLR and IL-1-family cytokines, in the pathogenesis of IRI-induced IM. Methods and Results: C57BL/6 (WT) mice (20-24g, n=6-7) and Knockout mice to TLR2 (TLR2-/-) or MyD88 (MyD88-/-) were given either saline or IRI (75 mg/kg i.p/4 days). On day 7, weight loss, diarrhea, blood leukocyte and bacterial count were assessed. Following euthanasia, ileum samples were obtained for myeloperoxydase (MPO) assay, morphometric analysis, NFκB immunohistochemistry, IL-1β dosage and COX2 gene expression. Kruskal Wallis/Dunn’s test or ANOVA/Bonferroni’s test were used for statistical analysis. P<0.05 was accepted. (CEPA 99/10). Irinotecan induced a markedly weight loss, diarrhea, leukopenia, increased bacteremia, MPO activity, morphometric alterations, NFκB immunostained, as well as increase IL-1β level and COX2 expression in intestinal samples of WT animals versus saline-injected group (P<0.05). On the other hand, IRI-injected to TLR2-/- and MyD88-/- mice showed a milder (P<0.05) weight loss, diarrhea (0[0-1]; 0[0-1] respectively), lower neutrophil infiltration (1888±545; 1310±443, respectively), bacterial clearance, increase villus length (149±6.8; 178.6±6.2), reduced NFkB immunoexpression (31.5±8.2; 13.2±5.1) and COX2 expression (0.95±0.2; 3.62±1.2), as well as IL-1β level (0.85±0.1; 1.11±0.1) when compared with IRI-administered WT mice (diarrhea: 2[1-3]; MPO:4725±1231; villus length: 122.1±5.7; NFkB: 85±2.2; COX2 expression: 21.2±5.9 and IL-1β level: 3.9±1.2) (P<0.05). In addition, knockout to TLR2 (P<0.05), but not to MyD88 (P>0.05), ameliorated IRI induced-leukopenia. Conclusions: This study suggests the participation of TLR2 and MyD88 in the pathogenesis of IRI-induced intestinal mucositis. Financial support: CNPq/CAPES/FUNCAP.
ISCHEMIC HINDLIMB REVASCULARIZATION IN TYPE 1 DIABETIC AND HIGH FAT OR HIGH REFINED CARBOHYDRATE DIET-INDUCED INSULIN RESISTANCE TYPE 2 DIABETIC MICE: A COMPARATIVE STUDY
LUIZA DIAS DA CUNHA LIMA (PG)1; LENDRO CEOTTO FREITAS LIMA (PG)2; CAMILA PEREIRA ALMEIDA (IC)3; KATIA MICHELLE FREITAS (PG)4; ROBSON AUGUSTO SOUZA DOS SANTOS5; ADALIENE VERSIANI FERREIRA6; MAURO MARTINS TEIXEIRA7; SERGIO HENRIQUE SOUSA SANTOS8; LUCIOLA DA SILVA BARCELOS9.
1,2,3,9.IMMUNOPHARMACOLOGY GROUP, DEPARTMENT OF PHYSIOLOGY AND BIOPHYSIC, UFMG, BELO HORIZONTE - MG - BRASIL; 4,8.DEPARTMENT OF PHARMACOLOGY, UFMG, BELO HORIZONTE - MG - BRASIL; 5.DEPARTMENT OF PHYSIOLOGY AND BIOPHYSIC, UFMG, BELO HORIZONTE - MG - BRASIL; 6.BASIC NURSING, NURSING SCHOOL, UFMG, BELO HORIZONTE - MG - BRASIL; 7.IMMUNOPHARMACOLOGY GROUP, DEPARTMENT OF BIOCHEMISTRY AND IMMUNOLOGY, BELO HORIZONTE - MG - BRASIL.
Introduction: Diabetes Mellitus (DM) has been described as the main 21st century global epidemic. DM is characterized by high blood glucose levels and/or insulin resistance and can lead to permanent damage to organs. Peripheral arterial disease (PAD) is a chronic disease that progressively impairs the arterial circulation of lower limbs. Diabetic patients have higher relative risk to undergo lower limb amputation than non-diabetic patients with PAD. Here, we compared the effects of streptozotocin (STZ)-induced type 1 DM and either high fat or high refined carbohydrate dietinduced insulin resistance type 2 DM on ischemic hindlimb revascularization.
Methods and Results: 8 weeks old C57BL/6 male mice were divided into five groups that received three differents diabetogenic diets (n=6-8 animals/group). HC group received a high refined carbohydrate diet and HF40 and HF60 groups received high fat diet with 40% and 60% of lipids, respectively. Control and STZ groups received regular diet. Hindlimb ischemia was induced by permanent occlusion of the left femoral artery (FAO) after three months of feeding. Diet feeding last until two weeks after surgery. Hindlimb blood flow was evaluated by laser Doppler perfusion imaging. Glucose-Tolerance and Insulin Sensitivity tests were performed one day before FAO. All animals fed high fat or high refined carbohydrate diets showed increased body weight gain (D body weight (g)) (Control 6.6±0.9 vs HC 11.0±1.2* vs HF40 12.9±1.4*** vs HF60 12.0±2.1***), glucose intolerance (area under the twohour blood glucose response curve (AUC)) (C 17574±903 vs HC 21119±669* vs HF40 21679±743** vs HF60 28947±963***) and insulin resistance (C 10935±320 vs HC 14848±662* vs HF40 16603±697** vs HF60 17256±1654**) when compared to the control group on regular diet. STZ group showed only glucose intolerance (C 17574±903 vs STZ 43689±2952***). Diet-induced insulin resistant T2DM mice (except HC diet) showed lower blood flow recovery in the ischemic limb than STZinduced T1DM diabetic mice when compared to control group (ischemic /contralateral ratio) (C 0.92±0.06 vs HC 0.68±0.06* vs HF40 0.41±0.01*** vs HF60 0.39±0.09*** vs STZ 0.7±0.05**, at day 14 after FAO).
Conclusion: Our results suggests that high fat diet-induced insulin resistance T2DM impairs ischemic hindlimb revascularization more preeminently than high refined carbohydrate diet-induced T2DM or STZ-induced T1DM in mice.
Financial support: FAPEMIG, CNPq, CAPES, INCT-Nanobiofar.
KETAMINE INDUCES PERIPHERAL ANTINOCICEPTION IN RAT BY OPIOID AND CANNABINOID SYSTEM ACTIVATION.
RENATA CRISTINA MENDES FERREIRA1; PATRICIA PAIVA LIMA2; CELSO QUEIROZ JUNIOR3; MARCELO CALIARI4; VINCENZO DI MARZO5; IGOR DIMITRI GAMA DUARTE6; THIAGO ROBERTO LIMA ROMERO7.
1,2,3,4,6,7.UFMG, BELO HORIZONTE - MG - BRASIL; 5.INSTITUTE OF BIOMOLECULAR CHEMISTRY, NAPOLI - ITÁLIA.
Introduction: Ketamine has a peripheral analgesic component, but the base of this mechanism is not completely elucidated. The aim was obtain pharmacological evidences for the involvement of opioid and cannabinoid system in the peripheral antinociceptive effect induced by ketamine. Methods and Results: The rat paw pressure test was used and hyperalgesia was induced by intraplantar injection of prostaglandin E2 (2 µg/paw). The imunohistochimic and the HPLC chromatography were performed to verify the participation of β-endorphin and endocannabinoids, respectively, in the ketamine-effect. All drugs were administered intraplantar of Wistar rats (n=4). Ketamine 80 µg/paw elicited a local inhibition of hiperalgesia (14.0 ± 1.2; p<0.05). The enkephalinase inhibitor bestatin 400 µg/paw increased the peripheral antinociceptive effect of ketamine 20 µg/paw (30.0 ± 4.3; p<0.05). The µ-δ opioid antagonists clocinnamox
40 µg/paw (94.1 ± 4.4), naltrindole 60 µg/paw (75.9 ± 10.3) and the CB1 cannabinoid antagonist AM251 80 µg/paw (95.0 ± 6.1) antagonized the antinociceptive effect induced by ketamine (p<0.05), but not by the κ-opioid receptor antagonist NOR BNI 100 µg/paw or the CB2 cannabinoid antagonist AM630 100 µg/paw. The anandamide amidase inhibitor MAFP 2 µg/paw (16.6 ± 3.3) and the anandamide reuptake inhibitor VDM11 20 µg/paw (9.1 ± 1.6) increased the peripheral antinociceptive effect of ketamine low dose (p<0.05). In addition, the imunohistochimic evidenced an increase of β-endorphin in the keratinocyte area after the ketamine-injection and the dosage of endocannabinoids indicated that ketamine induces a selective release of anandamide in the peripheral site. Conclusion: The results suggest that ketamine induces peripheral antinociceptive effect by β-endorphin and anandamide release to activate the selective µ-δ opioid receptors and CB1 cannabinoid receptor. Financial support: CNPq, CAPES and FAPEMIG.
LACK OF GALECTIN-3 PROTECTS MICE THAT UNDERWENT POLYMICROBIAL SEPSIS
RAPHAEL GOMES FERREIRA; DANIELE CARVALHO BERNARDO NASCIMENTO; PAULO HENRIQUE DE MELO; ALEXANDRE KANASHIRO; VANESSA DE FÁTIMA BORGES (PG); JOSÉ MAURÍCIO SEGUNDO CORREIA MOTA; FERNANDO DE QUEIRÓZ CUNHA; JOSÉ CARLOS ALVES-FILHO.
UNIVERSITY OF SÃO PAULO, RIBEIRÃO PRETO - SP - BRASIL.
Introduction: Sepsis is a systemic inflammatory response initiated after recognition of the infectious agent by the host, leading to activation of mechanisms of cellular and humoral defense. Galectin-3, a protein molecule capable of binding the polysaccharides expressed on the membrane of different cell types, has important immunoregulatory functions and participates in the development of inflammatory response. Methods: To assess the participation of galectin-3 in the development of systemic inflammatory response after sepsis, galectin-3 knockout (Gal-3 KO) and wild type (WT) BALB/c mice were subjected to cecal ligation and puncture (CLP). For this, an incision was made on the abdomen, the cecum was exposed and ligated below the ileocecal junction, and a single puncture was made through the cecum, mice were stitched and received basic support (hydration). 6, 12 and 24 hs after CLP, mice were euthanized and peritoneal fluid, blood and lung were collected. We evaluated neutrophils migration in peritoneal fluid using a Neubauer chamber and lung neutrophils sequestration using a myeloperoxidase assay. Lung tissue was also used to assess cytokines production by ELISA. Furthermore, blood serum was used to assess markers of tissue damage in liver (by aspartate aminotransferase activity), kidney (by blood urea nitrogen levels), and heart (by CK-MB activity). Finally, colony-forming units (CFU) were counted from peritoneal fluid and blood. Animal ethical Commission, Protocol No. 098/2012. Results: Gal-3 KO mice showed greater resistance to polymicrobial sepsis characterized by high survival rate and migration of neutrophils to the focus of infection 6 and 12 h after CLP. when compared with WT, KO mice showed fewer CFU counted in the peritoneal fluid and blood in all time periods evaluated. Moreover, Gal-3 KO mice showed lower markers of damage in kidney (6 and 24 h), heart (6 h) and liver (24 h) and low neutrophils sequestration in the lung 6 h after CLP. Accordingly, Gal-3 mice also showed lower levels of IL-6 and IFN-γ production in the lung. Conclusion: These data suggest that galectin-3 is an important mediator of inflammatory response triggered during sepsis. The lack of galectin-3 leads to high neutrophil number in peritoneal fluid, associated with reduction of local and systemic bacterial load, less systemic inflammation and consequently, reduction in tissue damage that is directly related to improvement of survival rates.
Financial support: CNPq, FAPESP
LASSBIO-1247 IS AN ORAL EFFECTIVE DRUG PROTOTYPE CANDIDATE TO TREAT RHEUMATOID ARTHRITIC PAIN
EWERTON ALVES PORTELA DOS SANTOS; FERNANDO RODRIGUES DE SÁ ALVES; CLEVERTON KLEITON FREITAS DE LIMA; CARLOS ALBERTO MANSSOUR FRAGA; ELIEZER JESUS DE LACERDA BARREIRO; ANA LUISA PALHARES DE MIRANDA.
FEDERAL UNIVERSITY OF RIO DE JANEIRO - UFRJ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: TNF-α plays crucial role in pathogenesis and pain associated to rheumatoid arthritis (RA), including cell chemotaxis (Nat Rev Imm,2:364-71,2002). Thus, the reduction of TNF-α level is an important strategy to treat RA. Previous study showed that LASSBio-1247 decreases TNF-α in vitro, has a relevant anti-hypernociceptive effect and reduces MPO level in a carrageenan model (Unpublished). The present study aimed to deepen the anti- hypernociceptive and anti-inflammatory properties of LASSBio-1247 and to evaluate it in RA model. Methods & Results: Animal protocols were approved by UFRJ ethical animal care committee (FARMACIA02). Results are expressed in mean±standard error (ANOVA-oneway, n=6-8 animals/group; in vitro n=3;*p<0.05). To MTT test and TNF-α production, macrophages were stimulated by LPS and incubated with LASSBio-1247. This compound showed an IC50=13µM on the TNF-α production and the inhibition cannot be associated to cell death. In addition, LASSBio- 1247 inhibited 55%* the LPS-induced thermal hypernociception that is mediated by TNF-α, IL-1β and IL-6 (Int Imm,4:901-9,2004) and inhibited by 70%* the hypernociception induced by capsaicin, a response mediated by the p38MAPK and TRPV1 activation (Pain,113:51-60,2005). In order to evaluate TRPV1 antagonism, calcium-imaging assay showed LASSBio-1247 at 10µM could not reduce Ca+2 influx in HEK293 cells expressing hTRPV1 after capsaicin stimulation. In the mBSA-induced delayed-type hypersensibility (RA model) (Eur J Pain,12:1059-68,2008), LASSBio-1247 at 100µmol/kg orally twice/day (5 days) reversed hypernociception at several points of the acute and chronic phases* in mechanical evaluation, it didn’t induce stomach damage in an optical observation. Preliminary results indicate LASSBio-1247 can reduce TNF-α level at plasma and synovial fluid from mBSA treated animals around 50%*. Moreover, it reduced the total number of leukocytes from the articular washes (42%*). Besides having inhibited TNF-α production, LASSBio-1247 also interferes directly in chemotaxys, since it inhibited neutrophil chemeotaxis in Boyden chamber by 45%*. (J Immun,170:2688-94,2003). Conclusion: The anti-hypernociceptive and anti-inflammatory effects of LASSBio-1247 could be in part due to the decrease of TNF-α and direct reduction of chemotaxis, leading to a decrease of cell migration. These results point out LASSBio-1247 as a promising prototype of a drug candidate to treat RA.Financial support-CAPES INCT-INOFAR FAPERJ
LEAVES OF NEPETA CATARIA (CATNIP) INDUCE INCREASE OF THE TUMOR GROWTH OF MAMMARY ADENOCARCINOMA CELLS
JOSÉ RENILDO CARVALHO1; EDILAINE SUDRÉ MARCELINO NASCIMENTO2; FABIANA TOSHIE KONNO3; DANILO CABRAL4; MARIA MARTHA BERNARDI5; ELIZABETH CRISTINA PÉREZ6.
1,2,3.UNIVERSIDADE PAULISTA, SÃO PAULO - SP - BRASIL; 4,5,6.UNIVERSIDADE PAULISTA,, SÃO PAULO - SP - BRASIL.
Introdution: Catnip is a perennial herb and member of the Mint family Labiatae that is known for it's ability to get cats high. In humans, is commonly used in herbal tea blends related to sleep, stress reduction, and relaxation. Recently studies have shown antitumor effects of catnip. However, the effects of catnip leaves in the development of breast cancer have not yet been investigated. Therefore, the aim of this study was to evaluate the role of catnip leaves in tumor growth of 4T1 mammary adenocarcinoma cells.
Methods and results: We used 20 female mice BALB/c divided into control and experimental groups. The control group was maintained on a conventional diet and the experimental group received ration with 10% of catnip leaves. 4T1 mammary adenocarcinoma cells (2x104 cells) were injected in the mammary region and tumor growth was measured every other day until complete 30 days. The results showed that animals in the experimental group had significantly greater tumor growth compared to the growth of the control groups.
Conclusion: The results suggest that catnip leaves induce increase of the tumor growth of 4T1 mammary adenocarcinoma cells.
Financial support: UNIP
LEAVES OF NEPETA CATARIA (CATNIP) REDUCE METASTASES OF MAMMARY ADENOCARCINOMA CELLS
EDILAINE SUDRÉ MARCELINO NASCIMENTO; JOSÉ RENILDO CARVALHO; FABIANA TOSHIE KONNO; DANILO CABRAL; MARIA MARTA BERNADI; ELIZABETH CRISTINA PÉREZ.
UNIVERSIDADE PAULISTA, PROGRAMA DE PÓS-GRADUAÇÃO, SÃO PAULO - SP - BRASIL.
Introduction: Cancer is the name given to the set of pathologies that are characterized by loss of control of cell proliferation and gain the ability to invade surrounding tissues forming secondary tumors or metastases. The second most common type worldwide, breast cancer, is considered the main cause of death in the female population due to its high invasiveness. Recent studies conducted in order to evaluate the antitumor effect present in plants have shown that aromatic herbs like catnip, could be used for the treatment of cancer. However, the effect of leaves catnip on metastatization of mammary adenocarcinoma cells has not been investigated. Thus the aim of this work was to investigate the influence of catnip on metastasis formation of mammary adenocarcinoma cells.
Methods and results: Twenty female mice BALB/c were divided in control and experimental groups. The control group was maintained on a conventional diet and the experimental group received ration with 10% of catnip leaves. 4T1 mammary adenocarcinoma cells (2x104 cells) were injected in the mammary region. After 30 days mice were sacrificed and lung, spleen and liver were surgically removed to evaluate of spontaneous metastases and histological analysis. Results of macroscopic analysis showed that mice in the experimental group had fewer nodules metastatic when compared to the control group.
Conclusion: These data suggest, for the first time, that catnip leaves reduce the metastatic behavior of mammary adenocarcinoma cells in murine model.
Financial support: UNIP
LEPTIN POTENTIATES LIPID MEDIATORS EXPRESSION IN PRIMARY CULTURE OF PULMONARY ENDOTHELIAL CELLS ACTIVATED BY LPS: THE ROLE OF ERK1/2, JNK AND SAPK
ALEKSANDRO MARTINS BALBINO1; REBÉCA M GASPARIN2; LEILA A DOS SANTOS3; LILIAM FERNANDES4; MARISTELLA A LANDGRAF5; RICHARDT G LANDGRAF6.
1,2,3,4,6.UNIVERSIDADE FEDERAL DE SÃO PAULO, DIADEMA - SP - BRASIL; 5.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL.
Introduction: Vascular endothelium is closely related with the circulatory control, and has an important participation in cellular and molecular events which occurred during immune system reactions and tissue injuries. Leptin is a hormone mainly synthesized by adipose tissue; it is involved in various biological systems, acting in the food intake control and energetic metabolism, modulate immune response, hematopoiesis and lymphopoiesis.
Objectives: Characterization of primary culture of pulmonary endothelial cells, production of inflammatory mediators
(PGE2,LTC4,LTB4) in these cells, stimulated or not with LPS and/or leptin, as well as the evaluation of the signaling events regulating these processes.
Methods and Results: Male C57Bl/6 mice were euthanized and lung tissue samples were isolated under sterile conditions. These cells were characterized by immunofluorescence using ULEX and von Willebrand factor, which is a traditional marker of endothelial cells, and also by flow cytometry using antibodies CD105(endoglin), CD106(V-CAM) and CD45(hematopoietic cells). After the characterization, these cells were stimulated or not with LPS(1µg/mL) and/or leptin(10ng/mL), for 6h to evaluate the production of inflammatory mediators such as PGE2,LTB4 and LTC4/D4 by EIA and phosphorylation of ERK1/2, JNK and SAPK by western Blot. Cells were positive for all markers used, except for
CD45. The leptin stimulus did not alter levels of the inflammatory mediators. LPS increased PGE2 levels (319%) and leptin addition potentiated it by 30%. Interestingly, we found that neither LPS nor leptin were able to alter leukotrienes levels, but when administered together, LPS and leptin increased the LTB4(293%) and LTC4/D4(374%) expression. Leptin alone did not induce changes in ERK1/2, JNK and SAPK phosphorylation, and LPS increased the JNK and SAPK phosphorylation. Leptin addition increased the ERK1/2, JNK and SAPK phosphorylation induced by LPS.
Conclusion: Our preliminary results suggest that leptin plays an important pro-inflammatory role in the cultures of mice primary endothelial cells, since this hormone potentiated the LPS effect. This effect seems involve ERK1/2, JNK and SAPK phosphorylation.
Financial support: FAPESP (2010/01404-0, 2011/09947-5, 2012/51104-8) and CNPq.
LEUKOCYTE MIGRATION INTO THE LUNG OF MALE RATS AFTER INTESTINAL ISCHEMIA AND REPERFUSION IS DOWNREGULATED BY ESTRADIOL DUE TO REDUCTION OF ICAM-I EXPRESSION
FERNANDA YAMAMOTO RICARDO DA SILVA1; ANA CRISTINA BREITHAUPT FALOPPA2; EVELYN THAÍS FANTOZZI3; MARIA BEATRIZ BERNARDEZ AMORIM4; RICARDO MARTINS DE OLIVEIRA FILHO5; BERNARDO BORIS VARGAFTIG6; WOTHAN TAVARES DE LIMA7.
1,3,4,5,6,7.USP/ICB, SÃO PAULO - SP - BRASIL; 2.LIM 11 - HCFMUSP, SÃO PAULO - SP - BRASIL.
Introduction: Sequestration of neutrophil into lung and the increase of lung microvascular permeability are repercussions of the systemic inflammatory response caused by intestinal ischemia and reperfusion (I/R) and characterize the acute lung injury (ALI). Female sex hormones interfere with immune response and immune cells are responsive to estradiol. In this context, the role of estradiol on ALI has been receiving considerable attention. Here, we investigated the effect of the acute treatment with estradiol on male rats, prior to intestinal I/R in order to reduce the lung and intestinal inflammation. Methods and Results: Upon anesthesia male rats were subjected to Intestinal I/R by occlusion (45-min) of superior mesenteric artery (SMA), followed by 2-h of intestinal reperfusion. Groups of rats received one single dose (280 μg/kg, i.v.) of 17β estradiol (E2), 30 min after the SMA occlusion. Lung myeloperoxidase activity (MPO) and Evans blue dye extravasation (EB) were used as index of the neutrophil recruitment and microvascular permeability, respectively. The expression of adhesion molecules (ICAM-1, PECAM-1 and VCAM) were evaluated by immunohistochemistry. Our results showed that the lung MPO activity and EB dye extravasation were increased by intestinal I/R and that E2 treatment was able to prevent these effects (MPO: i-I/R: 2.9 ± 0.9 vs E2+i-I/R: 2.1±0.12 p<0.05; EB: i-I/R: 172 ± 18 vs E2+i-I/R: 86.6±10.5, p<0.05). Regarding the expression of adhesion molecules, E2 treatment reduced ICAM-1 (i-I/R: 35.8 ± 3.6 vs E2+i-IR: 22.9 ± 4.4), whereas did not affect PECAM-1 but decreased the basal levels of VECAM found in lung of intestinal I/R. Conclusion: Our results indicate that intestinal I/R trigger inflammatory mechanisms involving increase of ICAM-1 expression that are downregulated by estradiol, restraining therefore the cell traffic into the lungs. Financial support: FAPESP and CNPq.
LEUKOTRIENE B4 DICTATES INFLAMMATION IN TYPE 1 DIABETES
LUCIANO FILGUEIRAS1; ANA ELISA FERREIRA2; FLAVIA SISTI3; SOUJUAN WANG4; ZHUO WANG5; SONIA JANCAR6; CARLOS HENRIQUE SEREZANI7.
1,6.DEPARTMENT OF IMMUNOLOGY, INSTITUTE OF BIOMEDICAL SCIENCES, UNIVERSITY OF SAO PAULO, SAO PAULO - SP - BRASIL; 2,3.DEPARTMENT OF PHARMACOLOGY, SCHOOL OF MEDICINE, UNIVERSITY OF SAO PAULO, RIBEIRAO PRETO - SP - BRASIL; 4,5,7.DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY, INDIANA UNIVERSITY SCHOOL OF MEDICINE, INDIANAPOLIS - ESTADOS UNIDOS.
Luciano Filgueiras*#, Ana Elisa Ferreira*&, Flavia Sisti*&, Soujuan Wang*, Zhuo Wang*, Sonia Jancar#, C Henrique Serezani*. *Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis. #Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil. &Department of Pharmacology, School of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil.
Introduction: In type 1 diabetes (T1D) macrophages produce high levels of pro-inflammatory cytokines that contributes to T1D morbidity. Activation of toll-like or IL1 type receptors drives inflammation in a manner dependent on MyD88 recruitment. Here we investigated the hypothesis that LTB4 contributes to TD1 morbidity by enhancing the responsiveness to cytokines acting on these receptors.
Methods: Peritoneal macrophages (PMs) from streptozotocin-induced T1D WT C57Bl/6, 5-lipoxygenase (5-LO)-/- or -/- LTB4 receptor 1 (BLT1 ) mice were used to determine the mRNA (qPCR) and protein expression (immunoblotting) in T1D mice. In vitro and in vivo treatments included insulin and the 5-LO inhibitor AA861. Chip assay was done to characterize the mechanisms involved in MyD88 expression. Sepsis was induced by colon ligation and pucture(CLP)
Results: PMs from T1D mice express higher levels of MyD88 and its transcription factor STAT-1 than PMs from control mice. 5-LO expression and LTB4 synthesis was also enhanced in PMs and in the serum from T1D mice. In vitro and in vivo insulin treatment restored LTB4 and STAT-1/MyD88 levels. Both genetic and pharmacologic inhibition of LTB4/BLT1 axis abolished MyD88/STAT-1 expression in T1D. ChIP assay analysis showed that LTB4 promoted
STAT-1 binding to MyD88 promoter, and high AP-1/cJun drives basal STAT-1 in a LTB4-dependent manner.
Speculating that enhanced LTB4-mediated MyD88 expression accounts for enhanced lethality in sepsis we found that both genetic and pharmacologic 5-LO inhibition enhanced T1D mice survival compared to WT or untreated T1D animals. Conclusion: LTB4 is the major driven force involved in inflammation by enhancing MyD88 levels and therefore the TLR/IL1R responsiveness in a manner dependent on c-Jun-mediated STAT-1 expression. These effects also contribute to high mortality associated to sepsis in T1D.
Financial support: NIH (R00HL103777-03); Showalter Foundation and FAPESP, Brazil.
LIGATURE-INDUCED PERIODONTITIS POTENTIATES RAT MESENTERIC ARTERY RELAXATION TO EXOGENOUS NITRIC OXIDE
FLAVIA NETO DE JESUS1; CAMILLA FERREIRA WENCESLAU2; GISELE KRUGER COUTO3; SIMONE APARECIDA TEIXEIRA4; SORAIA KÁTIA PEREIRA COSTA5; LUCIANA VENTURINE ROSSONI6; MARCELO NICOLÁS MUSCARÁ7.
1,4,5,7.DEPARTMENT OF PHARMACOLOGY, INSTITUTE OF BIOMEDICAL SCIENCES - UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL; 2,3,6.DEPARTMENT OF PHYSIOLOGY & BIOPHYSICS, INSTITUTE OF BIOMEDICAL SCIENCES - UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL.
Introduction: It is now evident that periodontal disease has systemic consequences. We have previously described that NO participates of some of the effects of periodontal disease on remote organs such as heart and kidney, and that endothelial dysfunction and decreased contractile response to norepinephrine in vitro also occur in aorta. We thus decided to study the in vitro vasomotor response of the resistance mesenteric artery from rats with periodontitis.
Methods and Results: The protocol was approved by the local Ethics Committee (CEUA-ICB 170, book 2/113, 2011). Male Wistar rats (10-14 wk-old) were anesthetized and bilateral periodontitis was induced by placing a cotton ligature around the lower first molars (sham animals had the ligature immediately removed). After one week, the rats were killed and the mesenteric bed was dissected. The vessels were mounted on a wire myograph to evaluate the in vitro response to KCl, acetylcholine (ACh), phenylephrine (Phe), sodium nitroprusside (SNP), and also in the presence of the NOS inhibitor L-NAME, and the COX inhibitors indometacin, SC560 (COX-1) and NS398 (COX-2). Potency (pD2) and maximal response (Emax) values were calculated. No differences between the groups were found in terms of artery diameter, KCl- or Phe-induced contraction tension, or ACh-induced relaxation. However, SNP induced a more potent relaxing response in the vessels obtained from the animals with periodontitis in comparison with the Sham group (pD2: 8.1±0.4 vs. 7.0±0.1; P<0.05), and the pre-incubation of the vessels with SC560 reduced the potency of ACh-induced relaxation in the periodontitis group (pD2: 7.0±0.2 vs. 8.6±0.6; P<0.05). In addition, q-PCR analysis revealed increased iNOS gene expression in the animals with periodontitis (P<0.001).
Conclusion: During the early phase of the bilateral ligature-induced periodontitis in rats, functional changes may occur in the mesenteric artery smooth muscle, as evidenced by the increased endothelium-independent relaxant response to exogenous NO. Whether this response is related to iNOS upregulation and/or COX-1 metabolites, still remains to be investigated.
Financial Support: FAPESP, CNPq and CAPES.
LIPID DROPLETS ARE SITES OF THE MTOR PATHWAY IN COLON CANCER CELLS
NARAYANA FAZOLINI1; RICHARD HEMMI VALENTE2; JOÃO P.B VIOLA3; CLARISSA M MAYA-MONTEIRO4; PATRICIA TORRES BOZZA5.
1,2,4,5.FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL; 3.INCA, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Enhanced lipid droplet biogenesis with functions in inflammatory mediator production and cell proliferation has been described in adenocarcinoma of colon. However, the composition and functions of lipid droplets in cancer cells are still largely unknown. Lipid droplets were shown to compartmentalize a variable array of proteins depending on the cell and stimuli. Signaling-associated proteins including PI3K, ERK1, ERK2, p38 and PKC have been demonstrated to compartmentalize within lipid droplets, suggesting a key role for this organelle as a cytoplasmic domain with roles in intracellular signaling. The mTOR pathway has been described to play an important role on tumor growth. This pathway is up regulated in colon cancer cells, suggesting mTOR as an attractive target for cancer therapy. Here we aimed to determine the role of lipid droplets in colon cancer cells as sites for mTOR pathway localization and its role in signaling compartmentalization. Methods and results: CACO-2 cells were subject to sucrose gradient subcellular fractionation. Buoyant lipid droplet fractions purity was ascertained by the lack of LDH cytosolic contamination and lipid droplet enrichment was confirmed by the presence of the lipid droplet structural protein ADRP. By Western blot, mTOR and P70S6K proteins were shown to localize in lipid droplet fractions. The mTOR pathway localization in lipid droplets was evaluated by indirect immunofluorescence microscopy in intact colon cancer cells. For differential proteomics, proteins contained in the LD fractions isolated from Caco-2 cells were digested with trypsin and peptides were identified by orbitrap XL. Conclusion: Our findings place lipid droplets as dynamic and functional active organelles that are intracellular sites for mTOR pathway localization and might potentially have implications to the proliferative phenotype in adenocarcinoma of colon. Financial support: CNPq, FAPERJ, INCT-Cancer, CAPES-SUS.
LOW LEVEL LASER THERAPY INHIBITS INFLAMMATORY HYPERALGESIA AND NEURONAL ACTIVATION INDUCED BY BOTHROPS MOOJENI VENOM IN MICE
NIKELE NADUR ANDRADE1; CAMILA SQUARZONI DALE2; VICTORIA REGINA SILVA OLIVEIRA3; STELLA REGINA ZAMUNER4.
1,3,4.UNIVERSIDADE NOVE DE JULHO, SÃO PAULO - SP - BRASIL; 2.UNIVERSIDADE DE SÃO PAULO, SÃO PAULO - SP - BRASIL.
Introduction: Bothrops snake venoms induce severe local fast-developing effects such as pain, edema, hemorrhage and necrosis. Antivenom treatment reverses systemic effects induced by Bothrops snake venoms but is ineffective in neutralizing the local effects. Herein we evaluated the therapeutic potential of Low-Level Laser Therapy (LLLT) in affecting nociceptive behavior and neuronal activation induced by Bothrops moojeni venom (BmV) in mice.Methods and Results: Mechanical hyperalgesia and allodynia (Takasaki et al., J Pharmacol Exp Ther. 296:270, 2001.) and thermal hyperalgesia (Hargreaves et al., J. Pain. 32:77, 1988) of the hind paw were assessed in male Swiss mice (20- 25 g). Animals received a sub-plantar injection of crude BmV (1.0 μg diluted in 50 µl of sterile saline) and were evaluated for nociceptive behavior at 1, 3, 6 and 24 h after BmV injection. Control animals were injected with sterile saline and submitted to the same protocols. LLLT was applied at 30 min and 3 h after BmV injection (15 sec, 685 nm, 30 mW, 2.2 J/cm2, irradiated area of 0.2 cm2). Neuronal activation was evaluated by Fos immunoreactivity (Fos-IR) in slices of the spinal cord of mice at the 6th h of nociceptive evaluation. BmV induced significant mechanical hyperalgesia and allodynia when compared with baseline measures taken before the tests (of about 50 % of increase on nociceptive threshold for all evaluated times). LLLT inhibited both mechanical hyperalgesia and allodynia of mice in all evaluated times (of about 60 %). Same results were observed for thermal hyperalgesia. Regarding neuronal activation, BmV induced a significant increase of Fos-IR in the dorsal horn of the spinal cord of mice that decreased around 61% after laser treatment. Conclusions: LLLT inhibits mechanical and thermal hyperalgesia in mice probably by a direct action on spinal nociceptors thus suggesting that phototherapy may be clinically relevant in the treatment of the local effects induced by Bothrops venom. Financial support: FAPESP, 2012/09710-8, 2012/12428-2.
LUNG ALTERATIONS IN STREPTOZOTOCIN-INDUCED DIABETES RATS: EFFECTS OF CISSAMPELOS SYMPODIALIS EXTRACT TREATMENT
KARINA CARLA DE PAULA MEDEIROS1; FLÁVIO SANTOS SILVA2; TESSIO DAVID MEDEIROS3; ANYELE TAVARES PEREIRA4; RAUL HERNANDES BORTOLIN5; KARINA VERISSIMO MEIRA6; NAISSANDRA BEZERRA SILVA7; BENTO AZEVEDO ABREU8; ADRIANA AUGUSTO REZENDE9; ALEXANDRO MARINHO10; MARIA JOSE BARBOSA FILHO11.
1,2,3,4,5,6,7,8,9.UFRN, NATAL - RN - BRASIL; 10,11.UFPB, JOÃO PESSOA - PB - BRASIL.
Introduction: Diabetes Mellitus (DM) is a chronic endocrine metabolic disorder characterized by hyperglycemia. Chronic complications affect many organs, including the lungs, reflected by abnormalities in lung function and morphological changes. Cissampelos sympodialis is a plant used in northeastern Brazil for the treatment of respiratory diseases. The present study was designed to evaluate the effect of the Cissampelos sympodialis extract (CSE) in the lungs of streptozotocin-induced diabetes rats. Methods and Results: We used 30 Wistar rats, and DM was induced by injecting streptozotocin (40mg/kg iv). CSE (400 mg / kg, po) was administered daily starting 35 days after disease onset. Clinical, biochemical profile and lung histological were analyzed in this study. Clinically, diabetic animals showed podipsia, weight loss, hyperglycemia and increased lipid profile; CSE treatment decreased cholesterol and triglyceride. The analysis of the histological slides in lung tissue of rats with DM revealed presence of an inflammatory process in lung, an increase in the extracellular matrix, as evidenced by the presence of fibrosis, as well as an increase in the thickness of the alveolar-capillary membrane, and CSE treatment was apparently able to reverse these histopathological findings. Conclusion: The Cissampelos sympodialis extract acute treatment was able to reduce the lipid profile, inflammation and contributed to lung tissue recovery in rats streptozotocin-induced diabetes. Financial support: CNPQ
MACROPHAGE CELLS AS MEDIATORS OF 15D-PGJ2–INDUCED ANTINOCICEPTION INTO THE TMJ
CRISTINA GOMES DE MACEDO1; JULIANA TRINDADE CLEMENTE NAPIMOGA2; LUIZ MARQUES DA ROCHA NETO3; MARCELO HENRIQUE NAPIMOGA4.
1,2,3.PIRACICABA DENTAL SCHOOL/UNICAMP, PIRACICABA - SP - BRASIL; 4.LABORATORY OF IMMUNOLOGY AND MOLECULAR BIOLOGY/SÃO LEOPOLDO MANDIC, CAMPINAS - SP - BRASIL.
Introduction: The 15d-PGJ2 have a potencial antinociceptive and antiinflamatory effect into temporandibular joint (TMJ) mediated by ppar-gama and kappa/delta opiod receptors. Thus the aim of the present work is to evaluate the interaction between PPAR-γ and κ /δ opioid receptors in15d-PGJ2-induced peripheral antinociception in the TMJ of rats. Methods and Results: Male Wistar rats (± 150 g, n=4-6/group) were pretreated (15 min) with an intra-TMJ injection of the PPAR-γ receptor antagonist GW9662 (3 ng/TMJ) followed by an ipsilateral intra-TMJ injection of 15d-
PGJ2 (100 ng/TMJ), or κ-opioid receptor agonist U50,488 (30 µg/TMJ), or δ-opioid receptor E6264 (10 µg/TMJ) 15 min prior to an ipsilateral intra-TMJ injection of 1.5% formalin. An additional group of animals was pretreated with the inducer of macrophage cells (1% Thioglycollate; 30 µl/TMJ/3 days) followed by an ipsilateral intra-TMJ injection of
15d-PGJ2 (100ng/TMJ) 15 min prior to an ipsilateral intra-TMJ injection of 1.5% Formalin. Animals’ nociceptive behavior was observed during 45 minutes and then they were killed by deep anesthesia and their periarticular tissue removed to evaluate the release of Dynorphin and β-Endorphin by ELISA. The intra-TMJ injection (15d-PGJ2, U50,488 and E6264) significantly reduced formalin-induced nociceptive behavior when compared with the vehicle (p<0.05: ANOVA, Tukey’s test). GW9662 could block only the 15d-PGJ2-induced antinociception suggesting that PPAR-γ has no effect on antinociception induced by κ/δ opioid receptors. ELISA analysis suggested that the 15d-PGJ2-induced antinociception resulted from the release of dynorphin and β-endorphin on periarticular tissues. Thioglycollate significantly increased the release of endogenous opioids (p<0.05: ANOVA, Tukey’s test). Conclusion: The 15d- PGJ2 induced TMJ antinociception resulting from the release of dynorphin and β-endorphin, partly mediated by macrophages.
Financial Support: CAPES- Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
MARCGRAVIACEAE-ORIGINATED COMPOUNDS DEMONSTRATE ANTIVIRAL AND IMMUNOMODULATORY ACTIVITIES AGAINST DENV-2 IN AN HUMAN HEPATOCYTE CELL LINE (HUH-7) AND PRIMARY HUMAN MONOCYTES INFECTED IN VITRO
LUCIANA GOMES FIALHO; RAIMUNDO SOUSA LIMA JÚNIOR; VAGNER PEREIRA DA SILVA; CÍNTIA MELLO; GLADYS CORRÊA; MARIA RAQUEL FIGUEIREDO; CLAIRE FERNANDES KUBELKA.
FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Immunoderegulatory disorders have been long treated by herbal medicine, modulating p. ex., cytokine production. However, little is known about the use of medicinal plants in dengue. The identification of compounds with immunomodulatory properties would be relevant for dengue treatment, since the disease severity is associated to an exacerbated production of the immunological response, mainly cytokines that may lead to hemodynamic and coagulation disorders (Cell and Mol. Life Sciences 67: 2773, 2010). Marcgraviaceae, a family of the Brazilian flora, present an anti-inflammatory effect. The aim of this work was to determine the antiviral and immunomodulator potential of a Marcgraviaceae sp. Methods and results: An hepatocyte cell line (HuH-7) was infected with DENV-2 (strain 16681) and incubated with a buthanolic fraction or four of its subfractions, derived all from a leave crude ethanolic extract of a Marcgraviaceae sp. A screening for their antiviral activity by detection of viral antigen (Ag DENV), using flow cytometry and the immunomodulatory activity, by ELISA detecting MIF levels, was performed using the supernatant of infected and treated cultures. The results of screening indicated that the buthanolic fraction and its subfraction 89-98 presented both activities (n=3), being selected to be better characterized. For this, the HuH-7 cells and primary human monocytes were evaluated, for the production of nitric oxide (NO) by Griess reaction, and viral protein NS1 detection, by ELISA. The immunomodulator action was evaluated by IL-8 measurement by ELISA as well. We observed that the buthanolic fraction and its subfraction 89-98 were also able to reduce NS1 production ratio, in both cell types (29.8 % of reduction in HuH-7 and 25.9% of reduction in monocytes). Furthermore, our preliminary results demonstrate that this fraction and its subfraction are stimulating NO production by infected HuH-7 cells and primary human monocytes. Still, it was possible to observe the IL-8 reduction (15.6% of reduction) in the hepatocyte cell line, induced by 89-98 subfraction. Conclusion: Compounds studied are good candidates for the development of herbal medicines for dengue treatment, since they generate an antiviral response and modulate the production of cytokines related to disease severity. They will be further studied to elucidate details in the mechanism of action. Financial support: PROEP-IOC & RPT 11D/FIOCRUZ; CAPES; CNPq and FAPERJ.
MAST CELLS AND HISTAMINE RELEASE CONTRIBUTE TO THE LOCAL REACTION INDUCED BY POTAMOTRYGON MOTORO STINGRAY VENOM IN MICE
LOUISE FAGGIONATO KIMURA1; JOSÉ PEDRO PREZOTTO-NETO2; MARTA MARIA ANTONIAZZI3; SIMONE GONÇALVES SILVA JARED4; ELIANA FAQUIM MAURO5; NICOLE ASSIS PEREIRA6; BIANCA DE CARVALHO LINS FERNANDES TÁVORA7; CATARINA TEIXEIRA8; MARCELO LARAMI SANTORO9; KATIA CRISTINA BARBARO10.
1,2,5,6,7,10.LABORATORY OF IMMUNOPATHOLOGY - INSTITUTE BUTANTAN, SÃO PAULO - SP - BRASIL; 3,4.LABORATORY OF CELL BIOLOGY - INSTITUTE BUTANTAN, SÃO PAULO - SP - BRASIL; 8.LABORATORY OF PHARMACOLOGY -INSTITUTE BUTANTAN, SÃO PAULO - SP - BRASIL; 9.LABORATORY OF PATHOPHYSIOLOGY - INSTITUTE BUTANTAN, SÃO PAULO - SP - BRASIL.
Introduction: Stingray accidents cause an intense pain followed by edema, erythema and necrosis formation. The therapy is based on administration of analgesic, antipyretic and anti-inflammatory drugs. The aim of this work was to verify the participation of mast cells (MC) and histamine on inflammatory reaction induced by injection of Potamotrygon motoro venom (PmV).
Methods and Results: Mice were pretreated with either cromoglycate (MC inhibitor), or promethazine, cimetidine, or thioperamide (histamine receptor antagonists) at selected time intervals before PmV injection. PBS treatment was used in control group. PmV (8 µg/ 30 µL) or PBS were injected into mouse footpad to evaluate edema formation (0.25, 0.5, 1, 4, 24 and 48 h) and cell recruitment (4 h). Presence of MC was confirmed by histological analysis. Pretreatment with cromoglycate diminished edematogenic activity in all periods analyzed, reaching 62 % of inhibition at 30 min. A decrease of edema forming was also verified in mice treated with promethazine or thioperamide after PmV injection. Moreover, treatment with cromoglycate, promethazine, or thioperamide diminished local PmV-induced leukocyte infiltration, mainly of neutrophils (15, 20 and 30 % respectively). Cimetidine neither affected edema nor cell influx induced by PmV. Differential cell count demonstrated that cromoglycate, promethazine, or thioperamide were responsible for the decrease in neutrophil influx. PmV (8 µg) also induced degranulation of PT-18 (mouse MC line) and RBL-2H3 (rat basophilic leukemia) cell lineages with no effect on cells viability.
Conclusion: This is the first report demonstrating participation of mast cells and histamine in the local inflammatory response induced by freshwater stingray venom. The use of antihistaminic drugs may be a new strategy to ameliorate the symptoms of stingray envenomation.
Supported by FAPESP (grant #2012/00166-3), CAPES and INCTTox.
MECHANISMS UNDERLYING THE PROTECTIVE EFFECT OF H2S DONORS AGAINST CARRAGEENAN- INDUCED KNEE JOINT SYNOVITIS
FILIPHE DE PAULA NUNES MESQUITA; LEANDRO RODRIGUES; BRUNA CAETANO DOS SANTOS; CRISTIANE ISABEL SILVA ZANONI; JULIANA FLORENZANO MARTORELLI; KAREN TIAGO DOS SANTOS; SIMONE APARECIDA TEIXEIRA; MARCELO NICOLÁS MUSCARÁ; SORAIA KÁTIA PEREIRA COSTA.
UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL.
Introduction: Controversy exists as to whether H2S has a direct protective or deleterious effect on arthritis. Although we have evidence from our previous study that exogenous H2S delivered to the knee joint produces a significant anti- inflammatory and anti-nociceptive effect against carrageenan (CGN)-induced joint synovitis (Br J Pharmacol;159(7):1463-74,2010), the mechanisms involved are unknown.
Methods and Results: To investigate this, synovitis was induced by intra-articular injection of CGN (3%, 50µl/cavity, n=8) into the knee joint of Wistar rats previously treated with a classical H2S donor, Lawesson reagent [LR, 3.6 µmol/cavity; n=8]. A separate set of rats with synovitis and treated with LR was also pretreated with glibenclamide + (antagonist at the sulfonylurea receptor 1 (SUR1) regulatory sub-unit of ATP-sensitive K (KATP) channels, 40mg/kg, i.p. n=8), capsazepine (transient receptor potential vanilloid 1 antagonist TRPV1, 25 μg/kg, s.c., n=8), verapamil (L- type Ca2+ channel blocker, 25 mg/kg, i.p. n=8) or its vehicle. Both nociceptive (Secondary Tactile Allodynia) and inflammatory parameters (Joint swelling and MPO acitivity) were assessed 4h later. Animal procedures were approved by the local Ethics Committee and injections were performed under isoflurane anaesthesia. As expected, LR significantly attenuated CGN-induced nociceptive response of the ipsilateral hindpaw (-33,6±10 and -7.7±10 Δg for control and LR) and related inflammatory process: joint swelling (3,4±0,9 and 1,7±1,0 Δcm for control and LR) and MPO activity (124±35 and 57±18 unity of MPO/ g tissue for control and LR) . Suppression of CGN-induced synovitis + and nociception by H2S donor was not reversed by the blockade of K (KATP), TRPV1 or L-type Ca2+ channels. CGN- induced knee oedema and nociception was suppressed by capsazepine (1,39±0,66 Δcm for oedema and -5,8±15 Δg for secondary tactile allodynia) or verapamil (2,2± 0,9 Δcm for oedema and 10,3±12,8 Δg for secondary tactile allodynia) alone to the same extent as by LR. Compared with control group, determinants of oxidative stress (glutathione S-transferase and glutathione reductase) in the rat synovial membrane with synovitis were similar among groups.
Conclusions: These findings demonstrate that H2S donor LR-induced protective effect against CGN-mediated 2+ synovitis and referred allodynia does not seem to require activation of either KATP and L-type Ca channels or TRPV1 in this model. The protective involved mechanism of LR against CGN-induced synovitis remains to be elucidated.
Financial support: CAPES, CNPq, FAPESP (2011/16645-5), Irene Maria Gouvea and Maria Alice Barreto
METHYL GALLATE INHIBITS THE INFLAMMATORY REACTION IN ZYMOSAN-INDUCED ARTHRITIS IN MICE
LUANA BARBOSA CORREA1; TATIANA ALMEIDA PÁDUA2; MARIA DAS GRAÇAS HENRIQUES3; ELAINE CRUZ ROSAS4.
1,2,4.LABORATÓRIO DE FARMACOLOGIA APLICADO DE FAR-MANGUINHOS, FUNDAÇÃO OSWALDO CRUZ, FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL; 3.LABORATÓRIO DE FARMACOLOGIA APLICADO DE FAR- MANGUINHOS; INCT/IDN/CDTS, FIOCRUZ,, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune disease morphologically characterized by infiltration of inflammatory cells and hyperplasia of synovial tissue. This transformed tissue expands and mediates destruction of bone and cartilage (Nature. 423:356-61, 2003). The current therapies for RA involves NSAIDs or glucocorticoids, however there are still an urgent need of novel therapy, including molecules from natural products (IJPSR. 2:1116-34, 2011). Methyl gallate (MG) has been found in a wide variety of plants and previous results demonstrated its anti- inflammatory activity different models. Our preliminary results showed that oral treatment with MG has anti- inflammatory action in the model of paw edema and pleurisy induced by zymosan in mice. In pleurisy, the MG decreased cellular accumulation, protein extravasation, the production of LTB4, IL-6 and CXCL-1/KC. In this study we evaluated the effect of MG in mice experimental arthritis.
Methods and Results: Arthritis was induced by intra-articular injection (i.a.) of zymosan (zym. - 500 µg / cav.) in the femoral-tibial joint of male C57BL/6 mice 1 h after oral pretreatment (p.o.) with MG (7 mg / kg); control animals received sterile saline (25 µL, i.a.) and dexametasone (10 mg / kg) was used as reference drug. The synovial edema was evaluated in millimeters (mm) by measuring the diameter of the joint using a digital caliper 6 and 24 hours after stimulation. All protocols were approved by the Fundação Oswaldo Cruz animal Welfare Committee.The synovial cavity was washed with PBS / EDTA (300 µl). The i.a. injection of zymosan induced significant increase in the knee diameter and leukocyte influx, 6 and 24 h after stimulation. Oral administration of MG inhibited the synovial joint edema (6 h - 23,5 %; 24 h - 49,1 %), and neutrophils migration (6 h - 59,7 %; 24 h - 70,1 %). The pretreatment with MG was able to reduce significantly the production of TNF-α (45,8 %), IL-6 (53,1 %) IL-1β (60,4 %) and CXCL-1/KC (47,8 %) at 6 h after injection zym and IL-6 (45,2 %) and IL-1β (50 %) at 24 h after injection zym. The results are expressed as mean ± SEM using from 6 to 8 animals.
Conclusion: Taken together, our results demonstrate that MG has an anti-inflammatory effect in arthritis experimental induced by zymosan in mice.
Financial support: CNPQ
MICE LACKING ANNEXIN-A1 DISPLAY LITTERS SIZE AND SEX RATIO DISTORTION
ISABEL DAUFENBACK MACHADO1; JOSÉ ROBERTO SANTIN2; MARIA ANGÉLICA PERES3; CAMILLA MOTA MENDES4; JOÃO DIEGO AGOSTINI LOSANO5; MAURO PERRETTI6; SANDRA HELENA POLISELLI FARSKY7.
1,2,3,4,5,7.UNIVERSITY OF SÃO PAULO, SAO PAULO - SP - BRASIL; 6.WILLIAM HARVEY RESEARCH INSTITUTE, LONDON - REINO UNIDO.
Introduction: Recent studies have revealed Annexin-A1 (ANXA1) as an important signaling in a variety of other system. We have been observed that ANXA1-/- mice present distortion on the sex ratio of offspring at birth with altering litter size. These data lead us to screening the differences between ANXA1-/- and wild type (WT) on sperm analysis and profile of cytokines in seminal fluid and uterine fluid after copulation.
Material and Results: Male adult Balb/C ANXA1-/- or WT mice were anesthetized to collect the seminal vesicle and epididymis. Sperm released into 245 μl of NaCl 0.9% (10 min, 37oC) was evaluated by Computer-assisted sperm analysis (CASA). Female adult Balb/C ANXA1-/- or WT mice were anesthetized to collect uterine fluid 12 hours after copulation to quantify IL-6, IL-10, IFN-γ, TNF-α and MCP-1 by flow cytometry (CBA). ANXA1-/- delivered higher number of pups per litter and female pups than WT mice. Data obtained on CASA analysis did not show any alteration on sperm quality and motility. Levels of IL-10 were drastically reduced in the seminal vesicle fluid of ANXA1-/- mice, without any alteration in pro-inflammatory cytokines. However, uterine fluid from the ANXA1-/- animals presented elevated and reduced levels of pro-inflammatory cytokines and IL-10 secretion, respectively.
Results and Conclusions: Data obtained show that the seminal and uterine environment of ANXA1-/- animals presents different levels of cytokines, which may be crucial to distort the sex ratio and litters size. This hypothesis has been investigated.
Financing: FAPESP (10/08402-2 and 10/16828-0).
MORITA-BAYLIS-HILLMAN ADDUCTS SHOWN IN VITRO ANTI-INFLAMMATORY ACTIVITY ASSOCIATED WITH IL-1, IL-6 AND NITRIC OXIDE REDUCTION
GLAUCIA VERÍSSIMO FAHEINA-MARTINS1; JACQUELINE ALVES LEITE2; CLAUDIO GABRIEL LIMA-JÚNIOR3; MÁRIO LUIZ ARAÚJO DE ALMEIDA VASCONCELLOS4; SANDRA RODRIGUES MASCARENHAS5; DEMETRIUS ANTONIO MACHADO ARAÚJO6.
1.CESED- CENTRO DE ENSINO SUPERIOR E DESENVOLVIMENTO-UFPB, JOÃO PESSOA - PB - BRASIL; 2,3,4,5,6.UFPB, JOÃO PESSOA - PB - BRASIL.
Introduction: Morita-Baylis-Hillman Adducts (MBHA) are a novel group of the synthetic molecules that have demonstrated many biological activities in literature as antiparasitary against Plasmodium falciparum, Leishmania amazonensis and Leishmania chagasi parasites. Furthermore, MBHA molecules showed antimitotic activity on sea urchin embryonic cells, suggesting a potential anticancer effect. Our group has demonstrated that these molecules induce a potent cytotoxicity on human tumor cells such as, acute and chronic leukemia, breast and colon cancers, and several lines of evidence suggest a strong association between chronic inflammation and increased susceptibility or progression of cancer. However, little is known about the mechanisms induced by MBHA in inflammatory process and its relation with anticancer activity. Aim: This study aimed to investigate a possible anti-inflammatory activity of MBHA (A2CN, A3CN and A4CN) in lipopolysaccharides (LPS)-stimulated RAW264.7 cells. Materials and methods: RAW264.7 cells were incubated at 5 x 104 cell/well in 96-well plates for one hour. Then, cells were cultured with MBHA isomers (A2CN, A3CN and A4CN) at (0 μM, 2.5 μM, 5 μM, 10 μM and 20 μM) in the presence of 1 μg/mL LPS for 24h. Nitric oxide (NO) production was measured using Griess reagent and pro-inflammatory cytokines such as, interleukin-1 (IL-1β) and interleukin-6 (IL-6) were assayed using ELISA kits. Results and Discussion: A2CN, A3CN or A4CN compounds which were co-incubated with LPS reduced the cell viability at all tested concentrations. Treatment using those compounds without LPS also showed the same response. Therefore, co-incubation with A2CN, A3CN or A4CN molecules added with LPS inhibited NO production at lowest concentration (p<0.001) and also decreased IL-1β and IL-6 levels (p<0.001). Conclusion: At subcytotoxic concentrations, the A2CN, A3CN and A4CN adduct isomers inhibited NO, IL-1β and IL-6 production induced by LPS. We conclude that these MBHA molecules showed anti-inflammatory properties.
Support: CNPq and CAPES
N-ACYLHYDRAZONE DERIVATIVE LASSBIO-294 SUPPRESSES LUNG INFLAMMATION CAUSED BY INTRANSAL SILICA IN MICE.
YAGO AMIGO PINHO JANNINI DE SÁ1; TATIANA PAULA TEIXEIRA FERREIRA2; ANA CAROLINA SANTOS DE ARANTES3; BIANCA TORRES CIAMBARELLA4; ELIEZER JESUS DE LACERDA BARREIRO5; CARLOS ALBERTO MANSSOUR FRAGA6; MARCO AURÉLIO MARTINS7; PATRICIA MACHADO RODRIGUES E SILVA8.
1,2,3,4,7,8.OSWALDO CRUZ INSTITUTE/FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL; 5,6.FEDERAL UNIVERSITY OF RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Silicosis is an occupational disease caused by prolonged inhalation of dust containing free silica particles and is characterized by an intense pulmonary inflammation with formation of fibrotic nodules. There is no effective treatment for fibrotic diseases until now. In this study we investigated the effect of the 1,3-N-acylidrazonic benzodioxolic derivative LASSBio-294 on the experimental silicosis in mice. Methods and Results: Swiss-Webster mice were intranasally instilled with silica (10 mg) and analyzes performed after 28 days. Animals received daily administration of LASSBio-294 (1 - 25 mg/kg, p.o.) for 7 days, starting 21 days after stimulation with silica. Lung function was evaluated by invasive plethysmography (Buxco System) and classical histological techniques were used including H&E and picrus Sirius staining. Quantification of collagen deposition and of cytokine/chemokine generation was made by Sircol and ELISA, respectively. Lung fibroblast proliferation was evaluated in vitro by means of 3H thymidine incorporation. Therapeutic treatment of silicotic mice with LASSBio-294 (1, 5 and 25 mg/Kg) inhibited the increased lung resistance and elastance as well as airways hyperreactivity to methacholine, when compared to silicotic group. Granuloma formation was sensitive to treatment with the compound. Values of granuloma area were 37.5% ± 7.2%; 20.5% ± 3.4%; 14.4% ± 4.6%; and 17.4% ± 3.1%; (mean ± SEM; n=7, p<0.05), in silica and silica- stimulated mice treated with 1, 5 and 25 mg/kg of LASSBio-294, respectively. In addition, collagen deposition and the increased expression α-smooth muscle actin positive cells were also suppressed by the LASSBio-294. The generation of proinflammatory and profibrotic cytokines (TNFα) as well as chemokines (MCP-1, KC and TARC) in the lungs of silicotic mice was sensitive to LASSBio-294. The in vitro analysis revealed that lung fibroblasts from silicotic animals were also sensitive to previous incubation with LASSBio-294. Conclusions: Our results show that compound LASSBio-294 inhibited the inflammatory response and fibrogenesis, including granuloma formation, caused by silica particles in the lungs of mice, by a mechanism which may be dependent on its suppressive activity on some important target cells such as fibroblasts.
Financial Support: FIOCRUZ, CNPq, FAPERJ. INCT-INOFAR, PRONEX (Brazil)
NASAL INSTILLATION OF CISSAMPELOS SYMPODIALIS EXTRACT OR ITS ALKALOIDS INHIBIT ALLERGIC AIRWAY INFLAMMATION
GICIANE CARVALHO VIEIRA1; LAÉRCIA KARLA DIEGA PAIVA FERREIRA2; HERMAN FERREIRA COSTA3; TAMIRES SENA4; CLAUDIO ROBERTO BEZERRA DOS SANTOS5; MARCIA REGINA PIUVEZAM6.
1.LABORATÓRIO DE IMUNOFARMACOLOGIA, DEPARTAMENTO DE MORFOLOGIA, CENTRO DE CIÊNCIAS DA SAÚDE, UFPB, JOÃO PESSOA - PB - BRASIL; 2,3,4,5,6.LABORATÓRIO DE IMUNOFARMACOLOGIA, DEPARTAMENTO DE FISIOLOGIA E PATOLOGIA, CCS, UFPB, JOÃO PESSOA - PB - BRASIL.
GICIANE CARVALHO VIEIRA(PQ)(1); LAÉRCIA KARLA DIEGA PAIVA FERREIRA(IC)(2); HERMAN FERREIRA COSTA(PQ)(2); TAMIRES SENA(IC)(2); CLAUDIO ROBERTO BEZERRA DOS SANTOS(PQ)(2); MARCIA REGINA PIUVEZAM(PQ)(2).
(1).Laboratório de Imunofarmacologia, Departamento de Morfologia, CCS, UFPB. João Pessoa, Brazil; (2).Laboratório de Imunofarmacologia, Departamento de Fisiologia e Patologia, CCS, UFPB. João Pessoa, Brazil.
Introduction: Cissampelos sympodialis Eichl. (Menispermaceae) is a plant used in Northeastern Brazil to treat respiratory diseases such as asthma. Drugs administrated by nasal instillation are good options to treat respiratory illness due to local and fast action, low systemic exposition and side effects. The goal of this study was to evaluate nasal instillation of leave extract (AFL) and its alkaloids warifteine (WAR), methylwarifteine (MWA) and milonine (MIL) on ovalbumina (OVA) sensitized BALB/c mice. Methods and Results: Mice (n=4) were twice sensitized (i.p.) with OVA and, one h before OVA challenged, the animals were treated with nasal instillation with 40 mg/kg of AFL or 2 mg/kg of WAR, MWA or MIL. AFL, WAR, MWA or MIL treatments inhibited significantly (P<0.05) inflammatory cell migration mainly eosinophils (0.07±0.02; 0.26±0.07; 0.25±0.04; 0.26±0.08 respectively) into the bronchoalveolar lavage (BAL) as compared to non-treated animals (0.46±0.12). The cell infiltration into the lung tissue was inhibited by AFL, WAR and MWA (8.24±0.32; 10.04±0.25; 5.98± 0.17) as compared with non-treated animals (14.26±0.66; P<0.05). Unexpected, MIL treatment promoted increasing of inflammatory cells in the lung (16.70 ±0.78). Additionally, the treatments inhibited significantly (P<0.05) the production of mucus by goblet cells of the bronchial epithelium and decreased significantly (P<0.05) the number of TCD4+ and TCD8+ cells into the lung cavity. AFL or MWA treatments inhibited significantly (P<0.05) the IL-13 production however only AFL treatment decreased OVA-specific IgE titer. Conclusion: Therefore nasal instillation of AFL or its alkaloids down regulated biological parameters associated with respiratory allergy. Despite of further studies are still required until an herbal medicine of Cissampelos sympodialis can be indicated, these results support evidences that AFL could be potentially useful to treat of asthma symptoms.
Financial support: CNPq; CAPES.
NEW EXPERIMENTAL MODEL OF COMBINED IRINOTECAN AND 5-FLUOROURACIL-INDUCED INTESTINAL MUCOSITIS IN MICE
VENÚCIA BRUNA MAGALHÃES PEREIRA (PG)1; DEYSI VIVIANA TENAZOA WONG2; ANIELLE TORRES MELO (PG)3; EUDMAR MARCOLINO ASSIS-JÚNIOR (PG)4; GERLY ANNE DE CASTRO BRITO5; RONALDO ALBUQUERQUE RIBEIRO6; ROBERTO CÉSAR PEREIRA LIMA-JÚNIOR7.
1,2,3,4,6,7.DEPHARMENT OF PHISIOLOGY AND PHARMACOLOGY. FEDERAL UNIVERSITY OF CEARA, FORTALEZA - CE - BRASIL; 5.DEPHARMENT OF MORPHOLOGY. FEDERAL UNIVERSITY OF CEARA, FORTALEZA - CE - BRASIL.
Introduction: Intestinal mucositis (IM) is a common side-effect (80%) of first line anticancer regimens for colorectal cancer, which includes FOLFIRI (5-Fluorouracil [5-FU] + Leucovorin + Irinotecan [IRI]). Despite the clinical use of polychemotherapy for cancer treatment, the pathogenesis of IM has been largely investigated in animal models that use these drugs given alone. However, the course of IM may vary according to the drug and regimens employed. Then, we aimed to develop a new experimental model of IM induced by the combination of IRI and 5-FU in mice. Methods and Results: C57BL/6 mice (20-25g, n=6) were injected with saline (5 mL/kg, i.p), IRI (30 or 45 mg/kg, i.p), 5-FU (25, 37.5 or 50 mg/kg, i.p) or IRI+5-FU for 4 days. Survival rate was obtained. On day 7, diarrhea, weight loss, and blood leukocyte count were registered. Following animal euthanasia, ileum samples were collected for histopathological analysis, myeloperoxidase activity (MPO, neutrophils/mg protein), TNF-a and IL-6 levels (pg/mg tissue). Kaplan-Mayer log rank test, Kruskal Wallis/Dunn’s or ANOVA/Bonferroni’s test was used for statistical analysis. P<0.05 was accepted. (CEPA 76/11). The best dose combination able to induce IM with no important mortality on day 7 was 5-FU (37.5 mg/kg)+IRI (45 mg/kg) (100% survival), which was then used for subsequent studies. However, on day 9, 0% survival was seen for this group (P<0.05 vs saline). IRI+5-FU induced (p<0.001) diarrhea (2[0-3]), weight loss (86.7±3.9), and leukopenia (7.3± 2.3) versus saline group (0[0-1]; 101.1±0.6; 215.5± 54.1, respectively) or each drug given alone (5-FU: diarrhea (0[0-1]), weight loss [92.6±2.7], and leukopenia [30.4± 13.4]; IRI: diarrhea (0[0-1]), weight loss [94.8±2.1], and leukopenia [49.2± 5.5]). In addition, IRI+5-FU induced inflammatory cells infiltration, and loss of villi and crypt architecture (4[3-4]), increased in MPO activity (14641±1598), TNF-a (3.2±0.9), IL-6 (1.4±0.5) tissue levels versus saline-injected group (0[0-1], 5747±1155; 0.7±0.2; 0.3±0.1) or the drugs injected alone (5-FU: Intestinal damage (2.5[2-3]), MPO [3788±1212], TNF-a [0.7±0.2], IL-6 [0.2±2.3]; IRI: Intestinal damage (1[0-2]), MPO [3580±1613], TNF-a [0.4±0,2], IL-6 [0.07±0.05]) (P<0.05). Conclusion: We developed a new experimental model of IM induced by the combination of IRI+5-FU in mice, which opens perspective for a more appropriate knowledge concerning the pathogenesis of IM. Financial Support: CNPq/CAPES/FUNCAP.
NEW METHOD FOR EVALUATION OF ARTICULAR DISABILITY IN EXPERIMENTAL ARTHRITIS: INVESTIGATION THE ROLE OF GLIAL CELLS
ANDREZA URBA DE QUADROS; MIRAM DAS DORES MENDES FONSECA; LARISSA GARCIA PINTO; SERGIO HENRIQUE FERREIRA; THIAGO MATTAR CUNHA.
RIBEIRÃO PRETO MEDICAL SCHOOL - UNIVERSITY OF SAO PAULO, RIBEIRAO PRETO - SP - BRASIL.
Introduction: The evaluation of articular nociception and disability is a challenge. The methods available so far have been limited and subject to analyzer influence. Searching for help in this problem, the purpose of this work was standardize the use of dynamic weight bearing (DWB), as a new device, for assessment of articular nociception and disability in experimental models of arthritis. In this system, the animal can walk freely and without analyst interference by 5 minutes in a platform with sensors, which capture the difference between the weight distribution of each animal limb. In rheumatoid arthritis (RA) the pain is severe in 60% of patients (CORBACHO & DAPUETO, 2010). Is a multifactorial pain and needs more researches that provide new treatments possibilities. One of them may is going to the control of glial cells activity. The involvement of glial cells in the chronic pain and disability in experimental models of arthritis have been reported, but there isn't description of this contribution in function of the time.
Objectives: Standardize DWB for evaluate articular nociception and disability in animal models of arthritis and investigate the role of glial cells in this process on function of the time.
Methods and Results: Animal care and handling procedures were in accordance the Animal Ethics Committee of the University of Sao Paulo. In AIA model DWB showed reduction until 43%±2,5% between 7 and 24 hours after the intrarticular challenge with mBSA. The disability proved was predominantly inflammatory, once four different anti- inflammatory drugs (indomethacin, dexamethasone, etoricoxib and infliximab) totally recovered the articular function. After that, there is a relation between pain and disability, since the treatment with morphine also restored the articular function. Employing pharmacological (minocycline and fluorocitrate) and molecular techniques, as real time PCR and Western Blotting (expression of GFAP and Iba1) was showed that there is a previous activation of satellite cells followed by astrocytes and microglia. The intrathecal treatment with IL-1Ra and infliximab, showed that this cells act by IL-1β and TNFα release in spinal cord and dorsal root ganglia, long term sensitizing the neurons.
Conclusion: The DWB is an effective and predictable method to study pain and disability in AIA model. Glial cells participate as both induction and maintenance of pain and disability in AIA model.
Financial support: CNPq and FAEPA
NITRIC OXIDE AND PROSTAGLANDINS INVOLVEMENT ON GASTROPROTECTIVE EFFECT OF MAYTENUS RIGIDA. IN ANIMAL MODEL OF GASTRIC LESIONS
SAMUEL MATEUS PEREIRA FILHO1; ÂNGELA MAGALHÃES VIEIRA2; LÍVIA CUNHA RIOS3; JONAS CAVALCANTE LEMOS4; JORDÂNIA MARQUES DE OLIVEIRA5; ALICE RAMOS DE FREITAS6; MIRNA MARQUES BEZERRA7; DEBORA DA SILVA FREITAS8; HELLÍADA VASCONCELOS CHAVES9; VICENTE DE PAULO TEIXEIRA PINTO10; MARIA BERNADETE DE SOUSA MAIA11; GERARDO CRISTINO FILHO12; ANTONIO ALFREDO RODRIGUES E SILVA13.
1,2,3,4,5,6,7,8,9,10,12,13.FEDERAL UNIVERSITY OF CEARÁ - SOBRAL CAMPUS, SOBRAL - CE - BRASIL; 11.FEDERAL UNVIERSITY OF PERNAMBUCO, RECIFE - PE - BRASIL.
Introduction: Maytenus rigida Mart. (Celastraceae) is a known species of northwest of Brazil popularly used to treat inflammatory diseases, rheumatism, renal problems and gastric disorders. Objective: investigate the participation of nitric oxide and prostaglandins on Maytenus rigida aquous extract (AE) gastro-protective effect in male Swiss mice. We used an ethanol-induced gastropathy experimental model. Methods and results: fasted mice (6/group) were pre- treated with AE (100, 200 or 400 mg/Kg, p.o.) 1 h before the ethanol (0.2 ml/animal) challenge. Control groups were treated with saline (0.5 ml/kg, p.p.) or ranitidine (80 mg/kg, p.o.), respectively. Different pharmacological tools (misoprostol, indomethacin, L-NAME, L-arginine) were added in different essays to clarify possible action mechanisms of AE. The animals were euthanized 30 min after ethanol administration to remove their stomachs. The excised stomachs were macro and microscopically analyzed. The protective effect of misoprostol (50 mg/kg, p.o.) was reversed by indomethacin (10 mg/kg, p.o.) administration. The damage induced by ethanol, in presence or absence of indomethacin, was significantly reduced (P < 0.05) in the group treated with AE (200 mg/kg) (20.7±3.6 versus 4.9±1.2 and 24.7±2.8 versus 5.2±2.2 % damaged area, respectively). Simultaneous administration of ethanol and L-NAME (20 mg/kg, i.p.) produced hemorrhagic bands that were absents in L-arginine treated (600 mg/kg, i.p.) groups. The AE (200 mg/kg) gastro-protective effect (1.9±0.5 versus 20.1±4.0) was reversed by previous treatment with L-NAME (1.9±0.5 versus 25.7±5.0). Conclusions: AE presents gastro-protective effects, corroborating with its popular use. The effects involve nitric oxide liberation, but do not depend on prostaglandins.
Financial Support: CAPES, CNPq, FUNCAP, UFC.
NORADRENALINE INJECTION INDUCES PERIPHERAL ANTINOCICEPTION BY ADRENERGIC, OPIOIDERGIC AND CANNABINOIDERGIC SYSTEM INTERACTION
JAYANE LAIS QUINTAO; WILLIAM ANTÔNIO GONÇALVES.
UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.
JAYANE LAÍS DIAS QUINTÃO(PG)1, WILLIAM ANTÔNIO GONÇALVES1, VINCENZO DI MARZO2, THIAGO ROBERTO LIMA ROMERO1 and IGOR DIMITRI GAMA DUARTE 1
1Laboratório de Dor e Analgesia, Instituto de ciências Biológicas, UFMG, Belo Horizonte, MG.
2Institute of Biomolecular Chemistry, Napoli, Italy.
Introduction: Knowing that noradrenaline is involved in intrinsic control of peripheral pain under interaction with the immune system, the aim of this study was verified the opioid and the cannabinoid system participation in the peripheral antinociceptive effect of NA. Methods and Results: The rat paw pressure test was used and hyperalgesia was induced by intraplantar injection of PGE2 (2 µg/paw). All drugs were administered intraplantar of Wistar rats (n>4).
NA (80 ng/paw) elicited a local inhibition of hyperalgesia. The α2 adrenoceptor antagonist yohimbine (20 µg/paw;
101.6±0.9), the α1 adrenoceptor antagonist prazosin (2 µg/paw; 101.6±0.9), the β adrenoceptor antagonist propranolol (600 ng/paw; 98.3±1.6), the opioid receptor naloxone (100 µg/paw; 96.5±3.0), the µ-opioid receptor antagonist clocinnamox (40 µg/paw; 93.7±1.5), the δ-opioid receptor antagonist naltrindole (60 µg/paw; 97.1±4.5) and the CB1 cannabinoid antagonist AM251 (80 µg/paw; 106.6±2.3) antagonized the antinociception effect induced by NA
(p<0.05), but not by the κ-opioid receptor antagonist NOR BNI (100 µg/paw; 15.9±2.1) and CB2 cannabinoid antagonist AM630 (100 µg/paw; 14.2±1.6) (p<0.05). Reboxetin (30 µg/paw; 3.5±1.4), a selective noradrenaline reuptaker, the anandamide amidase inhibitor MAFP (2 µg/paw; 10.0±4.0) and the anandamide reuptake inhibitor VDM11 (20 µg/paw; 5.8±2.5) increased the peripheral noradrenaline antinociception in low dose (p<0.05). The enkephalinase inhibitor bestatin potentiated the antinociceptive effect of NA (p<0.05). In additional, the dosage of endocannabinoids (AEA, 2-AG, PEA and OEA) indicated that noradrenaline induces a selective AEA release in the peripheral site with an increase twice in the lipid extract dosage. Conclusion: The results provide evidences that NA probably induces peripheral antinociceptive effect by activation of adrenoceptors in the resident immune cells to release β-endorphin or anandamide that activates selectively µ, δ-opioid receptors or CB1 cannabinoide receptor in peripheral site.
Financial support: CNPq, CAPES and FAPEMIG.
NOVEL CARTILAGE-PROTECTIVE ACTIVITIES IN PRO-RESOLVING EXUDATES
MAGDALENA K KANEVA1; PRASHANT MORI2; ADRIAN MOORE3; MAURO PERRETTI4.
1,4.WILLIAM HARVEY RESEARCH INSTITUTE, BARTS AND THE LONDON SCHOOL OF MEDICINE, QMUL, LONDON - REINO UNIDO; 2,3.UCB CELLTECH, SLOUGH - REINO UNIDO.
Introduction: We hypothesized that tissue protective activities might be produced during the resolution phase of an experimental acute response. Application of an integrated approach with experimental exudates, proteomic analyses and bioactions with human chondrocytes led to the identification of novel tissue protective mediators.
Methods: Rat pleural exudates (24h post-carrageenan) were subjected to size exclusion chromatography and run for mass spectrometry. C28/I2 chondrocytes were grown in 3D high-density micromass (1), incubated with the fractions (optimal dilution 1:30) in the presence or absence of interleukin(IL)-1b (20 ng/ml) or osteoarthritis synovial fluids (OA SF; 1:100) for 48h. Alpha-1-antitrypsin (ATT), gelsolin or hemopexin (0 – 30µg/ml) were tested against catabolic stimulation. Sulphated glycosaminoglycans (GAGs) deposition was assessed using Alcian Blue (AB) staining. Gene expression was quantified by qPCR. Experiments were run 3 times. One-way ANOVA was used for statistical analyses.
Results: Whilst large molecular weight (MW) fractions inhibited (≤60%) GAG deposition, low MW fractions were strongly anabolic with >60% increase in GAG deposition mirrored by ~25% increase in COL2A1 and ACAN gene expression (p<0.05). Low MW fractions reverted the catabolic effect of IL1b. Proteome analysis of rat exudates revealed 66 proteins including hemopexin, gelsolin and AAT. Exogenous AAT abrogated the effect of IL1b on MMP13 (8-fold increase over control; p<0.001). Gelsolin and Hemopexin also afforded anabolic properties.
Incubation of chondrocyte micromasses with OA SF augmented IL6 and MMP13, with concomitant downregulation of COL2A1 and ACAN, gene products (p<0.01). At 10µg/ml, ATT attenuated these effects (p<0.01). The AB assay for GAG deposition confirmed the chondroprotection afforded by these proteins, with GAG deposition being disrupted by OA SF (20% of control) and marked reversal (to ~80% of control) with ATT, gelsolin and hemopexin (p<0.01).
Conclusion: The strategy for identification of novel chondroprotective activities in resolving exudates elicited new mediators like AAT, gelsolin and hemopexin as opportunities for new discovery programs.
Funded by a collaborative project between UCB Celltech and QMUL.
1. Greco et al., PMID: 21946086
OPPOSING EFFECTS OF TOLL-LIKE RECEPTOR (TLR)-4 IN SPINAL CORD INJURY AND BRAIN TRAUMA
EMANUELA ESPOSITO; IMPELLIZZERI DANIELA; CAMPOLO MICHELA; PATERNITI IRENE; SALVATORE CUZZOCREA.
UNIVERSITY OF MESSINA, MESSINA - ITÁLIA.
BACKGROUND: Our knowledge of Toll-like receptor (TLR) expression and function in the central nervous system (CNS) has greatly expanded, with new data revealing that TLRs also have an impact on non-infectious CNS diseases/injury. In particular, TLRs recognize a number of endogenous molecules liberated from damaged tissues and influence inflammatory responses during tissue injury and autoimmunity. TLR signalling elicits the production of cytokines, enzymes and other inflammatory mediators that can have an impact on several aspects of CNS homoeostasis and pathology. However, TLRs can exert either beneficial or detrimental effects in the CNS, which probably depend on the context of tissue homoeostasis or pathology. Here, we examined the effects of targeted deletions of TLR-4 on spinal cord and brain trauma. METHODS: The lumbar spinal cords from C57BL/10ScNJ WT mice, and C57BL/10ScNJ TLR-4 knockout (KO) mice were studied after the application of an aneurysm clip to the dura via a four-level T5-T8 laminectomy, replicating the persistence of cord compression commonly observed in human SCI. Moreover, controlled cortical impact injury was performed on TLR-4KO and respective WT mice. Mice were sacrificed at 24 hours after SCI or brain trauma, and spinal cords or brains were processed for immunohistochemistry, western blotting, cell culture, and reverse transcriptase PCR. RESULTS: The results showed that TLR-4 expression in the CNS may have opposite effects on the stability of spinal cord and brain after damage. Infarct size, neurological deficits and neuronal damage were significantly attenuated in TLR-4KO mice compared with WT animals following brain trauma. On the contrary, deficiencies in TLR-4 signalling exacerbated post-traumatic accumulation of CNS macrophages that was accompanied by functional impairment and excessive tissue pathology. CONCLUSION: These studies implicate TLRs in exerting detrimental effects following brain trauma, while TLRs may represent a secondary adaptation to attenuate inappropriate immune responses following spinal cord acute injury. Although TLR-4 signalling also induces macrophage activation within the spinal cord, the nature of the TLR stimulus may dictate neuroprotective compared with neurodestructive outcomes.
PARTIAL CHARACTERIZATION OF CAMELID NANOBODIES AGAINST BOTHROPSTOXIN-II, A MYOTOXIN FROM BOTROPHS JARARACUSSU
MICHELE PEREIRA DA SILVA; NIDIANE DANTAS REIS PRADO; SORAYA DOS SANTOS PEREIRA; ANDERSON MAKOTO KAYANO; ANDREIMAR MARTINS SOARES; RODRIGO GUERINO STABELI; CARLA FREIRE CELEDÔNIO FERNANDES.
FUNDAÇÃO OSWALDO CRUZ, PORTO VELHO - RO - BRASIL.
Introduction: Envenomation by snakebites represents a relevant public health problem in tropical countries due to their severity and sequeale. In Brazil, about 73.5% of reported accidents are caused by snakes of the Bothrops genus. The bothropic venom consists of several bioactive peptides and proteins that can cause systemic and local effects, as blood coagulation, hemorrhage, edema, and tissue necrosis. Snakebite therapy is performed using equine hyperimmune serum, which demonstrates low efficacy in reversing the local effects of envenoming. Camelids produce functional immunoglobulins devoid of light chains, in which the antigen binding site is formed by the single domain called VHH or nanobody. Besides their small size, high solubility, and better ability to recognize sites normally inaccessible to conventional antibodies, nanobodies are not affected by variations in temperature and pH, which are important advantages in field treatment. So, this work aimed to produce and characterize VHHs against the bothropstoxin II (BthTX-II), a myotoxin isolated from the Bothrops jararacussu venom.
Metodology and Results: To produce nanobodies, the phage display technology was used. VHHs regions were isolated by PCR using peripheral lymphocyte cDNA obtained from one camelid previously immunized with BThTX-II. The amplicons were recombined into a phagemid using TG1 E. coli strain to construct a phage antibody immune library. After infection by M13K07 helper phage, VHHs were displayed fused to phage coat protein III and the selection step was performed with immobilized BThTX-II. Six clones recognized BThTX-II specifically by ELISA. After purification by affinity chromatography, all clones were able to recognize specifically venoms obtained from snakes of the Bothrops genus. Further experiments aiming to verify the capability of selected VHHs to neutralize BThTX-II and the venom activity are being performed.
Conclusion: The identified VHHs could be a new tool to help the treatment of envenoming caused by B. jararacussu, or by other snakes of the Bothrops genus.
Financial support: CNPq , CAPES
PATHOPHYSIOLOGY OF REDUCED SALIVARY FLOW RATES IN HAMSTERS WHICH UNDERWENT 5-FU TREATMENT AND EXPERIMENTAL ORAL MUCOSITIS
LUANA ESCHHOLZ BOMFIN1; DANIELLE ABREU FOSCHETTI2; INGRID SAMANTHA TAVARES FIGUEIREDO3; RENATA FERREIRA LEITÃO4; GUTENCILDA COLARES VASCONCELOS5; GERLYANNE CASTRO BRITO6.
1,2,4,5,6.UNIVERSITY OF CEARA, FORTALEZA - CE - BRASIL; 3.UNIVERSITY ESTÁCIO DO CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Xerostomia (manifestation of reduction in salivary flow) and oral mucositis (OM) are common and important oral manifestations associated with chemotherapy treatment (CT) which can affect the quality of life of patients. The pathophysiology of these two events is not fully understood. This study aims to evaluate morphophysiological changes in major salivary glands (parotid, sublingual and lingual) of hamsters undergoing OM induced by 5-Fluorouracil (5FU) chemotherapy. Methods and results: OM was induced by mechanical trauma on the cheeks of hamsters according to a pre-established model. We also verified the eficacy of 1400W (an inhibitor of the enzyme nitric oxide synthase - NOSi) in prevention on OM and on reduction of saliva. The animals were divided into saline group (S), trauma group (T), 1400W group and 5FU injected group plus mechanical trauma (5FU). The amount of saliva was measured using absorbent paper cones for the use in endodontics and salivary glands were removed for analysis on the 4th and 10th experimental day. The results demonstrated reduced salivary flow on 4th day after 5-FU injection and this redution was prevented by NOSi inhibitor 1400W on the 4th day. The analysis for toluidine blue staining showed a significant increase of total and degranulated mast cells on T group in submandibular salivary glands compared to S group both in 4th (n=10, p<0.0001; 8.83±0.48; 8.66±0.46 being total and degranulated mast cell respectively) and 10th (n=10, p<0.0001; 7.33±0.53 8.63±0.50, respectively) day. Mallory thicrome staining demonstrated increased number and area of blood vessels (p <0.05), observed in submandibular gland of 5FU group compared to S group. Conclusion: The above results demonstrate a possible interference of 5-FU in the pathophysiology of the submandibular gland. Financial support: This project was funded by CNPq/Capes.
PDE4 INHIBITOR ROLIPRAM INHIBITS TNF PRODUCTION AND CARRAGEENAN-INDUCED PAW INFLAMMATION BY ELEVATING MKP-1 THROUGH CAMP-PKA-CREB PATHWAY
TUIJA MARIA HÖMMÖ; MIRKA LAAVOLA; TIINA KERÄNEN; MARI HÄMÄLÄINEN; EEVA MOILANEN; RIKU KORHONEN.
THE IMMUNOPHARMACOLOGY RESEARCH GROUP, UNIVERSITY OF TAMPERE AND TAMPERE UNIVERSITY HOSPITAL, TAMPERE - FINLÂNDIA.
Introduction: Mitogen-activated protein kinase phosphatases (MKPs) are a subgroup of dual specificity phosphatases that regulate the phosphorylation of MAPK. MKP-1 expression is regulated by cAMP-PKA-CREB pathway, and MKP-1 suppresses p38 MAPK activity and serves as an endogenous factor limiting inflammatory response. Phosphodiesterase (PDE) 4 plays an important role in coordination of cAMP signalling by rapidly hydrolyzing cAMP in the immediacy of its production. PDE4 inhibitors prevent the hydrolysis of cAMP leading to the elevation of cAMP levels. In this study, we investigated the effect of a PDE4 inhibitor rolipram on MKP-1 expression and inflammatory gene expression, and whether MKP-1 would be involved in the anti-inflammatory effects of rolipram.
Methods: J774 mouse macrophages and primary mouse peritoneal macrophages (PM) from wild-type (WT) and MKP-1(–/–) mice were used. MKP-1 levels were determined by qRT-PCR and Western blot, and TNF levels were measured by qRT-PCR and ELISA. In J774 cells, MKP-1 expression was silenced by siRNA. p38 MAPK and CREB activation was detected by Western blot.
Results: Down-regulation of MKP-1 increased p38 MAPK phosphorylation, and enhanced the production of inflammatory cytokines TNF and IL-6. Inflammatory gene expression was increased in PMs from MKP-1(–/–) mice as compared to those from WT mice. Rolipram increased cAMP levels, CREB phosphorylation and MKP-1 expression, and those effects were reversed by a PKA inhibitor. Rolipram inhibited the phosphorylation of p38 MAPK and it was returned by the PKA inhibitor. Rolipram inhibited TNF production in PMs from WT mice, while rolipram was clearly less effective to inhibit TNF production in PM from MKP-1 (–/–) mice. The effect of rolipram on the severity of carrageenan-induced paw inflammation was investigated. Carrageenan-induced paw oedema was markedly attenuated by rolipram in WT mice, while rolipram did not have any effect on paw oedema in MKP-1(–/–) mice
Conclusion: PDE4 inhibitor rolipram increased MKP-1 by a mechanism dependent on cAMP-PKA-CREB pathway. The inhibition of TNF production and acute inflammation in vivo by rolipram was mediated by MKP-1. The results suggest that MKP-1 is an important factor in mediating anti-inflammatory effects of PDE4 inhibitors.
Financial support: Academy of Finland, The Competitive Research Funding of the Pirkanmaa Hospital District and FinPharma Doctoral Program-Drug Discovery (FPDP-D).
PENTOXIFYLLINE ROLE IN THE TREATMENT OF NON-ALCOHOLIC FATTY LIVER DISEASE IN AN ANIMAL MODEL OF OBESITY.
SIMONE COGHETTO ACEDO1; ÉRICA MARTINS FERREIRA GOTARDO2; CAROLINE CANDIDA DE OLIVEIRA3; CINTIA RABELO E PAIVA CARIA4; PATRÍCIA FERNANDES ZANOTTI NAKAMITSU5; ALESSANDRA GAMBERO6.
1,4,5.UNICAMP, CAMPINAS - SP - BRASIL; 2,3,6.UNIVERSIDADE SÃO FRANCISCO, BRAGANÇA PAULISTA - SP - BRASIL.
Introduction: Obesity predispose to the development of non-alcoholic fatty liver diseases (NAFLD) that encompasses non-alcoholic steatohepatitis (NASH), fibrosis and also hepatic cancer. There are no therapeutics options for treatment of NAFLD, although many have been studied with generally modest benefit. Pentoxifylline (PTX), a xanthine derivative, has been utilized as a treatment for alcoholic fatty liver diseases (AFDL) and its effects are related to decrease mortality in these patients. The aim of this study was to evaluate the PTX effects upon metabolic and inflammatory alterations associated to obesity in mice.
Methods and results: Swiss mice were fed with high fat diet (HFD) for 12 and 24 weeks, controls received normal diet (ND). In the last two weeks, mice were treated with 100 mg/kg/day of PTX (ND PTX and HFD PTX). During the treatment we assessed the body weight, insulin tolerance test (ITT) and glucose blood levels. The weight of adipose tissue and liver was determined and liver biopsies were used for cytokines measurements (IL-10 and TNF-α) by ELISA. The body weight after PTX treatment was reduced in 12 and 24 weeks of HFD. It had been observed changes in the epididymal adipose tissue (4.7±0.3 and 4.0±0.3 % of body weight for HFD and HFD PTX, respectively; p<0.05, n=5), in perirenal adipose tissue (0.39±0.04 and 0.30±0.03 % of body weight for HFD and HFD PTX, respectively; p<0.05, n=5) and also in the liver (4.7±0.2 and 3.3±0.1 % of body weight HFD and HFD PTX, respectively; p<0.01, n=5) in animals treated with pentoxifylline maintained on a high fat diet for 12 weeks. However, there were no changes in metabolic parameters (glucose blood levels and ITT) after PTX treatment. Liver TNF-α in obese mice treated with PTX (12 and 24 weeks) was lower than non-treated mice (4.6±0.9 and 2.0±0.4 pg/mg protein of TNF-a for HFD and HFD PTX, 12 weeks, respectively, p<0.05, n=5; 20±3 and 11±0.7 pg/mg protein of TNF-α for HFD and HFD PTX, 24 weeks, respectively, p<0.05, n=5). IL-10, an anti-inflammatory cytokine, was not modified by PTX treatment.
Conclusion: These results suggest that PTX could have a protective role in liver during obesity associated to reduction of TNF-α. A reduction of adiposity was also observed and more studies are necessary for a better comprehension of this effect.
Financial support: FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil.
PHARMACOLOGICAL EVALUATION OF A NEW SERIES OF SULFONAMIDE DERIVATIVES DESIGNED AS MODULATORS OF LUNG INFLAMMATION
EVERTON TENORIO DE SOUZA1; VINICIUS DE FRIAS CARVALHO2; TATIANA PAULA TEIXEIRA FERREIRA3; ISABELLE KARINE DA COSTA NUNES4; BIANCA TORRES CIAMBARELLA5; LIDIA MOREIRA LIMA6; ELIEZER JESUS DE LACERDA BARREIRO7; MARCO AURELIO MARTINS8; PATRICIA MACHADO RODRIGUES E SILVA9.
1,2,3,5,8,9.OSWALDO CRUZ INSTITUTE/FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL; 4,6,7.LASSBIO/FEDERAL UNIVERSITY OF RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL.
Introduction: The design and synthesis of a new series of sulphonamide derivatives planned by structural modification of the prototype LASSBio-448 have been described. LASSBio-448 was shown to be a phosphodiesterase (PDE) 4 inhibitor with therapeutic index and potency higher than of the standard inhibitor (R,S)-rolipram. In this study we evaluated the pharmacological profile of a new series of sulfonamide derivatives both in vitro and in vivo systems. Methods and Results: A series of 13 derivatives was first screened for anti-PDE 4 isoenzyme activity by IMAPTM TR- FRET system. The standard PDE4 inhibitor rolipram was used as control. The compounds were screened on LPS- activated alveolar macrophages (AMJ2C11) and TNF levels quantified by ELISA. For in vivo system, A/J mice (n=6) were instilled i.n. with LPS and the compounds were administered by oral route (25 - 100 µmol/kg), 1 h before. Analyses were made 24 h after LPS and included: i) lung function (resistance and elastance) and airways hyperreactivity to methacholine and ii) total and differential leukocyte counts in the bronchoalveolar lavage (BAL). We showed that at the concentration of 10 μM LASSBio-1612, LASSBio-1628, LASSBio-1631 and LASSBio-1632 inhibited PDE41A and PDE4D3 activity, but not PDE4B1 and PDE4C. The other compounds tested had no effect. Incubation of macrophages with the compounds and rolipram (0.1-10 μM), 1 h before LPS, reduced TNF release. Only LASSBio-1632 inhibited the inflammatory response caused by LPS in vivo, including increased lung resistance and elastance as well as airways hyperreactivity. Neutrophil infiltration in the BAL was also suppressed by LASSBio-1632 (values of neutrophils are 80.2 ± 11.2 x 103 cells/BAL, 603.8 ± 71.3 x 103 cells/BAL; 343.8 ± 54.6 x 103 cells/BAL (mean ± SEM) in saline-, LPS- and LPS-treated mice, respectively). Rolipram abolished the LPS-induced inflammation as expected. Conclusion: Our results show that screening of a new series of sulfonamide derivatives led us to identify LASSBio-1632 as a compound which had the ability to inhibit: i) PDE4A and PDE4D3 isoenzyme activity, ii) activation of macrophages in vitro, and iii) lung acute inflammatory response caused by LPS in mice. Altogether, these data indicate that LASSBio-1632 seems to be a promising anti-inflammatory compound, though additional experiments are needed to clarify better its pharmacological profile.
Financial support: FIOCRUZ, CNPq, FAPERJ, INCT-INOFAR.
PHARMACOLOGICAL STUDY OF JUNGIA SELLOWII LESS. IN A MURINE MODEL OF PLEURISY INDUCED BY CARRAGEENAN
MARINA NADER; GEISON VICENTE; ALYNE MACHADO BARBOSA; MAIQUE WEBER BIAVATTI; TÂNIA SILVIA FRÖDE.
FEDERAL UNIVERSITY OF SANTA CATARINA, FLORIANÓPOLIS - SC - BRASIL.
Introduction: Jungia sellowii Less. is a native plant from Brazil, used in folk medicine to treat inflammatory diseases. The aim of this study was to evaluate the anti-inflammatory effect of the Crude Extract (CE) of this plant and its derived fractions, Buthanol (n-BuOH) and Aqueous (Aq), upon leukocytes, exudation, myeloperoxidase (MPO) and Adenosine-Deaminase (ADA) activities, and nitric oxide (NOx) levels, using a murine model of pleurisy induced by carrageenan (Cg).
Methods and Results: Dried leaves of J. sellowii Less. were macerated in ethanol/water at room temperature to obtain the CE, which was partitioned with buthanol and water solutions to obtain the n-BuOH and Aq fractions. Swiss mice were used through the experiments in accordance with methodology described in Brit.J.Pharmacol.183.811- 19.1996. The study was approved by the Committee for Ethics in Animal Research of UFSC (protocol number: PP00757). The animals were injected with Evans blue dye (25 mg/kg, i.v.) before receiving the herb treatment (10 min), to evaluate the exudation. Different groups of animals (n=5) were treated with CE (10-50mg/kg), n-BuOH (10- 50mg/kg) or Aq (1-25mg/kg) administered by intraperitoneal route, 0.5h prior to intrapleural injection of Cg (1%). The leukocytes and exudation were analyzed after 4h. To analyze MPO and ADA activities, and NOx levels other groups of animals were pretreated (0.5h) with CE (25mg/kg), n-BuOH (25mg/kg) or Aq (5mg/kg). These parameters were analyzed after 4h. Statistical differences between groups were determined by ANOVA, and Newman-Keuls pos-Hoc test. Values of P<0.05 were considered significant. CE (25-50mg/kg), n-BuOH (25-50mg/kg) and Aq (5-25mg/kg) inhibited leukocytes: CE:42.8±2.9% to 66.7±5.8, n-BUOH:29.8±4.4 to 42.8±5.2% and Aq:47.4±5.0% to 60.7±5.2, neutrophils: CE:40.3±3.4 to 65.8±6.0%, n-BuOH:40.0±5.9% to 78.2±4.6, Aq:45.9±5.2% to 59.7±5.2 and exudation: CE:31.25±3.8 to 51.4± 3.2%, n-BuOH: 58.2±4.0% to 76.5±3.6 and Aq:41.36±2.7 to 73.3±3.2%. CE (25mg/kg), n- BuOH (25mg/kg) and Aq (5mg/kg) inhibited MPO by 60.0±1.6%, 60.9±1.3% and 67.5±1.1% and ADA activities by 45.2±2.3%, 51.2±8.0% and 63.8±5.8% and NOx levels by 40.7±1.1%, 61.9±4.0% and 70.0±0.8%, respectively (P<0.05).
Conclusion: J. sellowi Less. showed an important anti-inflammatory activity by inhibiting leukocytes and exudation. These inhibitory effects were associated with the decrease of MPO and ADA activities, and NOx levels.
Financial support: CNPq and CAPES.
PHYTOCHEMICAL AND ANTIINFLAMMATORY EVALUATION OF CAPSICUM BACCATUM VAR. PENDULUM L. (SOLANACEAE)
ALEXANDRA GOMES DA SILVA ALLEMAND1; BIANCA LEONARDI2; ALINE ZIMMER3; PEDRO R.T. ROMÃO4; GRACE GOSMANN5.
1,2,3,5.UFRGS, PORTO ALEGRE - RS - BRASIL; 4.UFCSPA, PORTO ALEGRE - RS - BRASIL.
Introduction: Capsicum baccatum L. var. pendulum (Willd.) is the most consumed red pepper species in Brazil, which has been found to inhibit inflammatory process. However, the compounds responsible for this activity and its mechanisms are not fully characterized. Thus, in this study the anti-inflammatory activity of C. baccatum was evaluated through the nitric oxide (NO) production in vitro and through paw edema and peritonitis model in female swiss mice(Protocols approved by CEUA-UFCSPA License n. 172/13). Methods and Results: The dried and powdered fruits were submitted to reflux for 4h using 70% ethanol to obtain the ethanol extract. Another portion was submitted to successive extraction in soxhlet using various organic solvents, separately, to obtain different extracts. The extracts are being submitted to phytochemical analysis and the content of the major chemical components will be determined by HPLC. To investigate the effects of C. baccatum on NO production, peritoneal macrophages (2x105) were pre-treated with extracts for 24h, and then stimulated with LPS (100 ng/mL)/IFN-g (30 ng/mL) for 48h. The CB10 extract at concentrations of 100 and 300 µg/mL suppressed the NO production by 37.5% and 77.2% respectively, while CB30 extract at 30, 100 and 300 µg/mL suppressed in order of 39.5%, 48.6% and 74.4% respectively. The ethanolic extract at 300 µg/mL (37.5%). To evaluate the anti-inflammatory activity, group of swiss mice (n=6) were orally treated with PBS, extracts (100 mg/kg orally) or dexamethasone (Dex, 2 mg/kg) 1 h prior to inoculation of Cg (500 μg, intraplantar in the right hindpaw for the edema evaluation or 500 μg i.p. for cell migration analyses). After 4 h, paw edema was calculated by a pachymeter and the number of neutrophils recruited to peritoneal cavity by microscopic differential count. The Cg induced significant increase in paw edema, which was inhibited by Dex, but not by extracts of C. baccatum. The neutrophil recruitment induced by Cg (5x106/cav) was inhibited by Dex in 69%, and by aqueous and CB30 extracts in order of 33.8% and 46.6% respectively. CB30 extract was able to inhibit the neutrophil migration in a dose-dependent way (25, 50 and 100 mg/kg caused 34.1%, 37.5% and 42% of inhibition, respectively). Conclusions: C. baccatum extracts inhibited the neutrophil migration to peritoneal cavity induced by Cg and also reduced the NO production by LPS/IFN-g stimulated-macrophages. Financial support: CNPq, CAPES, FAPERGS
PHYTOL, A DITERPENE ALCOHOL, REDUCES INFLAMMATORY RESPONSE: INHIBITION OF NEUTROPHIL MIGRATION AND OXIDATIVE STRESS
FRANCISCA BEATRIZ DE MELO SOUSA1; RENAN OLIVEIRA SILVA2; SAMARA RODRIGUES BONFIM DAMASCENO3; NATHALIA SANTOS CARVALHO4; VALDELÂNIA GOMES SILVA5; FRANCISCO DE ASEVEDO MENDES DE OLIVEIRA6; CAMILA DE FÁTIMA CARVALHO BRITO7; IRISMARA SOUSA SILVA8; MAISA DE SOUSA DOS SANTOS9; KAROLINE SABÓIA ARAGÃO10; ANDRÉ LUIZ DOS REIS BARBOSA11; RIVELILSON MENDES DE FREITAS12; JAND-VENES ROLIM MEDEIROS13.
1,2,3,4,5,7,8,9,11,13.BIOTECHNOLOGY AND BIODIVERSITY CENTER RESEARCH (BIOTEC), FEDERAL UNIVERSITY OF PIAUÍ, PARNAÍBA - PI - BRASIL; 6,12.POSTGRADUATE PROGRAM IN PHARMACEUTICAL SCIENCES, FEDERAL UNIVERSITY OF PIAUÍ, TERESINA - PI - BRASIL; 10.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Many drugs used as treatment for inflammatory process are associated with significant adverse effects and as alternative therapy many natural compounds with different mechanisms of action may be used to treat this process (Curr. Pharm. Des. 15:1212-1237, 2009). Based on this fact, the aim of this study was to investigate the effect anti-inflammatory of phytol in mice. Methods and Results: In the present study, approved by the local ethics committee (protocol no. 0066/10), male Swiss mice weighing 25–30 g, were used in classical models of inflammation. In models of paw edema measured by plethismometer (n = 6), the pre-treatment of mice with phytol significantly and dose-dependently reduced carrageenan-induced paw edema (0.061 ± 0.003 ml), with maximal inhibitory effect at a dose of 75 mg/kg (0.026 ± 0.002 ml). Phytol 75 mg/kg inhibited edema induced by compound 48/80 (0.108 ± 0.011 ml) to (0.038 ± 0.008 ml), histamine (0.082 ± 0.003 ml) to (0.030 ± 0.010 ml), serotonin (0.084 ± 0.008 ml) to (0.028 ± 0.005 ml), bradykinin (0.070 ± 0.009 ml) to (0.018 ± 0.06 ml) and PGE2 (0.069 ± 0.003 ml) to (0.030 ± 0.007 ml). For evaluation of neutrophil migration (n = 6), phytol 75 mg/kg, i.p., injected thirty minutes before of carrageenan, produced a significantly reduced the leukocyte recruitment (2.39 ± 0.62 x 103 cells/ml) and neutrophil migration (0.42 ± 0.45 × 103 cells/ml) to the peritoneal cavity, as compared to carrageenan group (total leukocyte 4.83 ± 0.99 × 103 cells/ml and neutrophil recruitment 3.55 ± 0.32 × 103 cells/ml). The levels of TNF-α and IL-1β were evaluated using the ELISA (n = 6), and the pre-treatment with phytol 75 mg/kg i.p significantly reduced TNF-α (106.8 ± 32.81 pg/ml), IL-1β (607.1 ± 147.6 pg/ml) as compared to carrageenan group (TNF-α, 208.7 ± 39.22 pg/ml and IL1-β, 1073 ± 180.1 pg/ml). The levels of GSH, MDA and MPO were measured by spectrophotometry (n = 6). The administration of carrageenan induced an increase in MDA (25.17 ± 4.47 nmol/ml) and decreased GSH levels (59.73 ± 8.19 µg/ml) concentrations in peritoneal fluid and also increased MPO activity (9.85 ± 0.96 U/mg) in paw tissue. The pre-treatment with phytol 75 mg/kg i.p significantly reduced MDA (10.73 ± 1.75 nmol/ml) concentrations, MPO activity (3.21 ± 0.82 U/mg) and increased GSH levels (94.05 ± 17:17 / ml). Conclusion: Phytol reduces the inflammatory response and oxidative stress in mice by reducing neutrophil migration.
Financial support: CNPq and FAPEPI
PIONEERING EXPERIMENTAL MODEL OF STEATOHEPATITIS INDUCED BY IRINOTECAN: A CANCER CHEMOTHERAPEUTIC
MARCELO LEITE VIEIRA COSTA; KAROLINE SABOIA ARAGÃO; PAULO ROBERTO CARVALHO DE ALMEIDA; ANIELLE TORRES DE MELO; CARLOS DIEGO HOLANDA LOPES; CIBELE BARRETO MANO DE CARVALHO; ROBERTO CÉSAR PEREIRA LIMA JÚNIOR; RONALDO DE ALBUQUERQUE RIBEIRO.
UNIVERSIDADE FEDERAL DO CEARA, FORTALEZA - CE - BRASIL.
Introduction: Nonalcoholic steatohepatitis (NASH) is a toxicity that may affect one in every twelve patients treated with irinotecan (IRI)-based cancer chemotherapy regimens. In spite of the unknown pathogenesis of IRI-induced NASH, there is no animal model in the literature. Then, we aimed to develop an animal model of this disease and also to verify the liver and gut immunoexpression of Interleukin- 1b (IL-1b), inducible nitric oxide synthase (iNOS) and toll- like receptor-4 (TLR4) and bacterial translocation in these animals. Methods and results: Swiss male mice (25g) were divided into groups (n=8) and were injected with saline (5mL/kg, i.p.) or IRI (25, 50, 75 or 100 mg/kg, i.p, thrice a week). The animals were killed at weeks 1, 3, 5, 7 and 9. Serum levels of protein and hepatic enzymes ALT, AST were measured. Blood samples were also aseptically collected for bacterial count. Liver samples were collected for total lipids content and myeloperoxidase activity assay. Histopathologic damage was scored and IL-1b, iNOS and TLR4 immunoexpression were also performed in duodenum and liver samples. Student’s T-test or Mann-Whitney test was used for statistical analysis. P<0.05 was accepted. CEPA: 21/12. The optimal dose of IRI (50 mg/kg) increased at week 7 versus saline group (P<0.05): liver wet weight (1718±156.2 vs. 1110±66.1), ALT (95.99±4.13 vs. 44.58±12.03), AST (138.7±9.52 vs. 84.16±8.12), proteins (3.25±0.01 vs. 5.38±0.25), liver MPO (2.19±0.56 vs. 0.06±0.03), total lipids (1.07±0.12 vs. 0.27±0.13), histological injury (4[4-7] vs. 1[0-1]), and bacteremia (portal blood: 80% and systemic blood: 40% vs. saline group: 0%). IRI-injected group also showed a significant increase in IL-1 (2[1- 3]), iNOS (2[2-3]) and TLR4 (3[1-3]) immunostaining versus saline group (IL-1: 0[0-1]; iNOS: 1[1-2] and TLR4: 1[1-2]). Shortened villi and the increased crypt depth were seen in gut samples of IRI group (4[3-4]) vs. saline group (0[0-0]). IL-1 (2[1-3]), iNOS (1[1–2]) and TLR4 (2.5[0–3]) expression in intestinal slices were also increased (P<0.05) in IRI group vs. saline group (IL-1: 0[0-1]; iNOS: 0[0-1]; TLR4:1[0–2]). Conclusion: IRI induces steatohepatitis in mice, mimicking the histological changes found in clinical setting. In addition, since we found a bacteremia and increased pro-inflammatory markers immunoexpression both in liver and intestinal samples, the role of these mediators in the pathogenesis of IRI-induced NASH need to be established. Support: FUNCAP/CAPES.
POLYSACCHARIDE EXTRACTS OF XIMENIA AMERICANA BARKS INHIBIT INFLAMMATORY NOCICEPTION
KAIRA EMANUELLA SALES DA SILVA1; LÍVIA DE PAULO PEREIRA2; ALANA DE FREITAS PIRES3; LUIS EDUARDO ALVES DAMASCENO4; FRANCISCA CRISTIANE NOGUEIRA5; ANA MARIA SAMPAIO ASSREUY6; MARIA GONÇALVES PEREIRA7.
1,2,6,7.INSTITUTO SUPERIOR DE CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA - CE - BRASIL; 3,4.1INSTITUTO SUPERIOR DE CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA - CE - BRASIL; 5.FACULDADE DE EDUCAÇÃO, CIÊNCIAS E LETRAS DO SERTÃO CENTRAL, UNIVERSIDADE ESTADUAL DO CEARÁ, QUIXADÁ - CE - BRASIL.
Introduction: There is an increasing effort in the search for natural molecules to control painful states, including compounds extracted from plants. The general immunomodulatory role of plant polysaccharides is already well described, but its mediation in the nociception process is scarce. Barks of Ximenia americana have been used in folk medicine for the treatment of headache, gastric and back pain. Experimentally, its aqueous extracts have shown antinociceptive effect. Here it was evaluated the antinociceptive effect and toxicity of polysaccharide extracts obtained from X. americana barks in mice. Methods and Results: Total polysaccharides of X. americana (TPL-Xa) were extracted according to Yoon et al. (2002) and analyzed for the carbohydrate and protein contents. Male Swiss mice (20-25 g) received TPL-Xa by intravenous (i.v.; 0.1, 1 and 10 mg/kg) or oral (p.o.; 100 mg/kg) routes and tested in the models of Formalin, Writhing, Hot plate and von Frey. Toxicity was assessed after the fourteen-day treatment with TPL-Xa (10 mg/kg; i.v.) by the variation of body/organ mass and hematological/biochemical parameters. Protocols were approved by the UECE Ethical Committee (CEUA n°12783679-9). Mean ± SEM (n=6-8), ANOVA and Bonferroni's test (p<0.05). TPL-Xa extraction revealed 8.1% yield, 40% carbohydrate and 6.5% protein. Pre-treatment of animals with TPL-Xa (i.v.) inhibited nociception in the second phase of formalin by 50% (0.1 mg/kg) and 93% (10 mg/kg) and in other models that evaluate inflammatory pain: von Frey (by 35% at the 2nd and by 71% at the 3rd hour) and writhing test [69.1% (i.v. at 10 mg/kg) and 44% (p.o. at 100 mg/kg)]. Contrary, TPL-Xa showed no inhibitory effect in tail flick or hot-plate tests. Moreover, TPL-Xa subchronic treatment did not induce alterations in the following parameters: animals body mass, organs wet weigh (kidney, stomach, liver, heart, spleen), renal, hepatic or hematological markers. Conclusion: Total polysaccharides of X. americana present antinociceptive activity in mice models of inflammatory pain, without causing renal, hepatic or hematological toxicity, making this polysaccharides of interest in future approaches to treat painful conditions.
Financial Support: CAPES, CNPQ and FUNCAP
PRECLINICAL EFFICACY AND IMMUNOLOGICAL SAFETY OF FR104, AN ANTAGONIST ANTI-CD28 MONOVALENT FAB’ANTIBODY, IN HUMANIZED MICE MODELS
BERNARD VANHOVE1; CAROLINE MARY2; NAHZLI DILEK3; STEPHANIE LE BAS-BERNARDET4; GILLES BLANCHO5; NICOLAS POIRIER6.
1,4,5.ITUN-INSERM 1064, NANTES - FRANÇA; 2,3,6.EFFIMUNE, NANTES - FRANÇA.
Introduction
Antagonist anti-CD28 mAbs prevent T-cell costimulation and functionally differentiate from CTLA4Ig since they cannot block CTLA-4, PDL-1 and B7-mediated coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing immune tolerance after organ transplantation. So far, however, antagonist anti-CD28 antibodies showing clinically acceptable pharmacokinetic profile have not been developed. FR104 is a novel monovalent humanized Fab’ anti-CD28 antibody fragment that was pegylated to improve elimination half-life. We have investigated its mechanism of action, immunological safety and preclinical efficacy in humanized mouse models.
Methods and Results
FR104 dose-dependently prevented human T cell proliferation and IL-2 secretion in vitro. Following the guidelines edicted after the ‘Tegenero affair’, we showed that FR104 failed to induce human T cell proliferation and cytokines secretion (IFN-g, TNF-a, IL-2, IL-4, IL-6 and IL-8) in soluble, air-dried or wet-coated condition, even in the presence of anti-CD3 stimulation or when cross-linked with secondary antibodies or over a monolayer of human endothelial cells, whereas superagonist and divalent anti-CD28 antibodies showed such activities. Furthermore, in humanized NOD/SCID mice (7-10 week old female) adoptively transferred with human PBMC, superagonist and divalent anti- CD28 Abs elicited rapid cytokines secretion and human T-cell activation, whereas FR104 stayed antagonist (n=7 to 15/group). After few weeks, these humanized mice developed a florid graft-versus-host disease due to the proliferation of xenogeneic human T cells, which was completely prevented by short-term administration of FR104. This effect was dependent on the availability of CTLA-4 since co-administration of an anti-CTLA-4 antibody abrogated the therapeutic activity of FR104. Interestingly, administration of high doses of Belatacept or Abatacept was ineffective to prevent graft-versus-host disease, whereas a less intensive regimen of administration was partially effective. These experiments have been performed with the agreement #CEEA.2012.155 of the CEEA Pays de la Loire (France).
Conclusion
The anti-CD28 monovalent antibody FR104 is devoid of agonist activity in vitro and in vivo on human T cells and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA-4, as compared to B7 antagonists in autoimmune diseases and transplantation.
PRO-ANGIOGENIC EFFECTS OF PLASMA FROM SICKLE CELL DISEASE PATIENTS AND ANTIANGIOGENIC EFFECTS OF HYDROXYUREA: EVALUATION OF MAJOR PRO- AND ANTIANGIOGENIC FACTORS USING BIOPLEX® TECHNOLOGY
FLÁVIA CRISTINE MASCIA LOPES; SARA TERESINHA OLALLA SAAD; FERNANDO FERREIRA COSTA; NICOLA CONRAN.
SCHOOL OF MEDICAL SCIENCES, UNIVERSITY OF CAMPINAS – UNICAMP, CAMPINAS - SP - BRASIL.
Introduction Sickle cell disease (SCD) has a complex pathophysiology involving vascular inflammation, decreased nitric oxide (NO) bioavailability and endothelial activation. Angiogenesis is considered an inflammatory immunopharmacologic target since it increases the influx of immune cells and the production of inflammatory mediators as well as provides nutrients for the inflammatory process. Pro- and antiangiogenic molecules originate from cells (endothelial, stromal, tumor), blood and extracellular matrix. A proangiogenic state is currently associated with SCD. Hydroxyurea (HU) has been used for SCD therapy acting through several mechanisms. It often reduces leukocyte counts, increases NO and decreases inflammatory mediators. In this study, we quantify the major pro- and antiangiogenic factors in the plasma of SCD patients using BioPlex®. Methods and Results Patients (steady state) and controls were divided into three groups: HbSS phenotype not treated with HU (SSHU-), HbSS treated with HU (15-30mg/kg/day; SSHU+) and healthy control individuals (HbAA). Results are expressed as mean±SEM (N=8/group; Mann-Whitney test). Analyzing the pro-angiogenic factors in the SSHU-, we observed 9256.3±497.1pg/mL of angiopoietin-1, 238.2±9.4pg/mL of bFGF, 14.3±1.0pg/mL of PlGF and 284.7±20.5pg/mL of VEGF-D. These factors were elevated by 135.1%, 18.8%, 76.0%, 94.3%, respectively, when compared to HbAA. In the SSHU+, angiopoietin- 1, bFGF, PlGF, PDGF-AA, PDGF-BB and VEGF-D measured, respectively, 984.6±323.4pg/mL,179.2±8.3pg/mL, 10.1±0.5pg/mL, 219.8±69.8pg/mL, 395.1±144.6pg/mL and 109.0±10.6pg/mL. Additionally, angiopointin-1, bFGF, PDGF-AA, PDGF-BB and VEGF-D were decreased by 75.0%, 10.6%, 56.0%, 69.3%, 25.6% respectively, in the SSHU+ compared to HbAA. With regard to anti-angiogenic factors, endostatin levels were 51700.5±5198.6pg/mL in SSHU- (increased by 39.7% when compared to HbAA). However endostatin (69561.2±5759.3pg/mL) and thrombospondin (22536.4±3092.8pg/mL) were elevated in SSHU+, by 88.1% and 143.2%, respectively, when compared to HbAA. Results show that plasma from SCD patients was found to contain significantly increased levels of a number of pro-angiogenic factors. In addition, plasma from SCD patients treated with HU presented decreased levels of proangiogenic factors and increased antiangiogenic factors. Conclusion This study finds further evidence for a proangiogenic state in SCD and suggests that one of the effects of HU may be inhibition of angiogenesis.
PROTEASE INHIBITORS PROMOTES RESOLUTION OF ACUTE INFLAMMATION ASSOCIATED WITH INCREASED INTACT FORM OF ANNEXIN-A1
THAÍS DE CAUX1; JULIANA PRISCILA VAGO2; LUCIANA PÁDUA TAVARES3; VANESSA PINHO4; MAURO PERRETTI5; MAURO MARTINS TEIXEIRA6; LIRLÂNDIA PIRES DE SOUSA7.
1,2,3,4,6,7.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 5.- BARTS AND THE LONDON SCHOOL OF MEDICINE, LONDRES - REINO UNIDO.
Introduction: Annexin-A1 (AnxA1) is a glucocorticoid (GC)-induced protein of 37KDa that is known as a mediator of several GC functions. The intact form (37KDa) is considered a mediator of the anti-inflammatory and pro-resolutive actions of AnxA1. However, this protein may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3 (PR3), generating the 33kDa isoform. In this study, we have investigated the dynamics of AnxA1 expression and the role of synthetic (Sivelestat, Eglin) and natural (Elafin) inhibitors of protease on resolution of LPS-induced neutrophil inflammation. Methods and Results: This work was approved by UFMG Research Ethics Committee (CETEA). Male BALB/C mice were challenged by i.pl. (intrapleural) administration of PBS or LPS (250ng/cavity) at different times. We also studied the role of two specific neutrophil elastase inhibitors (Sivelestat, 5mg/kg, i.p; Elafin, 10 µg/kg, i.p), the inhibitor of elastase and cathepsin g (Eglin, 100µg/kg, i.p), and a pan-inhibitor of serine proteases (PMSF, 30 mg/kg, i.p) 4h after treatment with LPS. The cells were processed for viable and apoptotic leukocyte count and western blot analysis. The injection of LPS induced a time-dependent influx of neutrophils into the pleural cavity, which peaked at 8h and decreased at 48h. The cleaved form of AnxA1 was more detected at 8h, time of maximal neutrophil recruitment. Intact AnxA1 expression was recovered during the resolutive phase (48h). These observations occurred in agreement with the endogenous elastase activity. The treatment of 4h- LPS challenged mice with silvelestat inhibited elastase activity and decreased the neutrophil number into the pleural cavity. Such effect on elastase inhibition was associated with increased numbers of neutrophils with apoptotic morphology, caspase-3 cleavage and an increase of intact AnxA1 (37KDa). Resolution of neutrophilic inflammation (decreased number of neutrophils and increased number of apoptotic neutrophils) was also observed when mice were treated with Elafin and Eglin. PMSF caused a similar effect on neutrophil although it decreases mononuclear cells. Conclusion: Our results showed that protease inhibition resolves inflammation associated with an increase of the intact AnxA1 and neutrophil apoptosis, suggesting that therapeutical strategies that increase AnxA1 may be an alternative treatment to acute inflammation.
Financial Support: CNPq, PRPq-UFMG, FAPEMIG and CAPES.
PROTECTIVE EFFECT OF SILYMARIN (SIL) ON IRINOTECAN INDUCED NON-ALCOHOLIC STEATOHEPATITIS IN MICE
EUDMAR MARCOLINO DE ASSIS JUNIOR (PG)1; LEONARDO DA SILVA MOREIRA (IC)2; NATHALIA RIBEIRO PINHO DE SOUZA (IC)3; LARA RAISSA CAVALCANTE MALVEIRA (IC)4; CHRISTIANE MENDES GONÇALVES DE OLIVEIRA (IC)5; DEYSI VIVIANA TENAZOA WONG (PG)6; ANIELLE TORRES MELO (PG)7; VENUCIA BRUNA MAGALHÃES PEREIRA (PG)8; KAROLINE SABÓIA ARAGÃO (PG)9; PAULO ROBERTO CARVALHO DE ALMEIDA (P)10; RONALDO ALBUQUERQUE RIBEIRO (P)11; ROBERTO CÉSAR PEREIRA LIMA JÚNIOR (P)12.
1,2,3,4,5,6,7,8,9,11,12.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FEDERAL UNIVERSITY OF CEARÁ., FORTALEZA - CE - BRASIL; 10.DEPARTMENT OF PATHOLOGY AND FORENSIC MEDICINE, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Colorectal Cancer (CRC) is the 3th most prevalent neoplasic disease in the world. Irinotecan (IRI), a first line drug for CRC and its liver metastasis, has improved patients’s survival. However, its side-effects, including non-alcoholic steatohepatitis (NASH), may limit the course of treatment. IRI-based regimens have been associated with a 3.4-fold increased risk of NASH. However, NASH pathogenesis is unknown, one reason why no effective therapy is available. Silymarin (SIL) has shown to prevent fatty liver diseases, drug- and chemical-induced hepatic toxicity in animal models. Then, we aimed to study the effect of SIL on IRI-induced NASH. Methods and Results: Swiss male mice (n=8), divided into 6 groups, were injected with saline (SAL, 5ml/kg, i.p.), IRI (50 mg/kg, i.p.), SIL (150 mg/kg p.o.) or IRI (50 mg/kg i.p) + SIL (SIL 1.5, 15 and 150 mg/kg p.o) 3x/week/7 weeks. Blood samples were collected at week 7 to determine serum concentration of the hepatic enzymes ALT and AST (U/L). Animals were killed and the livers were removed to assess tissue damage (Kleiner’s scores), total lipid (mg/g tissue), IL-1β (pg/mg tissue) dosages, iNOS and 3-Nitrotyrosine (NTyr) immunoexpression. ANOVA/Bonferroni’s test or Kruskal Wallis/Dunn was used for statistical analysis. P<0.05 was accepted (CARE:21/12). IRI induced a significant (P<0.05) increase in serum ALT and AST, hepatic lipids accumulation, histopathological injury, neutrophil infiltration/field, IL-1b tissue level, iNOS immunostained cells/field (94.8±21.7, 103.7±6.1, 27.3±6.6, 6.5[5-7], 3.90±0.55, 18.4±4.5, 15.5±0.7 respectively) and NTyr marked area vs. SAL (ALT: 48.2±3.2, AST: 41.5±2.7, Total lipids: 7.7±0.9, Tissue damage: 1[0-3], neutrophil infiltration: 0.32±0.10; IL-1b: 10.8±0.6; iNOS: 0.0±0.2). SIL (1.5 mg/kg) prevented the increase in these parameters (ALT: 34.2±12.2, AST: 33.8±6.4, Total lipids: 11.45±1.795, Tissue damage: 3[2-6]) versus IRI group (P<0.05). Neutrophil infiltration was only prevented by SIL 15 mg/kg (2.3±0.4). Besides, SIL reduced the NTyr immunoexpression vs. IRI. However, IL-1b and iNOS expression were not affected by SIL pre-treatment (P>0.05) and even the higher dose of SIL was more deleterious. Conclusions: SIL prevented IRI-induced liver injury likely through the reduction of protein nitrosylation. Despite the reduction of neutrophil infiltration, SIL was unable to prevent the increase in local production of inflammatory markers.
Financial Support: CNPq/CAPES/FUNCAP.
PROTECTIVE EFFECT OF WARIFTEINE ON LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY IN MICE
THERESA RAQUEL DE OLIVEIRA RAMALHO1; MARIA TALITA PACHECO DE OLIVEIRA2; MARY DA COSTA3; MARCIA REGINA PIUVEZAM4.
1,2,4.UNIVERSIDADE FEDERAL DA PARAÍBA, JOÃO PESSOA - PB - BRASIL; 3.STATE UNIVERSITY OF NEW YORK AT OSWEGO, NEW YORK - ESTADOS UNIDOS.
Introduction: Warifteine (WT) is the major alkaloid of the plant Cissampelos sympodialis Eichl and It is a bisbenzylisoquinoline alkaloid with antispasmodic, anti-inflammatory and ant-allergic effects. The plant is used in the Brazilian Northeast for the treatment of inflammatory and allergic diseases. Acute lung injury (ALI) is a common clinical problem, which is characterized by disruption of lung epithelial and endothelial cells, alveolar permeability, edema, inflammatory cell influx, followed by severe hypoxemia, thus leading to morbidity and/or mortality. The goal of this study was to evaluate warifteine in prevent the experimental model of ALI. Methods and Results: Swiss female mice (n=6) were orally pretreated with 2 mg/kg or 10 mg/kg of WT 1h before challenging with 50μl of LPS (2.5 mg/mL, intranasal) or PBS. After 24h the bronchoalveolar lavage (BAL) was harvested to characterize leukocyte migration, cytokines levels, and total protein concentration by colorimetric assay. The experimental protocols were approved by the Ethics Committee on Animal Research/UFPB with protocol number 0309/11. Animals pretreated with 2 mg/kg of WT showed significant (p<0.05) reduction of total inflammatory cells (36%) and polymorphonuclear cells (61.4%) migration and also reduction of TNF-α levels (60.1%) on the BAL fluid. Furthermore, it was observed an increasing of the IL-10 levels (p<0.05, 88.6%). Warifteine at 10 mg/kg did not reduce the cell migration nor alter cytokine levels. Both doses tested were not able to reduce concentration of proteins in lung vascular permeability. Conclusion: The results indicated that WT, despite not reduce lung vascular permeability, is effective in modulating some ALI parameters such as decreasing of inflammatory cell migration, levels of proinflammatory cytokine and increasing the amount of an anti-inflammatory cytokine into the lung of the animals.
Financial Support. CNPq/CAPES/INCT Cancer.
PROTEIN FRACTION OF CALOTROPIS PROCERA LATEX REDUCES MECHANICAL HYPERNOCICEPTION: INVOLVEMENT OF INFLAMMATORY MEDIATORS AND CONSTITUTIVE NOS.
GISELE DE FÁTIMA PINHEIRO RANGEL; PATRICIA BASTOS LUZ; RACHEL SINDEAUX PINHEIRO; LUANA DAVID CARMO; TATIANA SANTOS COUTO; KAROLINE SABOIA ARAGÃO; FLÁVIO SILVEIRA BITENCOURT; NYLANE NUNES ALENCAR; MÁRCIO VIANA RAMOS.
FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
PROTEIN FRACTION OF CALOTROPIS PROCERA LATEX REDUCES MECHANICAL HIPERNOCICEPTION IN MICE: INVOLVEMENT OF NO, PGE2 and KC.
GISELE DE FÁTIMA PINHEIRO RANGEL¹; PATRÍCIA BASTOS LUZ¹; RACHEL SINDEAUX PAIVA PINHEIRO¹; LUANA DAVID CARMO¹; TATIANA SANTOS COUTO¹; KAROLINE SABOIA ARAGÃO¹; FLAVIO SILVEIRA BITENCOURT¹; NYLANE MARIA NUNES ALENCAR²; MÁRCIO VIANA RAMOS².
¹Departaments of Physiology and Pharmacology ²Biochemistry and Molecular Biology; Federal University of Ceará, Fortaleza-CE, Brazil.
Background and aims: Calotropis procera laticífera is a plant found in Asia, Africa and South America. Its latex is rich in proteins that have relevant pharmacological activities like anti-diarrhoeal, anticancer, antidiabetic, bacteriolytic, antiinflamatory and analgesic. The aim of this study was to evaluate the activity of latex proteins of Calotropis procera (LP) in models of acute pain induced by carrageenan (Cg). Methods: a) Swiss male mice were used (n=5, 25-30g). b) Mechanical hypernociception (MH) was evaluated by the electronic version of the Von Frey before (T0) and after (1, 3 and 5 hours) the injection of Cg (300 µg/paw). c) Mice were treated with LP (0.5, 5 and 50 mg/kg, i.v.) or indomethacin (5 mg/kg, i.p.) or saline (i.v. and i.pl.) 30 min before Cg. d) After 3 h of injection of Cg, subplantar tissue was collected for quantification of proinflammatory cytokines (TNF-α and IL-1β), PGE2 and chemokine (KC). e) To assess the involvement of NO, the animals were pretreated with L-NAME (30 mg/kg i.p.) and 1400W (1.5mg/kg i.v.). The protocols in this study are in agreement with the standards ethics establi shed by the Ethics Committee of the Federal University of Ceará under case number 61/11. Results: LP (5 mg/kg) reduced (p<0.05) HM in 25%, 55% and 46% and LP 50 mg / kg in 39%, 64% and 60% in the 1st, 3rd and 5th hour respectively, when compared to Cg. The dose of 5mg/kg LP reduced the concentration of TNF-α, IL-1β, KC, and PGE2 71%, 81%, 72% and 72% respectively, when compared to Cg. This antinociceptive effect of LP was reversed with L-NAME (nonspecific inhibitor of NO synthase). However, 1400W (selctive inhibitor of iNOS) don't interfere with the effect of LP. Conclusion: From these data we can suggest that the antinociceptive effect of the LP is closely related to the decrease inflammatory mediators (TNF-α, IL-1β, PGE2 and KC), besides the involvement of constitutive NOS.
Financial support: CAPES and CNPq.
PROTEINS EXTRACTED FROM ACACIA FARNESIANA (L.) WILLD. SEEDS DISPLAY ANTI-INFLAMMATORY AND ANTINOCICEPTIVE ACTIVITIES
THIAGO DE SOUZA LOPES ARAÚJO1; LHAÍS SUELEN SOARES LEAL2; RENAN OLIVEIRA SILVA3; CLEIDIVAN AFONSO DE BRITO4; NATRICIO VALE ALMEIDA5; DOUGLAS LIMA MACHADO6; JAND-VENES ROLIM MEDEIROS7; ANDRÉ LUIZ DOS REIS BARBOSA8; JEFFERSON SOARES DE OLIVEIRA9; CLÁUDIO ÂNGELO VENTURA10.
1,2,4,5,10.NÚCLEO DE PESQUISA DE BIODIVERSIDADE E BIOTECNOLOGIA (BIOTEC), UFPI, CAMPUS DE PARNAÍBA, PARNAÍBA - PI - BRASIL; 3,7,8.LABORATÓRIO DE FISIOFARMACOLOGIA EXPERIMENTAL (LAFFEX), UFPI, CAMPUS DE PARNAÍBA, PARNAÍBA - PI - BRASIL; 6,9.LABORATÓRIO DE BIOQUÍMICA DE MICRO-ORGANISMOS E PLANTAS (BIOMIC), UFPI, CAMPUS DE PARNAÍBA, PARNAÍBA - PI - BRASIL.
Introduction: Acacia farnesiana (L.) Willd. is a plant belonging to the Leguminoseae family. In northeastern Brazil its seeds are commonly sold as a therapeutic agent; however, there are no scientific investigations confirming the plant’s pharmacological activities. The present work aimed to evaluate the anti-inflammatory and analgesic activities of proteins obtained from A. farnesiana seeds and show the involvement of lectin in those activities. Methods and Results: This study was approved by the local ethics committee (protocol no 0066/10). Proteins were extracted from A. farnesiana seeds, and five different protein fractions (albumin, globulin, prolamin, acidic and basic glutelins) were obtained. The total quantity of soluble proteins, the protein pattern and the presence of hemagglutinating and proteolytic activities were investigated in all of the fractions. The globulin fraction (GLB) was evaluated for possible anti-inflammatory activities on paw edemas that were induced by carrageenan(Cg)(Swiss albino mice, 25-30g, n=6) and dextran (Dx)(Wistar albino rats, 170-200g,n=6). The paw tissue myeloperoxidase activity (MPO) of Cg-treated animals was measured. The anti-inflammation effect was also investigated using a peritonitis model induced by Cg. The analgesic activity of GLB was assessed by the acetic acid-induced writhing, hot plate and formalin tests(Swiss albino mice, 25-30g, n=6). GLB was the protein fraction richest in soluble proteins, and an analysis of the protein pattern revealed the presence of a prominent band of 30.0 kDa. The active hemagglutination evidenced the presence of lectin in the GLB fraction, and no proteolytic activity was found in it. Injections of GLB (1, 3, 10 or 30mg/kg, i.p.) reduced the paw edema induced by Cg in a dose-dependent manner, which was accompanied by a reduction of MPO (p<0.05). However, GLB (30mg/kg) was not able to reduce the edema triggered by Dx. Additionally, GLB (30mg/kg) administration following Cg-induced peritonitis reduced the neutrophil peritoneal counts. Pre-treatment with GLB (30mg/kg) reduced the abdominal constrictions induced by acetic acid as well as the paw licking time induced by formalin. However, it did not produce a significant antinociceptive effect in the hot plate test. Conclusion: Seeds from A. farnesiana are a source of proteins with anti-inflammatory and analgesic properties, which seemed to be promoted by the lectin present in its GLB protein fraction.
Financial Support: CNPq, FAPEPI.
PULMONARY FIBROBLASTS PRODUCE CCL3, CXCL2, LTB4 AND LTC4 AFTER CXCL12-STIMULATION VIA P38, MEK1/2, PI-3K AND JNK
TAIS MAROLATO DANILUCCI; SANDRA HELENA PENHA OLIVEIRA.
SCHOOL OF DENTISTRY OF ARAÇATUBA - FOA/UNESP – UNIV. ESTADUAL PAULISTA, ARAÇATUBA - SP - BRASIL.
Introduction: CXCL12 and their specific receptor CXCR4 plays a critical role in airway inflammation. In this study, we investigate its effect in CCL3, CXCL2, LTB4 and LTC4 production by pulmonary fibroblast and the intracellular signaling involved in the process. Methods and Results: CXCL12 (100 ng/mL) induced CCL3 (1635±40,8 vs control group 553±95,2 pg/mL), CXCL2 (9001±505 vs 2430±224 pg/mL), LTB4 (94±6,7vs 47,14±12,4 pg/mL) and LTC4 (18,4±1,1 vs7±1,5 pg/mL) production after 24 hours and that CXCL2 and LTC4 production is dependent of CCL3 production. Pulmonary fibroblasts constitutively expressed mRNACXCR4 (0,7±0) and CXCL12 up-regulated its expression (1,1±0); measure unit: CXCR4/β-actina. Western blot analysis showed that CXCL12 increased protein expression of CXCR4 (1,1±0 vs 0,7±0) and induced phosphorylation at S339 of the CXCR4 (0,8±0 vs 0,4±0). Constitutive CXCR4 mRNA expression was inhibited by anti-CCL3 (0,9±0) antibody or MK 886 (0,3±0) and CXCR4 mRNA-induced was inhibited by anti-CXCL2 (0±0) compared to CXCL12 group (1,8±0); measure unit: CXCR4/β- actina. Indeed pulmonary fibroblasts were pretreated with MK886, dexamethasone (Dexa) and loratadine (Lor). CCL3 was inhibited by Dexa (261±11) and Dexa plus Lor (85±10,2) in relation CXCL12 group (507±48). CXCL2 was also inhibited by Dexa (668±32) and Dexa plus Lor (200±61) in relation CXCL12 group (1353±111). LTB4 and LTC4 productions were inhibited respectively by MK886 (26±2,1; 9,4±0,4), Lor (33±3,7; 96,6±1), Dexa (26,2±3,2; 8±2) and Dexa plus Lor (29,6±2; 4,8±1,2) compared to CXCL12 group (81,4±4,5; 20,5±0,6); measure unit: pg/mL. We have identified p38, MEK1/2, PI-3K and JNK, intracellular signaling pathways play a role in CCL3, CXCL2 and LTB4 protein production by CXCL12/CXCR4 axis activation in pulmonary fibroblasts. Conclusion: Our results suggest that CXCL12 increased CCL3, CXCL2, LTB4 and LTC4 production in pulmonary fibroblasts activating different intracellular mechanism playing an important role in pro-inflammatory mediators production involved in the regulation of allergic process.
Financial support: CAPES and FAPESP (2011/06405-7).
PUNICA GRANATUM PREVENTS ALVEOLAR BONE LOSS BY REDUCING NEUTROPHIL MIGRATION
VILANA MARIA ADRIANO ARAÚJO1; LARICE KÉRCIA BRAZ MONTEIRO2; IRACEMA MATOS MELO3; MARIANA VASCONCELOS GUIMARÃES4; ALINE DANTAS DIÓGENES SALDANHA5; LUCIANA CARVALHO CÂNDIDO6; THAYANNE BRASIL7; ANA CARLA SILVA SANTOS8; GERLY ANNE CASTRO BRITO9; RENATA CARVALHO FERREIRA LEITÃO10; RONALDO ALBUQUERQUE RIBEIRO11; VILMA LIMA12.
1,7,11,12.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 2,3,4,6,8.FACULTY OF PHARMACY, DENTISTRY AND NURSING, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 5,9,10.DEPARTAMENT OF MORPHOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Periodontitis is an immunoinflammatory disease characterized by leukocyte infiltration and bone resorption. P. granatum (PNG) has showed anti-inflammatory and antioxidant properties. So, it was evaluated the alveolar bone and anti-inflammatory activity of PNG in periodontitis. Methods and Results: Periodontitis was induced in 48 female rats by a nylon-3.0 around the left upper 2nd molar, and contralateral as control. Groups of 6 rats received (vo) saline 0.9% (SAL) or PNG [60, 180 and 540(mg/kg)] daily until 11thd, when they were killed. Periodontitis was analyzed through macroscopy, histometry, histology (scores), immunohistochemical staining for TRAP and activity of myeloperoxidase (MPO). Seric dosages of bone alkaline phosphatase (BALP), hepatic transaminasis, urea, creatinin and leukogram were performed. Rats were weighted daily, and indexes of liver and kidney were made (CARE-UFC 70/11). The data were presented as mean±standard error of the mean or median. The ligature caused intense alveolar bone loss (SAL=5.30±0.29), that was reduced by PNG (60=34%; 180=34%; 540=42%; p<0.05), and corroborated by histometry (SAL=0.023±0.004; PNG 540=0.014±0.0008). The ligature caused reduction of BALP (SAL=57.9±31.8 U/l), when compared to baseline (97±2.8), but PNG 540 (45.1±3.2) did not prevent this drop. Interesting, the periodontitis induced an intense immunostaing for TRAP (Unchallenged= 0.0±0.0; SAL=0.016±0.004), but PNG did not reduced this staing (PNG 540=0.011±0.003; p>0.05). However, the intense leukocyte infiltration, followed by destruction of aveolar process, cementum and periodontal ligament and low level of the junctional epithelium induced by ligature [SAL=3(2-3)] was prevented (p<0.05) by PNG 540 [(2(0-2)] accompanied by reduction (p<0.05) of the MPO activity (Unchallenged=0.81±0.07; SAL=4.98±0.49; PNG 60=46%; 180=49%; 540=75%). Sistemically, PNG prevented (p<0.05) the leukocytosis observed in periodontitis (Baseline=12±0.7; SAL=18.5±2.8; PNG 540=10.2±1.9). SAL and PNG groups did not show any changes in seric dosages for liver and kidney analysis, as well as respectives indexes, when compared to baseline (p>0.05). Conclusion: In spit of presence of osteclasts, PNG prevented the bone loss by decreasing leukocyte infiltration, preserving the periodontal ligament and cementum, and MPO activity, without causing systemic changes, what support the accute anti-inflamatory effects expected for P. granatum.
Financial support: Capes; CNPq.
QUERCETIN INHIBITS INFLAMMATORY BONE RESORPTION IN A MOUSE PERIODONTITIS MODEL
HENRIQUE BALLASSINI ABDALLA1; JULIANA TRINDADE CLEMENTE NAPIMOGA2; CRISTINA GOMES DE MACEDO3; FABIANA FURTADO FREITAS4; RAFAEL NOBREGA STIPP5; WALDICEU APARECIDO VERRI JUNIOR6; MARCELO HENRIQUE NAPIMOGA7.
1,2,3,4,5.PIRACICABA DENTAL SCHOOL/UNICAMP, PIRACICABA - SP - BRASIL; 6.BIOLOGICAL SCIENCES CENTER/UEL, LONDRINA - PR - BRASIL; 7.LABORATORY OF IMMUNOLOGY AND MOLECULAR BIOLOGY/SÃO LEOPOLDO MANDIC, CAMPINAS - SP - BRASIL.
Introduction: It is currently known that quercetin (3,5,7,3’,4’-pentahydroxyflavone) is an antioxidant flavonoid molecule with potential anti-inflammatory action. It has been demonstrated that the administration of quercetin can inhibit the bone loss in a rat periodontitis model, although there is no information about the possible mechanisms. Thus, in the present study we have tested the effect of quercetin in bone loss and immunoinflammatory responses, using a mouse periodontitis model infected with Aggregatibacter actinomycetemcomitans. Methods and Results: Balb/c mice (20–25 g) received an oral delivery of 1 x 109 colony-forming units (CFU) of a diluted culture of A. actinomycetemcomitans JP2 in 100 µl of phosphate-buffered saline (PBS) with 2% carboxymethylcellulose, placed into the oral cavity with a micropipette. This procedure was repeated after 48 and 96 hours. The treatment with quercetin was initiated after the third inoculation of bacteria, and consisted of daily subcutaneous injections (during 15 days) of 100 mg/kg of quercetin diluted in 20% Tween 80 in saline. Evaluation of the extent of alveolar bone loss was assessed morphometrically by measuring the distance between the cement-enamel junction (CEJ) and the alveolar bone crest (ABC) of the first and second molars. Aliquots of each gingival sample were assayed by ELISA and Western Blot Analysis to determine the levels of IL-1β, TNF-α, IL-17 and the expression of RANKL and ICAM-1. The infected animals treated with quercetin at a dose of 100 mg/kg showed significantly lower bone resorption than the vehicle-treated infected animals (p < 0.05). Quercetin statistically inhibited the release of IL-1β, TNF-α, IL-17 and the expression of RANKL and ICAM-1 in comparison to infected mice (p < 0.05).
Conclusion: Quercetin was able to inhibit inflammatory bone resorption in a mouse periodontitis model.
QUINOLINE COMPOUNDS REDUCE INFLAMMATORY CYTOKINES AND SPONTANEOUS PROLIFERATION OF T-LYMPHOCYTES FROM HTLV-1-INFECTED INDIVIDUALS
LORENA ANA PINTO1; MILENA BOTELHO PEREIRA SOARES2; ALAIN FOURNET3; BERNARDO GALVÃO CASTRO4; MARIA FERNANDA RIOS GRASSI5.
1.GONÇALO MONIZ RESEARCH CENTER – CPQGM/FIOCRUZ, SALVADOR - BA - BRASIL; 2.2CENTER OF BIOTECHNOLOGY AND CELL THERAPY, SALVADOR - BA - BRASIL; 3.3FACULTÉ DE PHARMACIE, RUE J. B. CLÉMENT, CHÂTENAY-MALABRY - FRANÇA; 4,5.BAHIANA SCHOOL OF MEDICINE AND PUBLIC HEALTH, SALVADOR - BA - BRASIL.
Introduction: The Human-T Lymphocyte virus (HTLV) infection is considered one of the neglected endemic diseases. There is no specific treatment for those infected with HTLV. The identification of drugs that modulate the spontaneous proliferation may be important not only for the treatment of pathologies associated with viral infection, but also for understanding the pathogenesis of the disease. Here we evaluated the modulation of spontaneous proliferation of T cells by three different quinoline compounds.
Methods and Results: Peripheral blood mononuclear cells (PBMC) from HTLV-1-infected individuals (PBMC+) were cultivated in the presence of quinolone compounds. For evaluation of proliferation by 3[H]thymidine incorporation cells from 08 HTLV-1-infected individuals were pulsed for 16 hours with 3[H]thymidine, to measure its incorporation using a liquid scintillation beta-counter. The results were presented in counts per minute (cpm). Cell viability was measured by optical density in the presence of MTT. Cytokines were quantified by flow cytometry (FACSAria, Becton Dickinson, Mountain View, Calif.) and the results were analyzed using FlowJo software (Ashland, USA) and Graphpad Prism 5.0 software. The compounds showed no toxicity at the concentrations evaluated. The IC50 were 4.3 mM, 3.4 mM and 0.3 mM for quinolones I, II and III, respectively. The three compounds inhibited more than 80% of spontaneous proliferation in the highest concentration tested (50mM of quinolone I, 100mM of quinolone II and 50mM of quinolone III). We observed a 30% increase in the concentration of cytokine IL-17 in the culture supernatant in PBMC+ in the presence of 50mM of quinoline I (p = 0.01). When PBMC+ were cultured with 25 mM of quinoline III, we observed a 68% reduction in the concentration of cytokine IL-2 (p=0.006), and more than a 90% reduction in the concentration of cytokines IL-10 (p=0.02), TNF-α (p=0.0002) and IFN-g (p<0.0001). There was no difference in the cytokine concentrations in supernatants of PBMC+ cultured in the presence of quinoline II.
Conclusion: The compounds showed no toxicity and were able to inhibit the inflammatory cytokine and IL-10 of PBMC from HTLV-1-infected individuals. New assessments are being carried on to understand how the quinoline compounds act on cells by decreasing cell proliferation.
Financial Support: PAPES V/FIOCRUZ; CAPES - Pós Graduação em Biotecnologia e Medicina Investigativa
REDUCED NEURONAL ACTIVATION IN TRIGEMINAL GANGLION IN EARLY PHASE DIABETES
FABIANA FURTADO FREITAS1; MARIA CLAUDIA OLIVEIRA FUSARO2; AUGUSTO MUZILLI3; MARCELO HENRIQUE NAPIMOGA4; JULIANA TRINDADE CLEMENTE NAPIMOGA5.
1,3,5.PIRACICABA DENTAL SCHOOL/UNICAMP, PIRACICABA - SP - BRASIL; 2.FACULTY APPLIED SCIENCES/UNICAMP, LIMEIRA - SP - BRASIL; 4.LABORATORY OF IMMUNOLOGY AND MOLECULAR BIOLOGY/SÃO LEOPOLDO MANDIC, CAMPINAS - SP - BRASIL.
Introduction: Diabetes in early phase is known to result in orthodontic tooth movement and painful neuropathy caused by hypernociceptive inflammatory mediators. Thus this study aimed to evaluate whether this diabetes-induced orthodontic tooth movement increases the neuronal excitability in the trigeminal ganglion. Methods and Results: All experimental procedures and protocols were approved by the Committee on Animal Research of the University of Campinas (#2559-1). Male Wistar rats (± 150 g, n=4-6/group) were treated with an intraperitoneal injection of vehicle (normoglycemic – NG) or 75 mg/kg Streptozotocin (diabetic – DB). Twenty-eight days after the treatment, animals received an orthodontic appliance and the tooth movement was evaluated at days 0, 1, 3, 6 and 12. Immediately after the evaluation of the orthodontic tooth movement, the animals were terminally anesthetized and their trigeminal ganglion and gingival tissue removed and submitted to biochemical analysis (Western Blot and ELISA) to measure the release of glutamate, substance P (SP), calcitonin-gene-related peptide (CGRP), TNF-α, and IL-1β and the expression of the AMPA receptor. The DB rats showed an orthodontic tooth movement greater than that observed for the NG rats (p<0.05: Two-way ANOVA, Bonferroni’s test); Glutamate levels and the expression of AMPA receptors in the trigeminal ganglion were significantly reduced in the DB rats (p<0.05: Two-way ANOVA, Bonferroni’s test). Levels of TNF-α and IL-1β in the gingival tissue were significantly higher in the DB rats (p<0.05: Two-way ANOVA, Bonferroni’s test); levels of SP and CGRP in the same tissue were significantly reduced in the DB rats (p<0.05: Two- way ANOVA, Bonferroni’s test). Conclusion: The results suggest that although diabetes might enhance the inflammatory process, it might develop, even in its early stage, a neuropathy in the trigeminal system caused by a decrease in the neuronal excitability.
Financial support: FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo
REGULATORY T CELLS AND CD39 ARE ESSENTIAL FOR THE ANTI-ARTHRITIC EFFECTS OF METHOTREXATE IN ANTIGEN-INDUCED-ARTHRITIS
RAPHAEL SANCHES PERES1; FOO YEH LIEW2; JHIMMY TALBOT3; VANESSA CARREGARO4; RAFAEL FREITAS OLIVEIRA FRANÇA5; LARISSA GARCIA PINTO6; RENE DONIZETE OLIVEIRA7; JOÃO SANTANA SILVA8; THIAGO MATTAR CUNHA9; JOSÉ CARLOS ALVES-FILHO10; PAULO LOUZADA-JUNIOR11; FERNANDO QUEIROZ CUNHA12.
1,3,4,5,6,7,8,9,10,11,12.SCHOOL OF MEDICINE OF RIBEIRÃO PRETO, RIBEIRÃO PRETO - SP - BRASIL; 2.UNIVERSITY OF GLASGOW, GLASGOW - REINO UNIDO.
Introduction: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints. The first line pharmacotherapy for RA comprises the use of low doses of methotrexate (MTX), an anti-metabolic drug, described as an inhibitor of dihydrofolate reductase/folic acid metabolism. In addition to its anti-folate effect, MTX also dampens inflammation by maintaining high levels of extracellular adenosine (ADO) by a mechanism associated with ATP degradation by ectonucleotidases CD39 and CD73. Moreover, it is suggested that anti-arthritic effects of MTX in RA may be related with number/suppressive activity of regulatory T (Treg) cells. The aim of the present study was to investigate the role of Treg cells on the anti-arthritic effects of MTX in antigen-induced arthritis (AIA). Methods and results: mBSA-immunized C57BL/6 mice were pretreated with MTX (2 mg/kg) weekly for 5 weeks before challenge with mBSA (30 µg/cavity) at day 21 after first immunization. Articular hyperalgesia, neutrophil migration to the joint, plasma titers of anti-mBSA IgG and Treg cells frequency in the spleen were evaluated 7 h after the challenge. Additionally, for Treg cells depletion and CD39 inhibition, mBSA-immunized C57BL/6 mice pretreated or not with MTX were undergoing administration of anti-CD25 (250 µg/ml) or CD39 inhibitor (ARL67156, 1 mg/kg) three times per week during the immunization protocol. MTX-treated mice showed a reduction of arthritis development, evidenced by reduction of neutrophil migration into the joint and articular hyperalgesia. The effect of MTX-treatment was not due to impaired mBSA priming, since the plasma titers of specific anti-mBSA IgG were similar among the groups. Interestingly, MTX-treated mice also had an increase in the frequency of Treg cells (CD4+FoxP3+) in the spleen and Treg cells depletion reversed the anti-arthritic effects triggered by MTX. Moreover, ARL67156 treatment increased the arthritic severity in immunized mice not treated with MTX, and abolished the anti-arthritic effect of MTX as measured by neutrophil migration and articular hyperalgesia. The increased frequency of Treg cells induced by MTX was also abrogated by CD39 inhibition. Conclusion: These findings show that the anti-arthritic effects of MTX are Treg-dependent, and that CD39 is key to the expansion of MTX-induced Treg cell population in experimental arthritis. Financial Support: FAPESP (2012/ 10438-0), FAEPA
REVISITING THE MYCOBACTERICIDAL MECHANISM OF ISONIAZID IN A HUMAN GRANULOMA MODEL
LÍVIA HARUMI YAMASHIRO1; VERÔNICA VARGAS HOREWICZ2; MAGNO DELMIRO GARCIA3; MARINA SONCINI4; ANTONIO GIGLIOTTI ROTHFUCHS5; ANDRÉ BÁFICA6.
1,2,3,4,6.UFSC, FLORIANÓPOLIS - SC - BRASIL; 5.KAROLINSKA INSTITUTE, ESTOCOLMO - SUÉCIA.
Introduction: Following exposure to Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), infected individuals develop the granuloma, a cellular structure involved in controlling bacterial proliferation. Currently, several anti-Mtb drugs are available and the most important is isoniazid (INH), which inhibits bacteria’s cell wall formation through the mycolic acids synthesis inhibition. Clinical studies have demonstrated that 2 days of INH treatment are sufficient to significantly decrease lung bacterial loads in TB patients, although only low doses (nM range) are found within the granuloma. In contrast, such low doses do not directly inhibit Mtb growth in vitro. Therefore, we have undertaken experiments to investigate the mechanisms by which low doses of INH stimulate bacterial killing within the granuloma.
Methods and Results: To investigate the mechanisms by which INH inhibits Mtb survival during cellular infection, we have utilized an in vitro model in which human leukocytes acquire granuloma-like conformation upon bacteria infection. Interestingly, treatment with low concentrations of INH inhibited Mtb growth in granulomas from healthy PPD-negative, but not in PPD-positive donors (n=12). Pro-inflammatory cytokines such as TNF, IL-1beta and IL-6 showed no major differences in treated granulomas. Surprisingly, IL-8 was found to be decreased in INH-treated granulomas from PPD+ donors. Moreover, we observed that frequency of CD4+ or CD8+ T cells remained unaltered during infection, but the frequency of gd-T cells were diminished in the same cultured granulomas. In vitro primed T cells from PPD- donors were found more effective in controlling Mtb growth when compared to those from PPD+, suggesting the existence of a T cell population detrimental to the control of infection in PPD+ individuals.
Conclusions: These results suggest that INH enhances Mtb killing by directly modulating adaptive immune responses within the granuloma. Experiments to address this hypothesis are in progress.
Financial Sup Financial Support: CNPq, HHMI, NIH.
RHEUMATOID ARTHRITIS INDUCE PERSISTENT HYPERNOCICEPTION INTO RATS’ TMJ
RICARDO BONFANTE1; CRISTINA GOMES MACEDO2; HENRIQUE BALASSINI ABDALLA3; MARCELO HENRIQUE NAPIMOGA4; JULIANA TRINDADE CLEMENTE NAPIMOGA5.
1,2,3,5.PIRACICABA DENTAL SCHOOL, STATE UNIVERSITY OF CAMPINAS, PIRACICABA - SP - BRASIL; 4.LABORATORY OF IMMUNOLOGY AND MOLECULAR BIOLOGY, SÃO LEOPOLDO MANDIC INSTITUTE AND RESEARCH CENTER, CAMPINAS - SP - BRASIL.
Introduction: Inflammation of the temporomandibular joint (TMJ) induced by Rheumatoid Arthritis (RA) is known to cause persistent pain and distress. This study aimed to evaluate persistent inflammatory RA-induced hypernociception into the TMJ of rats. Methods and Results: Male Wistar rats were sensitized with an subcutaneously injection of Methylated Bovine Serum Albumin (mBSA, 500µg) in an emulsion containing phosphate buffered saline (PBS) and Freund’s Complete Adjuvant. mBSA dissolved in Freund’s Incomplete Adjuvant was injected in different sites in the back of the rat 7 and 14 days after the first immunization. Twenty-one days after the initial injection, TMJ-arthritis was induced in the immunized animals by an intra-articular injection of mBSA (10µg/TMJ/week) during 3 weeks. RA-induced TMJ hypernociception was assessed by the animals’ nociceptive behavior induced by an intra-articular injection of a low dose of formalin (0.5%) 24h, and 7 and 14 days after the last intra-articular mBSA injection. After behavioral assays, animals were terminally anesthetized and their periarticular tissue removed for leukocytes migration analysis and to biochemical analysis (Western Blot and ELISA) to measure the release of IL-12, IL-18 and expression of the ICAM-1. RA induced hypernociception into TMJ of rats 24h and 7 and 14 days after the last intra-articular injection of mBSA (p<0.05: ANOVA, Tukey’s test). The release of IL-12, IL-18 and the expression of ICAM-1 in periarticular tissue were significantly higher at day 14 when compared to that at 24h after the last intra-articular injection of mBSA (p<0.05: ANOVA, Tukey’s test). Conclusion: This new model allows to study the mechanism involved in the initiation and maintenance of pain induced by Rheumatoid Arthritis in the TMJ.
Financial Support: FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo
RIPARIN B, AN ALKALOID OBTAINED FROM ANIBA RIPARIA, DECREASED THE PAW EDEMA INDUCED BY SEVERAL MEDIATORS.
RENATA FORTES SANTIAGO; AMANDA MOREIRA FONTENELE; ISABELA DE SOUZA BRAÚNA; TARCISIO VIEIRA BRITO; JOSÉ SIMIÃO DA CRUZ JÚNIOR; JALLES ARRUDA BATISTA; HELIANA DE BARROS FERNANDES; JORDANA MAIA DIAS; RIVELILSON MENDES FREITAS; JAND-VENES ROLIM MEDEIROS; ANDRÉ LUIZ REIS BARBOSA.
UNIVERSIDADE FEDERAL DO PIAUÍ, TERESINA - PI - BRASIL.
RENATA FORTES SANTIAGO1; AMANDA MOREIRA FONTENELE1; ISABELA DE SOUZA BRAÚNA1; TARCISIO VIEIRA BRITO1; JOSÉ SIMIÃO DA CRUZ JÚNIOR1; JALLES ARRUDA BATISTA1; HELIANA DE BARROS FERNANDES1; JORDANA MAIA DIAS1; RIVELILSON MENDES FREITAS2; JAND-VENES ROLIM MEDEIROS1; ANDRÉ LUIZ REIS BARBOSA1.
1LAFFEX – Laboratory of Experimental Physiopharmacology, Biotechnology and Biodiversity Center Research (BIOTEC), Federal University of Piauí.
2Department of Pharmacy, Federal University of Piaui, 64.049-550 Teresina, PI, Brazil.
Introduction: Riparin is the name given to the pharmacological potential derived from amides isolated and synthesized, attributed to extracts of Aniba riparia (Nees) Mez Lauraceae plant typical of the Amazon. Through the Schotten- Baumann new derivatives were obtained theriparin A and B. However, there are few studies in support of the anti- inflammatory activity of Riparin B. Methods and Results: We used mice Mus musculus Swiss breed, males, two months old. The project was approved by the Ethics Committee on Animal Experimentation (EAEC), with protocol number 017/13.Mice were pretreated with intraperitoneal injection of Riparin B (1, 3 or 10 mg.kg-1). One hour later, carrageenan (Cg, 500 µg/paw, 50 µl) or dextran (Dxt, 500 µg/paw, 50 µL) and bradykinin (6nmol) or serotonin (1% w/v) was administered subplantar injection in the right paw. The paw volume was measured by pletismometry immediately before injection, and then 1, 2, 3, and 4 hours later by Cg or 30 min, 1, 2, 3, and 4 hours later, for the injection of Dxt and 30, 60, 90 and 120 min for bradykinin, and serotonin. Riparin B (10 mg.kg-1) significantly decrease the paw edema induced by Cg (1h: 0.0060 ± 0.002449 ml; 2h: 0.006667 ± 0.004216 ml) when compared with the only Cg group (1h: 0.0475 ±0.01181; 2h: 0.0800±0.01291). In edema induced by bradykinin, serotonin and dextran also can observer in the Riparin B group decrease in paw edema: bradykinin (30min: 0.0400 ± 0.008165; 60min: 0.0± 0.0); Serotonin (30 min: 0.0450 ± 0.007188; 60min: 0.0250 ± 0.008660) Dextran (30min: 0.0280 ± 0.01319; 1h: 0.01833 ± 0.005426) when compared with the groups bradykinin (30 min: 0.0700 ± 0.0100; 60min: 0.0575 ± 0.006292); Serotonin (30 min: 0.0880 ± 0.009695; 60 min: 0.05667 ± 0.007601); Dextran (30min: 0.1000± 0.01225; 1h: 0.0825± 0.008539). CONCLUSION: The study corroborated that given Riparina B reduced inflammation by decreasing acute paw edema induced by carrageenan, dextran, serotonin and bradykinin.
ROLE OF BRADYKININ B2 RECEPTOR IN ARTICULAR INFLAMMATION INDUCED BY STAPHYLOCOCCUS AUREUS INJECTION
ISABELA GONCALVES SIQUEIRA; DAIANE BOFF; VIVIAN LOUISE SOARES DE OLIVEIRA; MAURO MARTINS TEIXEIRA; FLAVIO ALMEIDA AMARAL.
UNIVERSIDADE FERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.
Introduction: Septic arthritis is an uncommon, but potentially fatal emergency. The most frequent organism associated with the disease is Staphylococcus aureus, which is isolated in 37–56% of cases. A joint becomes infected when an infectious agent enters into the articular cavity, where the pathogen triggers an inflammatory response, with the increase of inflammatory mediators and leukocyte influx. There is a compelling evidence linking bradykinin (BK) with the pathophysiological processes that accompany tissue damage and inflammation, especially the production of pain and hyperalgesia. The objective of this study is to investigate the role of Bradykinin B2 receptor in the pathogenesis of septic arthritis. Methods and Results: Experimental septic arthritis was induced by inoculation of S.aureus (107 CFU; 10 μL) into the right knee joint of C57/Bl6 (WT) and B2 knockout (B2-/-) male mice (n = 5-6 mice/group; 8 weeks old). All experiments were approved by the Institutional Ethic Committee for the use of animals in experimental research (protocol number: 236/2012 - CETEA/UFMG). Seven days after S. aureus injection, B2-/- mice presented an increase in the number of total cells when comparing with the WT mice (WT: V: 5±1.15; I: 49.5±11.12; B2-/-: V: 4.5 ± 2.12, I: 106 ±14.35). This increase was observed both for neutrophils (WT: V: 1.87±2.18; I: 35.92±7.26; B2-/-: V: 0±0, I: 81.16±11.39) and mononuclear cells (WT: V: 3.12±1.06; I: 13.57±4.21; B2-/-: V: 4.5±2.12, I: 24.84±4.88). When analyzing the number of bacteria in the joint, we could not perceive a significant difference between the WT and B2-/- mice (WT: I: 209.75±83.77; B2-/-: I: 202.75±91.70). Conclusion: The results presented above indicated that the bradykinin receptor B2 is an important regulator of inflammation in the context of septic arthritis, especially controlling the leukocyte migration to the articular cavity, although it does not seem to be involved in the control of bacteria in the joint.
Financial support: FAPEMIG, CNPq
ROLE OF CHEMOKINE RECEPTOR D6 IN THE MODULATION OF ARTICULAR INFLAMMATION FOLLOWING STAPHYLOCCOCUS AUREUS
VIVIAN LOUISE SOARES DE OLIVEIRA; DAIANE BOFF; MAURO MARTINS TEIXEIRA; FLAVIO ALMEIDA AMARAL.
UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Septic arthritis is an infection mostly associated with the colonization of Staphylococcus aureus in the joints. Apart from damage caused by the pathogen, the cell activation intensifies the inflammatory response and enhances tissue damage in arthritis. Cellular migration occurs due to a variety of chemotatic agents, mainly chemokines, through chemokine-receptor interaction. Beyond conventional chemokines receptors, there is a group with different intracellular machinery, the silent chemokine receptors, including the receptor D6, which recognizes inflammatory CC chemokines. A significant contribution of D6 in the resolution of inflammatory response is through its role as a chemokine scavenger, doing the internalization and degradation of the CC chemokine family members. The objective this work was investigated the role of D6 receptor in the pathogenesis of experimental septic arthritis. Methods and Results: Experimental septic arthritis was induced by intra-articular injection of S. aureus (107 CFU; 10 μL) in wild type (WT) and D6 deficient (D6-/-) mice (5-6 mice/group). D6-/- mice had decreased number of infiltrated cells into the joint (Neubauer chamber and histology) (103,80x104±90,45 cells/mL to 49,80x104±33,97 cells/mL), including neutrophil (61,30x104±18,28 cells/mL to 38,09 cells/mL), when compared to WT littermates seven days after infection. At this point, D6-/- mice also presented lower bacterial load at the joint compared to WT ones (916,00±190,58 CFU/joint to 253,80±171,81 CFU/joint). Corroborating the reduced inflammation of D6-/- mice following S. aureus infection, these mice had reduced hypernociception (electronic von Frey) compared to WT mice. Conclusion: Altogether, the results indicate that the absence of D6 receptor could regulate the number of infiltrated cells at the initial time points, which, in turn, reduced bacterial load and articular lesion-associated hypernociception. More studies should be performed to elucidate the mechanisms of these events.
Financial support: FAPEMIG, CNPq
ROLE OF INTEGRIN ALPHADBETA2 IN THE EARLY PHASE OF PULMONARY INFLAMMATION CAUSED BY SILICA PARTICLES IN MICE.
TATIANA PAULA TEIXEIRA FERREIRA1; VINICUS FRIAS DE CARVALHO2; ANA CAROLINA SANTOS DE ARANTES3; GUY ZIMMERMAN4; ADRIANA VIEIRA DE ABREU5; RENATO SÉRGIO BALÃO CORDEIRO6; MARCO AURÉLIO MARTINS7; HUGO CAIRE DE CASTRO FARIA NETO8; PATRÍCIA MACHADO RODRIGUES E SILVA9.
1,2,3,5,6,7,8,9.OSWALDO CRUZ INSTITUTE/FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL; 4.UNIVERSITY OF UTAH, UTAH - ESTADOS UNIDOS.
Introduction: Integrins are plasma membrane broadly distributed on different cell types and mediate critical functions, including adhesion, homing, signaling, and gene expression. Leukocyte integrins are required for host defense against invasion by pathogens and for wound healing and repair. AlphaDß2 (CD11d/CD18) is the most recently discovered integrin, appearing restricted to subsets of macrophages and shown to be regulated during macrophage differentiation in vitro. In this study, we investigated the contribution of alphaDß2 to the early phase of experimental silicosis in mice. -/- +/+ Methods and Results: alphaDß2 knockout (alphaDß2 ) and C57/Black 6 wild type (alphaDß2 ) mice were instilled intranasally with silica particles (10 mg). Analyses were performed on day 7 and included lung function by invasive plethysmography (Buxco System) and leukocyte infiltration in the bronchoalveolar lavage (BAL). Total and differential leukocyte counts were performed and classical histological techniques included H&E and Picrus Sirius staining. +/+ Collagen was quantified by Sircol method. Intranasal silica into alphaDß2 mice led to an increase in the leukocyte numbers in the BAL, mainly macrophages and neutrophils, phenomenon which paralleled with a marked leukocyte -/- infiltration and numerous granulomas presented in the lung tissue. The alphaDß2 mice exhibited a less intense macrophage infiltration in the BAL as well as reduced infiltration in the lung tissue. Attenuation of granuloma formation -/- and tissue collagen deposition was also detected in the alphaDß2 mice stimulated with silica. Values of granuloma +/+ area were 52.0% ± 0.3% and 30.0% ± 0.4% (mean ± SEM; n=8, p<0.05), in silica-stimulated alphaDß2 and -/- alphaDß2 mice, respectively. As attested by immunohistochemistry, expression of TGFß in the lung tissue of the -/- +/+ alphaDß2 mice was clearly reduced as compared to that of alphaDß2 mice. Interestingly, silica-stimulated mice -/- showed an increase in the basal levels of lung resistance and elastance, response clearly decreased in the alphaDß2 animals. Conclusion: Our findings indicate that alphaDß2 integrin seems to play a role in the early phase of lung inflammation caused by silica in mice and also offer evidence that macrophages are important and effective part of the process of immediate immunity responses such as silicosis.
Financial support: CNPq, PAPES/FIOCRUZ, FAPERJ (Brazil).
ROLE OF NEUTROPHILS IN THE PATHOGENESIS OF EXPERIMENTAL SEPTIC ARTHRITIS
DAIANE BOFF; VIVIAN LOUISE SOARES DE OLIVEIRA; ISABELA SIQUEIRA; MAURO MARTINS TEIXEIRA; FLÁVIO ALMEIDA AMARAL.
UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Septic Arthritis is the joint disease which occurs when the pathogen invade the joint causing infection. The main microorganism involved in that pathology is Staphylococcus aureus. The disease present high mortality and morbid, about 50% of patients have irreversible loss of joint. The most important cell involved in immune response to bacterial infections is the neutrophil. This cell is the first to arrive at the site of inflammation, thereby helping to combat infection by means of several enzymes and mediators. Neutrophils also accumulate at diseased sites leading to tissue damage. The CXC chemokines that signals via CXCR2 activate neutrophils and promote their adhesion to the endothelium. The objective of this work was to investigate the role of chemokine receptor CXCR2 in inflammation caused by S. aureus in an experimental model of septic arthritis. Methods and Results: Experimental septic arthritis was induced by intra-articular injection of S. aureus (107 CFU; 10 μL) in C57/Bl6 mice (5-6 mice/group). A group of mice were treated with an alosteric inhibitor (DF2156A) of CXCR2 receptor 1h after injection of bacteria and every 12h by 7 days. The treatment with DF2156A presented decreased number of total cells (V:82,2 ± 10,52; T:54 ± 8,49) and neutrophils into the inflamed joint when compared to non-treated mice(V: 63,99 ± 9,09; T: 31,30 ± 4,86). This reduced cellular migration in DF2156A-treated mice is associated to a lower TNF-α (V: 388,28 ± 122,15; T: 190,31 ± 33,13)and IL-1β (V: 497,58 ± 294,10; T: 180,35 ± 194,95) and hypernociception (V: 5,37 ± 0,87; T: 6,36 ± 0,85). However, DF2156A-treated group showed slightly increased in bacterial load at the joint when compared non-treated mice (V: 321,50 ± 152,63; T: 968 ± 509,15). Conclusions: Neutrophils are important cell involved in articular inflammation and degradation following S. aureus infection, instead of these cells is the main immune cells involved in clearance of bacteria in cavity. The inhibition of neutrophil migration could be used as a complementary therapeutic option to combat articular inflammation caused by S. aureus infection.
Financial support: CAPES, CNPq and FAPEMIG
ROLE OF PRO-INFLAMMATORY CYTOKINE INTERLEUKIN-17 ON HUMAN GLIOMA CELLS (U138)
MARINA PETERSEN GEHRING1; TALITA PEREIRA2; RAFAEL ZANIN3; MAURICIO REIS BOGO4; MARIA MARTHA CAMPOS5; FERNANDA BUENO MORRONE6.
1,3,6.1LABORATÓRIO DE FARMACOLOGIA APLICADA, PUCRS, PORTO ALEGRE - RS - BRASIL; 2,4.2LABORATÓRIO DE GENÔMICA E BIOLOGIA MOLECULAR, PUCRS, PORTO ALEGRE - RS - BRASIL; 5.3INSTITUTO DE TOXICOLOGIA E FARMACOLOGIA, PUCRS, PORTO ALEGRE - RS - BRASIL.
Introduction: Glioma is the most common and lethal type of primary brain tumor (Karrlander et al., PLoS One 4:e8536 – 2009) and has disproportionately high mortality rate of more than 70% of cases in two years after diagnosis (Filippi et al., 2011). Cytokine production acts as means of communication between the tumor and the immune system (Mantovani et al., 2008). The cytokine IL-17 is characterized as a proinflammatory cytokine that induces the release of secondary pro-inflammatory chemokines and growth factors (Ivanov and Linden, 2009). IL-17 action in cancer is still quite controversial. Results relative to a pro or anti-tumor activity of this cytokine are not unanimous (Zou and Restifo, 2010). Therefore, our study aims to investigate the role of cytokine IL-17 in human glioma cells (U138). Methods and Results: We analyzed IL-17 receptor expression on glioma cells through qPCR-RT. To evaluate the effects of IL-17 (0.01 to 100 ng/ml) on cell viability and proliferation it was performed MTT assay and cell counting. IL-17-induced glioma cytokines release (IL-2, IL-4, IL-6, IL-10, TNF, INF-γ and IL-17) and IL-17-induced intracellular signal transduction pathways (Akt, Erk and p38) were determined by flow cytometry. IL-17 cytokine involvement on glioma cells migration was analyzed using wound-healing assay. The results were obtained from at least two different experiments with at least three wells in each group. Data were analyzed by ANOVA, followed by Tukey–Kramer post test using GraphPad Software (San Diego, CA, U.S.A.). p values <0.05 were taken to indicate statistical significance. Our results confirmed that U-138 glioma cells express IL-17 receptor (IL-17R). However, the treatment with IL-17 did not show any changes in human glioma cells viability and proliferation. On the other hand, IL-17 increased the secretion of the immunosuppressive interleukin IL-10 by glioma cells after 24 h treatment (526.5 pg/3x104 cells ± 180.5), when compared to not treated cells (312.5 pg/3x104 ± 148.5). IL-17 also increased significantly glioma cell migration in vitro after 48 h (95.6% ± 1.8) when compared to not treated cells (80.0% ± 4.2). This occurs , possibly through Erk intracellular signal, since we observed a significant increase in Erk activation after IL-17 (10 ng/ml) treatment (MFI 9648 ± 429), when compared to not treated cells (MFI 7132 ± 412). Conclusion: Our data demonstrate that U-138 glioma cells express IL-17R. After IL-17 cytokine binding on its receptor, occurs an increase in IL-10 secretion and a significant induction of cell migration in vitro, probably via Erk. We are performing more experiments to understand the role of this cytokine in gliomas, since it can be used to identify, develop and/or refine therapies for cancer treatment.
Financial support: CAPES, FAPERGS, FINEP and PUCRS
ROLE OF PROSTANOIDS AND NEUTROPHILS IN THE BOTHROPS JARARACUSSU SNAKE VENOM-INDUCED PAW EDEMA
CARLOS WAGNER WANDERLEY1; CAMILA MEIRELLES SILVA2; DEYSI TENAZOA WONG3; KAROLINE SABÓIA ARAGÃO4; RAFAEL MATOS XIMENEZ5; RAIMUNDO CAMPOS PALHETA JR.6; RONALDO ALBUQUERQUE RIBEIRO7; ROBERTO CESAR PEREIRA LIMA JR8.
1,2,3,4,5,7,8.DEPT OF PHYSIOLOGY AND PHARMACOLOGY - FACULTY OF MEDICINE - FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 6.FACULTY OF VETERINARY MEDICINE - FEDERAL UNIVERSITY OF VALE DO SÃO FRANCISCO, PETROLINA - PE - BRASIL.
Introduction: In Brazil 25.000 snakebites occur annually, and 90.5% are related to Bothrops sp. Envenomation by snakes causes local pain, edema, hemorrhage, inflammatory reaction and tissue necrosis that cannot be reversed by anti-venom. The mechanisms of tissue damage are recognized to vary among snake venoms. The study of the mechanisms and mediators involved in the local inflammatory reaction associated to these venoms may help to establish the most adequate treatment. Then, we aimed to study the role of histamine, cyclooxygenase (COX), mast cells, afferent C fibers, and neutrophils in the Bothrops jararacussu (VBj)-induced paw edema in mice. Methods: Swiss female mice (25-27g, n=7) were injected either saline (0.15 ml/paw), VBj (0.125, 0.5, 2 and 8 µg/paw) or Loratadine (5 mg/kg, p.o., 1h before), Dexamethasone, (1 mg/kg, i.p., 1 h before), Indomethacine, (10 mg/kg, i.p., 30min before), Celecoxib (5 mg/kg, i.p., 30min before), Fucoidan (25 mg/kg, i.p., 30min before), compound 48/80 (6x0.6 + 2x1.2 mg/kg, i.p., 36, 24 and 12h before), Capsaicin (2x 25 + 50 mg/kg, i.p., 7 days before) which were injected before VBj. The resultant paw edema was measured hourly by plethysmography (paw volume %). Paws tissues were collected for the measurement of myeloperoxidase (MPO, U/mg tissue), IL-1 and TNF-α levels (pg/mg tissue) and COX-2 immunohistochemistry detection. Statistical analysis was performed with ANOVA/Newman-Keuls or Kruskal Wallis/Dunn’s tests. P<0.05 was accepted (CEPA: 62/12). Results: The VBj caused a dose and time dependent edema (P<0.05) which peaked at 0.5 h (74.5±5.8) and was stable up to 4.5 h (62.6±8.0) vs saline (0.5 h: 13.8±2.5 and 4.5 h: 0±0). Dexamethasone (positive control), indomethacin, celecoxib and fucoidan markedly inhibited edema formation (39.1±5.5; 32±3.4; 35.7±6.2; and 46.2±8.5, respectively) and MPO activity (21±2,4; 13,2±1,5; 19,1±3,4; 15,1±3,0, respectively) when compared with VBj group (edema: 76.24±6.0; MPO: 31.5±3.6, respectively). In addition, VBj increased local production of TNF-α and IL-1 (1.7±0.1 and 8.8±1.4) and COX-2 immunoexpression (3[3- 3]) versus saline group (TNF: 0.1±0.1; IL-1: 0.3±0.1; COX-2: 1[0-2]). However, VBj-induced edema and inflammatory reaction were not prevented by Loratadine, compound 48/80 or capsaicin treatment vs VBj group (P>0.05). Conclusion: The VBj induces an early-onset edema which seems to be dependent on prostanoids production and neutrophil migration. Financial Support: CNPq/FUNCAP/CAPES
ROLE OF THE NITRIC OXIDE (NO) IN THE LUNG FUNCTION AND AIRWAY HYPERREACTIVITY IN SILICOTIC MICE
DAVIDSON FURTADO DIAS; TATIANA PAULA TEIXEIRA FERREIRA; BIANCA TORRES CIAMBARELLA; ANA CAROLINA SANTOS DE ARANTES; MARCO AURÉLIO MARTINS; PATRÍCIA MACHADO RODRIGUES E SILVA.
INSTITUTO OSWALDO CRUZ, FUNDAÇÃO OSWALDO CRUZ (FIOCRUZ), RIO DE JANEIRO - RJ - BRASIL.
Introduction: Silicosis is a restrictive pulmonary disease caused by silica particle inhalation and is characterized by an inflammatory response followed by intense fibrosis and granuloma formation. In this study we aimed to investigate the involvement of NO in the lung function and airways hyperreactivity in silicotic mice. Morphological changes in the lung tissue were also evaluated. Methods and Results: Male wild-type C57BL/6 (iNOS+/+) and iNOS knockout (iNOS- /-) mice were used. Anesthetized animals received a single intranasal instillation of silica particles (10 mg) or 0.9% saline (control). The analyzes were made 7 and 28 days after silica provocation and included: i) lung function and airways hyperrreactivity to methacholine by invasive whole body plethysmography, ii) quantification of nitric oxide levels in the bronchoalveolar lavage (BAL) by Griess method and iii) morphological and morphometric analyses by classical histology (haematoxilyn-eosin). A temporal correlation with morphological changes in the lung tissue, reflected an intense inflammatory cell infiltrate, followed by progressive fibrosis and granuloma formation. Higher levels of nitric oxide (NO) and peroxynitrite as well as iNOS expression were detected in the BAL and lung tissue of silica-stimulated mice stimulated, respectively. iNOS-/- mice had the responses of increased basal levels of lung resistance and elastance and airways hyperreactivity abolished as compared to iNOS+/+ mice. The inflammatory and fibrotic granulomatous responses were also inhibited in iNOS-/- animals. Therapeutic treatment with the iNOS inhibitor 1400W (2 mg/kg, i.p.), for 5 consecutive days starting 23 days post-silica, suppressed changes in lung function and tissue fibrosis. Values of granuloma area reduced from 17.33% ± 5.16% to 4.18% ± 1.45% (mean ± SEM; n=7, p<0.05), in silica and silica treated-mice, respectively. Conclusion: Our results showed that decreased lung function and airways hyperreactivity directly correlated with NO and peroxynitrite generation as well as the iNOS expression in silicotic mice. The depletion of the gene encoding to or inhibition of iNOS enzymatic activity abolished the response of lung function and tissue fibrosis. Altogether, our findings indicate that NO is an important mediator in the context of silicosis and suggest that it can be considered a potential therapeutic target for the treatment of fibrotic diseases.
Financial support: FIOCRUZ, CNPq, FAPERJ.
ROLIPRAM AND OTHERS CAMP ELEVATING AGENTS INDUCES PKA-DEPENDENT ANNEXIN A1 EXPRESSION
KÁTIA MACIEL LIMA; THAÍS ROLLA DE CAUX; RAQUEL GREGÓRIO ARIBADA; JULIANA PRISCILA VAGO; ALINE FORTUNATO DO CARMO; LUCIANA PÁDUA TAVARES; FREDERICO MARIANETTI SORIANI; MAURO MARTINS TEIXEIRA; LIRLÂNDIA PIRES DE SOUSA.
UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.
Introduction: Annexin A1 (AnxA1) is a 37kDa protein induced by Glucocorticoids (GCs) that have been shown to possess powerful anti-inflammatory actions in a wide variety of models of acute and chronic inflammation. In this study, we investigated the ability of Rolipram, (a specific inhibitor of phosphodiesterase 4 - PDE4), db-cAMP (cAMP mimetic) and Forskolin (an adenylate cyclase activator) to modulate the AnxA1 expression using in vivo and in vitro assays. Methods and results: BALB/c mice were challenged by intrapleural (i.pl.) administration of LPS (250ηg/cavity) or PBS. Rolipram (6mg/kg) and the synthetic glucocorticoid dexamethasone (Dexa-2mg/kg) were administered systemically (i.p.). The cells in the pleural cavity were harvested after 4h of treatment with drugs and processed for total and differential leukocyte counts and western blot analysis. In vitro studies were performed using PMA-differentiated THP-1 cells (a human monocytoid cell line). The cells were treated with Rolipram, db-cAMP or Forskolin at different times and concentrations. Dexamethasone (1mM) was used as a control of AnxA1 induction by GC. Furthermore, to investigate whether the effect of Rolipram was via PKA, the best-known cAMP effector, we used two PKA inhibitors: H89 and Rp. Inflammatory cells extract from Rolipram and Dexa-treated mice showed that these anti-inflammatory drugs induced Anxa1 expression and prevented its cleavage. In vitro analysis showed that Rolipram induced AnxA1 expression at dose and time-dependent manner in both cell lines. Consistently with cAMP induction by Rolipram, we verified an increase of the phosphorylation of CREB (cAMP responsive element-binding protein), associated with the increase of AnxA1. db-cAMP and Forskolin was also able to induce AnxA1 expression at dose and time-dependent manner in THP-1 cells. In addition, the induction of AnxA1 by Rolipram was prevented by both PKA inhibitors used, suggesting the involvement of PKA in such induction. Conclusion: Our results showed that AnxA1 is induced by Rolipram and others cAMP elevating agents in vitro and in vivo and that this induction by Rolipram is PKA dependent. Financial support: CNPq, FAPEMIG and CAPES.
RUTIN IMPAIRS PANCREATIC INFLAMMATION DURING ACUTE PANCREATITIS INDUCED BY L-ARGININE IN MICE
FABIULA FRANCISCA ABREU; MARCELA SANTOS SANTANA; ALAN SANTOS OLIVEIRA; ANA CARLA ARAUJO SOUZA; ENILTON APARECIDO CAMARGO.
FEDERAL UNIVERSITY OF SERGIPE, SÃO CRISTOVÃO - SE - BRASIL.
Introduction: Acute pancreatitis (AP) is an inflammatory disease of the pancreas that generates systemic complications involving mainly the lung injury. In spite of many recent advances, the clinical management of AP is primarily supportive and still ineffective. In this context, new therapeutic alternatives are need and rutin, a flavonoid with well-known antioxidant and anti-inflammatory activities, presents a potential to treat AP. In this study we investigated the effect of rutin on the acute pancreatitis induced by L-arginine in mice.
Methods and Results: Pancreatitis was induced in male Swiss mice (25-30 g) by two injections of L-arginine (4 g/kg, 1 h apart). The treatment of animals with rutin, at the doses of 37.5, 75 or 150 mg/kg (p.o.), was performed at 24, 36, 48 and 60 h after induction. Euthanasia occurred after 72 h post-induction. The treatment with rutin, at the doses of 75 and 150 mg/kg, significantly reduced (p<0.05) the serum amylase concentrations (352±48 and 315±26 U/L, respectively), when compared with the vehicle-treated group (1006±232 U/L). A significant reduction of pancreatic myeloperoxidase (MPO) activity (p<0.001) was observed in all treated groups (0.47±0.08, 0.18±0.06 and 0.41±0.09 UMPO/mg tissue, respectively for the doses of 37.5, 75 and 150 mg/kg), when compared with the vehicle group (2.20±0.16 UMPO/mg tissue), but there was no significant change in the lung MPO activity of the groups that received rutin (3.8±0.4, 4.1±0.6, 4.0±0.4 UMPO/mg tissue), in comparison to the vehicle group (4.2±0.7 UMPO/mg tissue). Treatment with rutin also reduced (p<0.05) the concentration of MDA in pancreas (36±8.7, 55±16 and 57±14 pmol of MDA/mg tissue, for treatments with 37.5, 75 and 150 mg/kg of rutin, respectively), when compared with vehicle- treated mice (115±16 pmol of MDA/mg tissue). No alteration of MDA in lung was observed in mice treated with rutin (49±5, 45±5 and 47±6 MDA/mg tissue, for treatments with 37.5, 75 and 150 mg/kg respectively), in comparison to the vehicle group (46±8 pmol of MDA/mg tissue). Pancreatic edema index (wet/dry weight) was not altered by treatment with rutin (3.3±0.4, 3.7±0.4 and 3.5±0.4 for treatments with 37.5, 75 and 150 mg/kg respectively), when compared with the vehicle group (3.4±0.4).
Conclusion: These results suggest that the flavonoid rutin reduces pancreatic inflammation and may have potential to treat AP.
Financial support: FAPITEC/SE.
SACCHAROMYCES BOULARDII AMELIORATES GASTRIC DYSMOTILITY AND REVERTS THE INFLAMMATION PRESENTS IN INTESTINAL MUCOSITIS INDUCED BY 5-FLUOROURACIL IN MICE
PRISCILLA FERNANDA JUSTINO1; CECILIA MENDES MORAIS2; ALVARO FRANCO XAVIER3; ANDRE FERREIRA NOGUEIRA4; GABRIELA DO NASCIMENTO REBOUÇAS VIANA5; CAIO BRAGA MALVEIRA6; DEYSEN BEZERRA GIRÃO7; JULLIETE RAULINO ALCÂNTARA8; MARCELLUS HENRIQUE LOIOLA SOUZA9; RONALDO ALBUQUERQUE RIBEIRO10; RICARDO DE FREITAS LIMA11; EMMANOEL PRATA SOUZA12; PEDRO MARCOS GOMES SOARES13.
1,2,3,4,5,6,7,9,10,11.FEDERAL UNIVERSITY OF CEARA - DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FORTALEZA - CE - BRASIL; 8.STATE UNIVERSITY OF CEARA - DEPARTMENT OF HEALTH AND NUTRITION, FORTALEZA - CE - BRASIL; 12,13.FEDERAL UNIVERSITY OF CEARA - DEPARTMENT OF MORPHOLOGY, FORTALEZA - CE - BRASIL.
Introduction:Intestinal mucositis is a toxicity effect associated to 5-fluorouracil (5-FU) clinical use.In that toxicity effect is observed inflammatory and functional symptoms and your physiopathology is characterized by epithelial ulcerations, abdominal pain, nauseas and diarrhea.Saccharomyces boulardii (SB) is used widely in the treatment of gastrointestinal disorders associated with diarrhea.The possible mechanism gastroprotective of the SB upon the intestinal mucositis induced by 5-FU not is totally defined.Objective was to evaluate effect of SB in inflammatory and functional aspects of intestinal mucositis induced by 5-FU in mice.Methods and Results:Swiss male mice (25-30g) were treated with 5-FU (450mg/Kg,ip) or 5-FU+SB(800mg/Kg,daily for 3 days),other group received saline.After animals were sacrificed and sample of ileum (I) were collected for assessment histopathological, MPO activity (U/mg tissue) and cytokines concentrations (pg/mL) by ELISA.To determination of gastric emptying (retention fraction %) was administration phenol red (PR,0.75 mg/mL,300 µL) by gavage and after of the 20min the mice were sacrificed being the stomach and intestinal processed for measurement of PR concentrations by spectrophotometry.To determination of intestinal permeability was used the lactulose/mannitol(L/M) test.Statistical analysis (tests ANOVA followed by Bonferroni and p<0.05).(CEPA:Protocol 34/10).5-FU induced significant histopathological alterations that were reverted by the treatment with SB: C=0(0-1),5-FU=2.5(1-3),SB+5-FU=1(0-2). 5-FU induced an increase on the MPO(C=1.51±0.17;5-FU=14.09±2.99),TNF-α(C=667.80±89.20;5-FU=2496.00±320.10),IL-1β(C=1059.00±122.00;5- FU=2197.0±149.3),CXCL-1(C=512.3±113.7;5-FU=1991.0±837.5) and reduced IL-10(C=177.0±5.77;5- FU=84.54±8.32), that were reverted by SB treatment (SB+5-FU:MPO=3.67±0.85,TNF-α=888.3±157.5,IL- 1β=726.0±98.4,CXCL-1=66.3±50.5,IL-10=126.5±4.7). Besides, the treatment with SB improved delay in gastric emptying (C=25.21±2.55%;5-FU=54.91±3.43%;SB+5-FU=31.38±2.80%), gastrointestinal transit measured by center of mass (C=2.32±0.08;5-FU=1.83±0.06) and the ratio of L/M (C=0.54±0.03;5FU=2.12±0.77). SB restores the center mass and ratio of L/M (SB+5-FU=2.31±0.06;SB+5FU=0.62±0.02),respectively.Conclusion:Our results suggest that the treatment with SB reverted the inflammation and amelioration the transit and the permeability gastrointestinal in the intestinal mucositis by 5-FU in mice. Financial Support:Funcap,Capes,CNPq.
SAFETY AND BIOLOGICAL ACTIVITY OF FARAMEA ATLANTICA J.G. JARDIM & ZAPPI (RUBIACEAE) IN CARRAGEENAN-INDUCED RAT PAW EDEMA
KÁTIA ALVES RIBEIRO1; ANTÔNIO MATEUS GOMES PEREIRA2; DALYNE MENEZES TELES3; SAMUEL MATEUS PEREIRA FILHO4; DÉBORA DA SILVA FREITAS5; KARUZA MARIA ALVES PEREIRA6; ANDREZA MARIA LIMA PIRES7; LISSIANA MAGNA VASCONCELOS AGUIAR8; GERARDO CRISTINO FILHO9; MARIA ROSE JANE RIBEIRO ALBUQUERQUE10; HELLÍADA VASCONCELOS CHAVES11; ANTONIO ALFREDO RODRIGUES E SILVA12; MIRNA MARQUES BEZERRA13.
1,4,5,6,8,9,11,12,13.FEDERAL UNIVERSITY OF CEARÁ CAMPUS SOBRAL, SOBRAL - CE - BRASIL; 2,3,7,10.STATE UNIVERSITY VALE ACARAÚ, SOBRAL - CE - BRASIL.
Introduction: Rubiaceae Juss constitutes the fourth major family of angiosperms, comprising approximately 650 genera. Faramea atlantica species (Fa) belongs to the Farameas genus that includes 200 species, in which 123 are found in Brazil. The aim of this study was to evaluate the safety and biological activity of Faramea atlantica J.G. Jardim & Zappi (Rubiaceae) in carrageenan-induced rat paw edema. Methods: Five groups (n=6) of Male Wistar rats (180-200g) were treated with EHA (1, 10 or 100 mg/Kg; p.o.); sterile saline 0.9% or dexamethasone (1 mg/kg; s.c.), 60 min before Cg (500 ug/paw; 100 µL) injection in the subplantar region of rat paws. Edema was measured pletismographically 1, 2, 3 e 4 h after Cg injection. After the last measure the animals were euthanized under anesthesia and the subplantar tissues were excised and histologically (H&E). In other series of experiments two groups (n=10) of female Swiss mice were treated with EHA (10 mg/kg, p.o.) or sterile saline 0,9% daily for 14 days. On day 15 the animals were euthanized under anesthesia. Blood samples were collected to perform serum analyses (aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine and alkaline phosphatase. Organs were excised, weighted and processed to histopathological analyses (H&E). P < 0.05; ANOVA followed by Dunnet test. Results: Cg injection promoted intense paw edema, reaching a peak on the 3rd h. HAE previous treatment potentiated Cg inflammatory effect. In fact, HAE (1 mg/kg) increased (P < 0.05) the Cg edema when compared to Cg group. The toxicity assay did not reveal significant alterations on the ponderal evolution of the two mice groups. The blood parameters analyses did not demonstrated significant differences (P > 0.05) on the serum levels of ALT, AST, urea, creatinine and alkaline phospatase between groups. Conclusion: Although HAE was found to be safe it increased carrageenan-induced rat paw edema making it particularly useful instructural/functional studies of this class of Farameas genus. Financial Support: CAPES, CNPq, FUNCAP, UFC.
SALBUTAMOL INCREASES MKP-1 EXPRESSION AND INHIBITS THE PRODUCTION OF INFLAMMATORY MEDIATORS TNF AND NO IN ACTIVATED MACROPHAGES.
TIINA KERÄNEN; TUIJA HÖMMÖ; MARI HÄMÄLÄINEN; EEVA MOILANEN; RIKU KORHONEN.
THE IMMUNOPHARMACOLOGY RESEARCH GROUP, UNIVERSITY OF TAMPERE AND TAMPERE UNIVERSITY HOSPITAL, TAMPERE - FINLÂNDIA.
Introduction: Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a nuclear phosphatase that dephosphorylates (i.e. inactivates) p38 MAP kinase, and inhibits cytokine production and inflammatory response.
Salbutamol is a bronchodilating drug that activates β2-receptors and elevates cAMP levels. We have recently shown that a PDE4 inhibitor rolipram up-regulated MKP-1 expression by a mechanism dependent on cAMP-PKA-CREB pathway, and using MKP-1 knockout mice we found that MKP-1 mediated anti-inflammatory effects of rolipram in vivo. Therefore we investigated the effect of salbutamol on the expression of MKP-1 and the production of inflammatory mediators tumor necrosis factor (TNF) and nitric oxide (NO).
Methods: The effects of salbutamol on MKP-1 expression, p38 MAP kinase phosphorylation and the production of TNF and NO were investigated in primary mouse peritoneal macrophages (PM) and J774 mouse macrophages. MKP- 1 expression was determined by quantitative RT-PCR and Western blot, and p38 MAP kinase and CREB phosphorylation by Western blot. TNF and NO accumulated into the culture medium were measured by ELISA and Griess reaction, respectively.
Results: Salbutamol (1-1000 nM) alone and in combination with LPS (10 ng/mL) increased intracellular cAMP and, interestingly also MKP-1 expression in J774 macrophages in a dose-dependent manner. cAMP analog 8-Br-cAMP (100 µM) also enhanced LPS-induced MKP-1 expression. Increase in MKP-1 expression by salbutamol was reversed by a PKA inhibitor. Salbutamol (100 nM) inhibited LPS-induced production of TNF and NO. Salbutamol reduced LPS- induced phosphorylation of p38 MAP kinase. p38 MAP kinase inhibitor BIRB 796 (1 µM) inhibited TNF and NO production in J774 macrophages and TNF production in PMs.
Conclusions: Salbutamol increased the expression of MKP-1 and concomitantly inhibited the phosphorylation of p38 MAP kinase and the production of TNF and NO in mouse J774 macrophages and PMs. The results suggest that the anti-inflammatory effects of salbutamol on TNF and NO production are mediated by increased MKP-1 expression, which limits p38 MAP kinase pathway and, subsequently, inflammatory gene expression.
Financial support: Academy of Finland, The Competitive Research Funding of the Pirkanmaa Hospital Districk and National Doctoral Programme of Musculoskeletal Disorders and Biomaterials.
SELECTIVE INHIBITION OF N-TYPE VOLTAGE-GATED CALCIUM CHANNELS ATTENUATES CYCLOPHOSPHAMIDE-INDUCED IMMUNOLOGICAL CHANGES IN MICE
RODRIGO BRACCINI MADEIRA SILVA1; NATHALIA DENISE DE MOURA SPEROTTO2; EDINÉIA LEMOS ANDRADE3; ALESSANDRA HÜBNER DE SOUZA4; MARCUS VINICIUS GOMEZ5; JOÃO BATISTA CALIXTO6; FERNANDA BUENO MORRONE7; MARIA MARTHA CAMPOS8.
1,2,7,8.PUCRS, PORTO ALEGRE - RS - BRASIL; 3,6.UFSC, FLORIANÓPOLIS - SC - BRASIL; 4,5.UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Hemorrhagic cystitis (HC) is the most common form of cyclophosphamide (CPA)-induced bladder toxicity (Nat Rev Clin Oncol. 6:638-47, 2009). Voltage-gated calcium channels (VGCC) are pivotal regulators of the inflammatory process and have emerged as attractive therapeutic targets (Pain. 151:633-43, 2010). Therewith, in the present study, we investigated the effects of the selective N-type VGCC blocker Tx3-6 (J Pharmacol Exp Ther. 314:1370-7, 2005), isolated from the spider P. nigriventer, on macroscopic inflammation, cell migration and cytokine production in CPA-induced HC in mice, aiming to correlate these inflammatory parameters to functional cystometry alterations in this experimental model. Methods and results: Male Swiss mice (n = 6 per group) were used. All the experimental procedures were approved by the Local Ethics Committee (12/00292). HC was induced by a single intraperitoneal injection of CPA (300 mg/kg). Tx3-6 (50 pmol/site) was injected intrathecally, 2 h after CPA injection. The intrathecal injection of Tx3-6 significantly inhibited the edema scores and bladder wet weight in CPA-treated mice (37 ± 8% and 25 ± 6%, respectively). Moreover, Tx3-6 was able to significantly reduce the neutrophil migration elicited by CPA, according to evaluation of myeloperoxidase activity at 6 h after cystitis induction (65 ± 9%). Of note, the same treatment with Tx3-6 significantly diminished the bladder levels of the pro-inflammatory cytokines TNF-α and IL-1β, by 66 ± 11% and 58 ± 6%, respectively, at the same time-point of evaluation. The anti-inflammatory effects of Tx3-6 were allied to a significant improvement of several urodynamic parameters, such as micturition pressure, basal pressure, threshold pressure, intercontraction interval, bladder capacity and the voiding efficiency of bladder, as measured by in vivo cystometry, during 30 min, after 6 h of CPA injection. Conclusion: In summary, our results show, for the first time, that selective spinal inhibition of N-type VDCC attenuates CPA-induced immunological changes, what can be related to a general functional improvement of bladder function. These findings point out Tx3-6 from P. nigriventer spider as a potential strategy to control the pathogenic alterations observed in HC. Financial Support: PRPPG/PUCRS, CAPES-AUX-PE Toxinologia and FINEP/PUCRSINFRA #01.11.0014-00.
SELECTIVE INHIBITION OF N-TYPE VOLTAGE-GATED CALCIUM CHANNELS PREVENTS THE SCRATCHING BEHAVIOR INDUCED BY TRYPSIN IN MICE, VIA NEUTROPHIL-INDEPENDENT MECHANISMS
IZAQUE DE SOUSA MACIEL1; VANESSA MACHADO AZEVEDO2; FERNANDA BUENO MORRONE3; ALESSANDRA HÜBNER DE SOUZA4; MARCUS VINÍCIUS GOMEZ5; MARIA MARTHA CAMPOS6.
1,2,3,6.PUCRS, PORTO ALEGRE - RS - BRASIL; 4,5.UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Pruritus is a common symptom present in most skin diseases, and also in other pathological states, such as autoimmune disorders (Vet Dermatol 22: 121, 2011). Voltage-gated calcium channels (VGCC) have been recently implicated in the mouse model of ozaxolane-induced scratching (J Pharmacol Sci 115: 27, 2011). The toxin MVIIA derived from Conus magus or PhTx3.6 obtained from Phoneutria nigriventer spider are selective blockers of N- type VGCC, with recognized analgesic actions (Cell Mol Neurobiol. 33:59, 2013). Herein, we evaluated the effects of spinal treatment with PhTx3.6 and MVIIA on trypsin-elicited scratching behavior and myeloperoxidase (MPO) activity in mice.
Methods and Results: Male Swiss mice were used (8/group, 25-30 g). The experimental protocols were approved by the local Ethics Committee (12/00293). The animals received an intradermal (i.d.) injection of trypsin (200 μg/site, 50 μl), and were observed for 40 min. Scratching behavior was measured as the number of scratches close to the injected site. Mice received an intrathecal (i.t) injection of MVIIA (0.3 to10 ρmol/site) or PhTx3.6 (10 to 300 ρmol/site). The MPO assay was performed to assess neutrophil migration after behavioral tests. MVIIA significantly prevented trypsin-evoked scratching at the doses 1 and 10 ρmol/site (58 ± 6% and 66 ± 7%, respectively). The toxin PhTx3.6 markedly reduced trypsin-induced pruritus at the doses of 50, 100 and 300 ρmol/site (54 ± 14%; 49 ± 7% e 49 ± 6%, respectively). Time-course experiments revealed that MVIIA (10 ρmol/site) significantly prevented the scratches when administered at 1, 2 and 4 h (77 ± 6%; 79 ± 6% and 39 ± 11%, respectivity), but not at 6, 12 and 24 h before. Relevantly, the toxin PhTx3.6 (50 ρmol/site) significantly prevented pruritus when dosed at 1, 2, 4, 6 and 12 h (77 ± 6%; 65 ± 10%; 62 ± 5%; 47 ± 6% and 55 ± 9%, respectively), but not at 24 h. The assessment of MPO activity did not reveal significant differences among the experimental groups.
Conclusion: This study demonstrates, for the first time, the relevance of N-type VGCC in trypsin-elicited itching, via mechanisms unlikely dependent on neutrophil migration. Interestingly, the fraction PhTx3.6 from P. nigriventer displayed a long-term effect, when compared to MVIIA conotoxin, making it a potential tool for the study and control of pruritus-related conditions.
Financial support: PROPESQ/PUCRS, CAPES-AUX-PE Toxinologia, CNPq and FINEP/PUCRSINFRA #01.11.0014-00.
SELECTIVE PHARMACOLOGICAL INHIBITION OF CHEMOKINE RECEPTORS CXCR2 MODULATES THE IMMUNOLOGICAL CHANGES EVOKED BY PARAQUAT INTOXICATION IN RATS
KESIANE MAYRA COSTA; IZAQUE SOUSA MACIEL; MARIA MARTHA CAMPOS; MAURICIO REIS BOGO.
PUCRS, PORTO ALEGRE - RS - BRASIL.
Aim: Paraquat (PQ) is a widely used organic heterocyclic herbicide (Chin J Crit Care Med. 26:542-543, 2006), which has been correlated to huge neurotoxicity and lung inflammation via generation of reactive oxygen species (ROS) and formation of apoptosis-related molecules (Brain Res. 1011:170-176, 2004). The aim of this study was to evaluate the lung neutrophil migration and the total blood cell counts in a rat model of PQ-induced toxicity, as well as to assess the effects of treatment with the selective CXCR2 receptor antagonist SB225002 on these parameters. Methods and Results: Male Wistar rats were used (weighing 180 g at the beginning of experiments; N=4-8 per group). All the experimental protocols were approved by the local animal ethics committee (CEUA/12/00295). PQ-toxicity was induced by a total dose of 50 mg/kg, given by i.p. route, one injection of 10 mg/kg each three days, until 15 days (Free Radic Biol Med. 51:1428-1436, 2011). Control animals received saline solution (10 ml/kg) at the same schedule of administration. Separate groups of animals were treated with the selective CXCR2 antagonist SB225002 (1 or 3 mg/kg, i.p.), administered 30 min before each PQ injection (Eur J Pain. 14:23-31, 2010). On the 15th day, the animals were euthanized and the total blood was collected for differential hematological analysis using Giemsa staining. The lungs were collected for mieloperoxidase (MPO) assay, as indicative of neutrophil migration. Differential hematological analysis showed that saline control group displayed a prevalence of lymphocytes (85%), with a low percentage of neutrophils (15%), whereas PQ-intoxicated rats presented higher levels of neutrophils (65%), some lymphocytes (25%) and immature cells (10%). In the groups that received SB225002 (1 or 3 mg/kg) the hematological analysis demonstrated an equal proportion (about 50% each) of neutrophils and lymphocytes. The administration of PQ led to a significant increase of lung MPO activity, an effect that was prevented by SB225002, at the dose of 3 mg/kg, with an inhibition percentage of 47 ± 14%. Conclusion: Our data provide evidence that pharmacological inhibition of CXCR2 with the selective antagonist SB225002 was able to prevent the immunological changes elicited by PQ in rats. This antagonist might well represent a valuable strategy for treating PQ-intoxicated individuals. Financial support: ISM was recipient of fellowship from CAPES. MMC and MRB are productivity research fellows from CNPq.
SMALL MOLECULE SRC FAMILY KINASE INHIBITORS AS PONTENTIAL TREATMENT FOR IMMUNOLOGICAL DIESASES
CSABA SZÁNTAI-KIS1; LILIÁN ZSÁKAI2; ZSUZSANNA PATÓ3; ANNA SIPOS4; JENÖ MAROSFALVI5; ISTVÁN SZABADKAI6; FRIGYES WÁCZEK7; ZOLTÁN HORVÁTH8; ATTILA MÓCSAI9; ANDRÁS DANCSÓ10; LÁSZLÓ ÖRFI11; GYÖRGY KÉRI12.
1,2,3,4,5,6,7,8,11,12.VICHEM CHEMIE RESEARCH LTD., BUDAPEST - HUNGRIA; 9.DEPARTMENT OF PHYSIOLOGY, SEMMELWEIS UNIVERSITY, BUDAPEST - HUNGRIA; 10.EGIS PHARMACEUTICALS PLC, BUDAPEST - HUNGRIA.
Introduction: Autoimmune inflammatory diseases cause worldwide medical problem. Current therapies have serious side effects, are ineffective in some cases or too expensive. Src-family tyrosine kinases play important role in development of autoimmune and inflammatory diseases. Our objective was to find new scaffolds from Vichem’s NCL™ library against Src family kinases which can be developed further into novel anti-inflammatory drug candidates.
Methods and Results: At first we developed IMAP FP technology based in-vitro assays for screening inhibitors on Src, Hck, Fgr, Lyn kinases, then we assembled 1000 member compound focused sublibrary which was built around the core structures of the best hits of a previous screening on immune complex-induced in-vitro functions of leukocytes. We screened these compounds at 10 μM concentration on all four kinases then we determined IC50 values of the most effective ones. 14 compounds have lesser than 100 nM average IC50 on the four Src-family tyrosine kinases. The scaffolds of these compounds are: Pyrido[2,3-d]pyrimidin, Quinoline-3-carbonitrile, Pyrimidine- 2,4-diamine, Quinoline-4-ol, 3-(Phenyl-phenylamino-methylene)-1,3-dihydro-indol-2-one. In order to predict potentially effective new Hck inhibitor scaffolds we performed in silico pre-screening using the Schrödinger molecular modeling suite. We used the X-ray diffraction based model of human Hck to determine the docking scores of Vichem’s NCL™ compound library.
Conclusion: We identified novel inhibitors of the selected Src-family tyrosine kinases which have potential anti- inflammatory effect.
Financial support: The research leading to these results has received funding from the European Community's Seventh Framework Programme [FP7-2007-2013] under grant agreement n°HEALTH-F4-2011-282095-TARKINAID.
SODIUM NITROPRUSSIDE PREVENTS ALENDRONATE-INDUCED GASTRIC DAMAGE IN RATS: INHIBITION CYTOKINE PRODUCTION AND OXIDATIVE STRESS
RENAN OLIVEIRA SILVA1; LUCAS ANTONIO DUARTE NICOLAU2; LARISSE TAVARES LUCETTI3; ANA PAULA MACEDO SANTANA4; KAROLINE SABOIA ARAGÃO5; ANDRÉ LUIZ DOS REIS BARBOSA6; PEDRO MARCOS GOMES SOARES7; RONALDO ALBUQUERQUE RIBEIRO8; MARCELLUS HENRIQUE LOIOLA PONTE SOUZA9; JAND-VENES ROLIM MEDEIROS10.
1,2,6,10.FEDERAL UNIVERSITY OF PIAUÍ, PARNAÍBA - PI - BRASIL; 3,4,5,7,8,9.FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Alendronate is a bisphosphonate that can cause serious adverse effects in patients, including ulcers, gastric inflammation, nausea and abdominal pain, although the mechanism underlying these reactions remains unknown (Dig. Dis. Sci. 47:1665-1678, 2002). The aim of this study was to investigate the effect of sodium nitroprusside (nitric oxide donor) in pathogenesis of alendronate-induced gastric damage in rats and role of proinflammatory cytokines and oxidative stress in this effect. Methods and Results: This study was approved by the local ethics committee (protocol no 0067/10). Wistar albino rats (170-200g) were administered alendronate (30 mg/kg) by gavage for 4 days, either alone or following treatment with sodium nitroprusside (10 mg/kg, p.o.). On the last day of treatment rats were killed and stomachs were removed. The gastric damage was measured using a computer planimetry programme. Samples of stomach were also taken for histopathological assessment, glutathione levels (GSH), malonyldialdehyde concentration (MDA), myeloperoxidase (MPO) activity and cytokines levels (TNF-α and IL- 1β). Chronic oral administration of alendronate (n=6) induced macroscopic (38.7±7.2 mm2) and microscopic (edema, loss of epithelial cells and inflammatory infiltrate) gastric damage. However the sodium nitroprusside prevented the macroscopic (10.8±2.7 mm2) gastric damage and confirmed by histological evaluation (n=6). Similarly, the alendronate increased MDA levels (n=6) (121.1±4.3 nmol/g), MPO activities (n=6) (31.5±3.8 U/mg) and TNF-α and IL-1β concentration (n=6) (901.9±106.2 pg/ml and 2311.0±302.3 pg/ml, respectively) and decrease GSH levels (n=6) (180.3±21.9 µg/g). Treatment with sodium nitroprusside changed all biochemical parameters, MDA (86.6±3.7 nmol/g), MPO (17.7±2.6 U/mg), tissue level of TNF-α and IL-1β (31% and 39% inhibition, respectively) and GSH (471.6±45.27 µg/g). Conclusion: The present study suggests that sodium nitroprusside reduces alendronate-induces gastric damage by reduction cytokine levels, neutrophil infiltration and oxidative stress. Financial Support: CNPq, FAPEPI.
STRONTIUM RANELATE INDUCES BONE REPAIR OF RAT CALVARIA CRITICAL SIZE DEFECT VIA AXIS RANK-RANKL-OPG
MARIANA VASCONCELOS GUIMARÃES1; ANA CRISTINA MELLO FIALLOS2; IRACEMA MATOS MELO3; NICOLE MELO FIALLOS4; JOSÉ CARLOS VIANA RIBEIRO5; THAYANNE BRASIL6; KARINN SOARES ARAÚJO7; RENATA FERREIRA CARVALHO LEITÃO8; LUIS CARLOS SPOLIDORIO9; LÚCIO MITSUO KURITA10; VILMA LIMA11.
1,2,3,4,5,10.FACULTY OF PHARMACY, DENTISTRY AND NURSING, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 6,11.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 7,8.DEPARTMENT OF MORPHOLOGY, FEDERAL UNIVERSTIY OF CEARÁ, FORTALEZA - CE - BRASIL; 9.DEPARTMENT OF PHYSIOLOGY AND PATHOLOGY, ARARAQUARA SCHOOL OF DENTISTRY, UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL.
Introduction: The bone repair is a multifunctional process, involving a variety of cells and inflammatory mediators. Strontium ranelate (SrR) has stood out mainly by its dual mechanism of action to stimulate bone formation and to inhibit bone resorption. Thus, the aim of this study was to evaluate the bone repair induced by SrR in critical size defects (CSD) in calvaria of rats. Methods and Results: CSD (Diameter 8 mm) were made in the high of the calvaria of 72 anaesthetized male Wistar rats. Immediately upon the defect, groups of rats received into the CSD a single application of SrR (2.1 or 6.3 mg) or no treatment (NT control). Thereafter, rats were accompanied, and the SrR groups and respective controls were killed at 15, 45, 90 and 120d. Then calvaria samples were removed and CT scans by Cone Beam Computed Tomography (CBCT; mm²) were made. An extra group of NT rats (n=6) sacrificed at 0h was used as baseline for CBCT. Next, the same specimens followed to histology (HE) considering leukocyte and extracellular matrix counting, and immunohistochemical for RANKL and OPG were also performed. The data were presented as mean±standard error of the mean. CBCT showed that the defect area of NT CSD groups from 15 to 120d were not reduced (CSD 90d= 74.2±2.7; 120d=72.04±1.7; p>0.05), when compared to CSD newly induced (0h=78.6±0,96). SrR 6.3 group induced a bone formation evaluated by reducing (p<0.05) the defect area at 90d (67.8±2.3) and 120d (62.3±4.2), when compared to 0h group, but not when compared to its respective controls at 90d or 120d (p>0.05). Microscopically, NT CSD groups revealed an important leukocyte infiltration on 90d (NT=216±29) and 120d (NT=68±17.7), what was significantly reduced by SrR on 90d (SrR 2.1=114±21.8; SrR 6.3=76±5) and 120d (SrR 2.1= 30±7; SrR 6.3= 28±7.5). Also, NT CSD groups presented extracellular matrix at 90d (NT=270±23.2) and 120d (340±50.9), which was significantly increased by SrR at 90d (SrR 2.1=360±50.9; SrR 6.3=420±37.4) and 120d (SrR 2.1=420±58.3; SrR 6.3=480±58.3), respectively. In accordance with these results, the CSD of SrR 6.3 animals at 120d showed intense immunostaining for OPG and negative for RANKL, whereas the calvariae of control groups showed positive immunostaining only for RANKL. Conclusion: The local treatment with major concentration of SrR revealed its osteoinductive role conducing to bone repair accompanied by decreasing RANKL expression and increase OPG expression. Financial support: CAPES; CNPq.
STUDY OF RED PROPOLIS PROPERTIES IN VITRO AND ON LIGATURE-INDUCED PERIODONTITIS MODEL
CINTHIA FIGUEIREDO; VAGNER SANTOS; ANA CÂNDIDA ARAÚJO; CELSO QUEIROZ; MIRIAM LOPES; TATIANA RIBEIRO; ANDRÉ FARACO.
UFMG, BELO HORIZONTE - MG - BRASIL.
INTRODUCTION: Periodontitis is a common disease that affects supporting tissues and alveolar bone (Ann Periodontol. 4:1-6, 1999). In spite of already tested and used drugs against periodontitis, there is not one that shows effective pharmacological features like high penetrability.and less side effects (Acta Facult Med. 27:85-92,2010; J Am Dental Assoc. 137: 5S-9S, 2006) . Propolis has been used as a medicinal agent because of its analgesic, antiinflamatory, antimicrobial and healing properties (Ev Based Compl Altern Med. 2: 33-38, 2005). These related properties and its low toxicity indicate a possible propolis action against chronical periodontitis. In this study, we begin to evaluate properties of propolis that may support other future studies using the product against periodontal disease (Drug Dev Ind Pharm. 34:267-278, 2008). METHODS AND RESULTS: Growing cells were seeded (96-well plates) and exposure to ethanolic propolis extract in concentrations 0,06 to 0,25% for 72 hours (n=12) and the proliferation index determined by MTT assay (Canc Res. 50(12): 3681-3690, 1990). We noticed a statistically significant proliferation using the 0,25% concentration for all lines of cells (L929: 0,407±0,080 %v/v; CHO: 1,44±0,37 % v/v; and FIB:1,43±0,37 % v/v) against the control (0,265±0,04; 0,622±0,080; 0,619±0,56; p<0,05, ANOVA, Bonferroni’s post test). On ligature induced periodontitis model experiment, Wistar rats (n= 3) received a ligature in the the mandibular first molars. Seven days later, the ligatures were removed and we administrated red propolis gel on concentrations of 2 ; 5 and 10% and vehicle. The bone loss and inserction loss were determined by measure of pixels in the ImageJ program (results for bone loss: 1,73±0,52; 1,43±0,22; 0,94±0,42; 0,94±0,15 and for inserction loss: 3,42±0,59; 3,09±0,69; 2,49±0,16; 2,42±0,16, for control; 2; 5 and 10% dosis, respectively, in mm) and we found a tendency of reduction for bone loss (no statistics significancy). CONCLUSION: More studies about red propolis against periodontitis are needed. Financial support: CAPES,FAPEMIG and CNPq.
STUDY OF THE INFLAMMATORY ACTIVITY OF DINOPONERA QUADRICEPS VENOM IN MICE
PALOMA LEÃO SOUSA1; YVES PATRIC QUINET2; JAQUELINE FREIRE VALE3; ALEXANDRE HAVT4; ALBA FABÍOLA TORRES5; ALICE MARIA COSTA MARTINS6; ANA MARIA SAMPAIO ASSREUY7.
1,2,3,7.UNIVERSIDADE ESTADUAL DO CEARÁ, FORTALEZA - CE - BRASIL; 4,5,6.UNIVERSIDADE FEDERAL DO CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Dinoponera quadriceps (Hymenoptera, Formicidae, Ponerinae) is a primitive and endemic ant of Northeastern Brazil, that uses its sting and associated venom gland to capture preys and for defense. Venom of Dinoponera is of clinical importance, since its sting causes intense local pain, accompanied by erythema and edema. Besides, the literature reports inflammatory effects of other Hymenoptera venoms. Here, the inflammatory effect of D. quadriceps venom (DqV) in mice was evaluated.
Methods and Results: DqV contains 64% protein with bands (15 and 100 kDa). DqV subplantar injection elicited edema at 5 µg/kg (3 fold), 50 µg/kg (4 fold) or 500 µg/kg (7 fold) from zero to 360 min compared to saline (AUC: 4323 ± 727.28193 µL). DqV (50 µg/kg) increased also vascular permeability (4 fold) first hour after induction (DqV: 7.7±2.8 vs saline: 3.5 ± 0.6 µg/g paw). The paw tissue histology showed moderate inflammatory focus caused by DqV (50 µg/kg) in the first hour of paw edema, but severe tissue changes (edema, inflammatory infiltrate and focal areas of hemorrhage) in the third hour. Intraperitoneal injection of DqV (50 µg/kg) stimulated neutrophil (7 fold) and mononuclear (1.4 fold) migration vs saline (neutrophil: 131.6 ± 56.5; mononuclear: 898.9 ± 86.8/mL). DqV edematogenic effect (AUC: 15888±609.0; saline: 2306±762.6 µL) was inhibited by dexamethasone (92%), thalidomide (82%), cyproheptadine (62%), AA861 (58%), celecoxib (34%) or L-NAME (34%), but the neutrophil migration (DqV: 616.0±34.5 neutrophil/mL) was only by dexamethasone (57%) and thalidomide (32%). DqV-elicited neutrophil migration at 50 µg/kg was potentiated 1.7 fold by the animals pre-treatment with 3% thyoglicolate (DqV: 601.7±29.4 vs
DqVTg: 1061±188.5 neutrophil/mL). Incubation of cultivated macrophages (RAW 264.7) with DqV (IC50: 35 µg/uL) did not change the regulation of IL-1β mRNA expression (DqV: 2.1 ± 0.6 vs PBS: 0.8 ± 0.1 µg/mL) or TNF-α (DqV: 1.3±0.2918 vs PBS: 1.1±0.0 µg/mL). Macrophages incubated with DqV (from 3.12 to 50 mg/mL) showed no significant decrease in cell viability or LDH measurements.
Conclusion: DqV possesses inflammatory properties, eliciting edema, increase in vascular permeability and neutrophil migration. These effects imply participation of histamine, serotonine, cytokines, leukotrienes, prostaglandins, nitric oxide and the positive influence of macrophage.
Financial support: FUNCAP
SUCCINATE AUGMENTS SUSCEPTIBILITY TO EXPERIMENTAL INTESTINAL INFLAMMATION
VANESSA BEATRIZ FREITAS ALVES1; PATRÍCIA REIS SOUZA2; JOSÉ CARLOS FARIAS ALVES FILHO3; CRISTINA RIBEIRO DE BARROS CARDOSO4.
1,2,4.1FACULDADE DE CIÊNCIAS FARMACÊUTICAS DE RIBEIRÃO PRETO, RIBEIRÃO PRETO - SP - BRASIL; 3.FACULDADE DE MEDICINA DE RIBEIRÃO PRETO, RIBEIRÃO PRETO - SP - BRASIL.
Introduction: Inflammatory Bowel Disease (IBD) is a multifactorial disease in which genetic basis, environmental factors and deregulated immune response act together for its development. Succinate is an intermediate metabolite of the citric acid cycle that can exert various functions in the body physiology. It is an endogenous ligand for GPR91, a receptor located on dendritic cells membranes, able to play signaling functions and pro-inflammatory activities, which possibly are involved in the exacerbated activation of the immune system in many diseases. Objectives: To assess the role of succinate in the pathogenesis of experimentally induced IBD. Methodology: Colitis (DSS 3%) was induced in C57BL/6 mice which were exposed to succinate at 0,025% or 0,054% in drinking water for 9 days, followed by drug withdraw and exposure to natural water for evaluation of tissue repair until the 15th experimental day. Mice were clinically evaluated for disease development. The gut inflammation was assessed by histopathology, eosinophil peroxidase (EPO), myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activity, which accounts for eosinophils, neutrophils and macrophages accumulation in the gut, respectively, and ELISA for IL-1β, IFN-у, TNF-α, IL-17 and IL-4 detection in the colon. Systemic alteration post succinate exposure was evaluated by differential counts of circulating leukocytes on days 9 and 15. Results: Succinate treated mice presented increased clinical disease score and higher mortality when compared to colitis not treated animals. Succinate 0,025% led to a decrease in circulating eosinophils on day 9 comparing to colitis mice and an increase in systemic neutrophilis when comparing to naïve controls. In addition, the monocyte subpopulation was increased in succinate mice (0,025%) within 15 days comparing to controls. The local MPO activity was also augmented in succinate 0,025% mice on day 9 as well as there was reduced NAG activity on day 15 when compared to controls. Although the production of IL-1β, TNF-α, IFN-у and IL-4 in the colon remained the same for all groups, IL-17 levels were elevated in mice treated with succinate at 0,054%, on day 15. Conclusion: Succinate may act as a metabolite able to augment the susceptibility to intestinal inflammation by linking physiology and immune system functions, thus contributing to the understanding of IBD pathogenesis.
Financial support: FAPESP/NAPDIN (Núcleo de Apoio à Pesquisa em Doenças Inflamatórias)
SULFATED FUCANS AND ITS IMMUNOPHARMACOLOGICAL MODULATION: A STUDY IN A HEPATIC DAMAGE MODEL
THUANE DE SOUSA PINHEIRO; LUIZA SHEYLA EVENNI PORFIRIO WILL CASTRO; MARILIA DA SILVA NASCIMENTO SANTOS; ALLISSON JHONATAN GOMES CASTRO; JOEDYSON EMMANUEL DE MACEDO MAGALHÃES; CELINA MARIA PINTO GUERRA DORE; ALMINO AFONSO DE OLIVEIRA PAIVA; EDDA LISBOA LEITE.
FEDERAL UNIVERSITY OF RIO GRANDE DO NORTE, NATAL - RN - BRASIL.
Introduction: Studies show that compounds isolated from brown algae, sulfated polysaccharides as fucans, exhibit different biological activities, including anti- inflammatory, anticoagulant and antitumoral activities. Thus, the present study aimed to show the sulfated fucans (SF) imunofarmacology extracted from brown seaweed Lobophora variegata and its effect during inflammation using a liver damage model.
Methods and Results: Induction of hepatic damage was induced with carbon tetrachloride (2 mL/kg body weight diluted in olive oil, 1:1 sc) in male Wistar rats (n=6) divided into five groups. Analysis of total bilirubin (TB), direct (DB) and indirect (IB), alanine-aminotransferase (ALT), aspartate-aminotransferase (AST) and γ-glutamyl-transferase (γ- GT) using kits and removal of liver tissue for histologic analysis. Doses of 25, 50 and 75 mg/kg reduced the dosage of total bilirubin. The enzymatic measurement showed that decreasing in ALT in 70.21 %, 72.14 % and 76.93 % compared to the positive control and using the same doses. AST with the same doses presented reductions of 19.55 %, 32.57 % and 44.58 % compared to the positive control group. The γ-GT at doses of 75 mg/kg showed decrease of 50 % compared to the positive control. These results were confirmed by histological analysis of slides of liver tissue as the parameter analysis of tissue damage.
Conclusions: SF showed potent effects on the modulation of hepatic damage in a dose-dependent profile, indicating that this compound may be a possible pharmacological agent.
Financial support: The authors would like to thank CNPq and the CAPES for the financial support.
SULFATED POLYSACCHARIDE FRACTION FROM RED SEAWEED GRACILARIA CORNEA PROTECTS AGAINST CARRAGEENAN INDUCED ACUTE INFLAMMATION IN RATS: COX-2, IL1Β AND TNFΑ MRNA EXPRESSION ANALYSIS
CHISTIANE OLIVEIRA COURA1; RICARDO BASTO SOUZA2; EDFRANCK DE SOUSA OLIVEIRA VANDERLEI3; IANNA WIVIANNE FERNANDES DE ARAÚJO4; ANNYTA FERNANDES FROTA5; JOSÉ ARIÉVILO GURGEL RODRIGUES6; HELLÍADA VASCONCELOS CHAVES7; NORMA MARIA BARROS BENEVIDES8.
1,2,3,4,6,8.BIOCHEMISTRY AND MOLECULAR BIOLOGY DEPARTMENT OF THE UNIVERSITY FEDERAL OF CEARÁ, FORTALEZA - CE - BRASIL; 5.MEDICINE FACULTY OF THE BIOTECHNOLOGY FROM MEDICINE FACULTY OF SOBRAL, SOBRAL - CE - BRASIL; 7.MEDICINE FACULTY OF BIOCHEMISTRY AND MOLECULAR BIOLOGY DEPARTMENT OF THE UNIVERSITY FEDERAL OF CEARÁ, SOBRAL - CE - BRASIL.
Introduction: Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used medications due to their efficacy for wide range inflammatory conditions. However, the prolonged use of NSAID may induce gastro- intestinal ulcers, bleeding, and renal disorders. Thus, there is intense interest in the discovery of new natural anti- inflammatory compounds with few collateral effects. Many biological activities have been reported to sulfated polysaccharides from marine algae. In this study, we evaluated the anti-edematogenic effects of sulfated polysaccharide fraction isolated from the red seaweed Gracilaria cornea (FI-Gc) in paw edema model induced by carrageenan (Cg) in rats and COX-2, TNFα and IL1β mRNA expression profile. Methods and Results: The total sulfated polysaccharides were extracted by enzymatic digestion and then purified by ion exchange chromatography on DEAE-cellulose column. The anti-inflammatory property was assayed using the carrageenan induced paw edema models in rats. One hour before Cg (500 µg/paw; 100 µ/L) injection in the paw, Male Wistar rats (n=6) were pre- treated with FI-Gc (3, 9 or 27 mg/Kg; s.c.), sterile saline or dexamethasone (1 mg/Kg; s.c). After, rats were euthanized and subplantar tissues were collected to determination of myeloperoxidase activity (MPO) and gene expression analyses. Total RNA was obtained by Trizol® kit. cDNA was synthesized by superscript® III reverse transcript. COX- 2, TNFα and IL1β mRNA expression were analyzed by qPCR and normalized with GAPDH (housekeeping).The results showed that groups treated with FI-Gc reduced significantly (p<0.05) paw edema induced by Cg, especially in the third hour, at all doses (3, 9 or 27 mg/Kg) by 51.8%, 60.9% and 61.7%, respectively, in comparison Cg group.These results were confirmed by MPO activity showing that all doses inhibited neutrophil accumulation in the paw tissue. The qPCR analyses showed a down-regulation in the TNFα (1.25±0.04 fold), COX-2 (0.93±0.01 fold) and IL1β (4.47±0.25 fold) mRNA expression profile in the group treated with FI-Gc (9 mg/Kg, s.c.) in comparison to Cg group (TNFα: 2.24±0.21; COX-2: 1.41±0.05; IL1β (25.5±0.36 fold, respectively). Conclusion: This study showed that sulfated polysaccharide fraction from Gracilaria cornea has anti-inflammatory effect and we suggest this effect occur through a negative modulation of COX-2, TNFα and IL1β gene expression.
This work was support by CAPES and FUNCAP
SULFATED POLYSACCHARIDE FROM RED SEAWEED SOLIERIA FILIFORMIS: NOCICEPTION AND INFLAMMATION EFFECTS ON EXPERIMENTAL MODEL OF ZYMOSAN-INDUCED ARTHRITIS IN THE RATS TEMPOROMANDIBULAR JOINT
IANNA WIVIANNE FERNANDES DE ARAÚJO1; HELLÍADA VASCONCELOS CHAVES2; RICARDO BASTO SOUZA3; DANIELLE ROCHA DO VAL4; AURILENE GOMES CAJADO5; JEDSON ANTONIO DE SOUZA ARAGÃO6; SUZANA CAPISTRANO TEXEIRA7; JOSÉ ARIÉVILO GURGEL RODRIGUES8; EDFRANCK DE SOUSA OLIVEIRA VANDERLEI9; KARUZA MARIA ALVES PEREIRA10; RODRIGO MARANGUAPE SILVA DA CUNHA11; MIRNA MARQUES BEZERRA12; NORMA MARIA BARROS BENEVIDES13.
1,3,8,9,13.DEPARTAMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 2.FACULTY OF DENTISTRY, FEDERAL UNIVERSITY OF CEARÁ, SOBRAL - CE - BRASIL; 4,6,7,10,12.FACULTY OF MEDICINE, FEDERAL UNIVERSITY OF CEARÁ, SOBRAL - CE - BRASIL; 5.FACULTY OF MEDICINE, FEDERAL UNIVERSITY OF CEARÁ VALE OF ACARAÚ, SOBRAL - CE - BRASIL; 11.BIOLOGICAL SCIENCE CENTER, STATE UNIVERSITY VALE OF ACARAÚ, SOBRAL - CE - BRASIL.
Introduction: Seaweed can serve as sources for structurally diverse bioactive compounds with valuable pharmaceutical and biomedical potentials. The aim of this study was to isolate a sulfated polysaccharide from the red seaweed Solieria filiformis (SP-Sf) and to analyze its nociceptive and inflammatory effects using experimental model of zymosan-induced temporomandibular joint (TMJ) arthritis in rats. Methods and Results: SP-Sf was extracted by enzymatic digestion, followed by ion exchange chromatography (DEAE-cellulose). We used male Wistar rats (200-220 g) (CEPA 80/10). Animals (n=6) were treated with SP-Sf (1, 3 or 9 mg/kg; s.c.) 1 h before induction of arthritis. Rats were anesthetized and received an intra-articular (i.art.) injection of zymosan (2 mg/40 μL) or saline (sham) into the left TMJ. Mechanical hypernociception in the TMJ was evaluated by measuring the threshold of force intensity that needed to be applied to the TMJ region until the occurrence of a reflex response of the animal. The force threshold value was recorded before the i.art. injections of either zymosan or vehicle and after 4 h. After 6 h, the rats were sacrificed under anesthesia and exsanguinated. The superficial tissues were dissected, the TMJ cavity was washed to collect the synovial fluid (total cell counting and myeloperoxidase (MPO) assay) and TMJ tissues were excised to perform, histological changes, immunohistochemistry (TNF-α, IL-1β and HO-1) and extraction of the RNA. TNF-α, IL- 1β and HO-1 mRNA expression were analyzed by qRT-PCR and normalized with GAPDH. Pretreatment with SP-Sf (1, 3 or 9 mg/kg) inhibited (p<0.05) the nociceptive response of rats (69%, 66.6% and 78.08%, respectively). The injection of zymosan resulted in a significant increase in the number of leukocytes (25.460±5.116) in the synovial fluid. SP-Sf (1, 3 or 9 mg/kg) no reduced (p>0.05) neutrophil accumulation, as demonstrated by MPO activity. Inflammatory cell influx was observed in the synovial membrane, the periarticular tissue, and the thickness in synovial membrane in the treated groups with SP-Sf. Immunohistochemical assay showed an increase in TNF-α, IL-1β and HO-1 expression, besides an increase in the relative mRNA expression of TNF-α, IL-1β and HO-1 by qRT-PCR in animals that received SP-Sf. Conclusion: Thus, these data suggest that this sulfated polysaccharide may be key tool by which to study the inflammatory processes associated with nociception.
Financial support: CNPq, CAPES and FUNCAP
SULPHATED POLYSACCHARIDE (PLS) FRACTION EXTRACTED FROM SEAWEED AGARDHIELLA RAMOSISSIMA DIMISHED PAW EDEMA INDUCED BY CARRAGEENAN, DEXTRAN, HISTAMINE AND SEROTONIN STIMULUS.
JALLES ARRUDA BATISTA1; EULINA GABRIELA NASCIMENTO DIAS2; TARCISIO VIEIRA BRITO3; RAFAEL SILVA PRUDÊNCIO4; RENAN OLIVEIRA SILVA5; RONALDO ALBUQUERQUE RIBEIRO6; MARCELLUS HENRIQUE LOIOLA PONTE SOUZA7; REGINA CÉLIA MONTEIRO PAULA8; LUCIANO SOUSA CHAVES9; MÁRCIA RUBIA MELO10; ANA LUCIA PONTE FREITAS11; JAND-VENES ROLIM MEDEIROS12; ANDRÉ LUIZ REIS BARBOSA13.
1,2,3,4,5,12,13.UNIVERSIDADE FEDERAL DO PIAUÍ - UFPI, PARNAÍBA - PI - BRASIL; 6,7,9,10,11.UNIVERSIDADE FEDERAL DO CEARÁ - UFC, FORTALEZA - CE - BRASIL; 8.UNIVERSIDADE FEDERAL DO CEARÁ- UFC, FORTALEZA - CE - BRASIL.
JALLES ARRUDA BATISTAA, EULINA GABRIELA NASCIMENTO DIASA, TARCISIO VIEIRA BRITOA, RAFAEL SILVA PRUDÊNCIOA, RENAN OLIVEIRA SILVAA, RONALDO ALBUQUERQUE RIBEIROB, MARCELLUS HENRIQUE LOIOLA PONTE SOUZAB, REGINA CÉLIA MONTEIRO PAULAD, LUCIANO SOUSA CHAVESC, MÁRCIA RUBIA MELOC, ANA LUCIA PONTE FREITASC, JAND-VENES ROLIM MEDEIROSA, ANDRÉ LUIZ REIS BARBOSAA.
ALAFFEX – Laboratory of Experimental Physiopharmacology, Biotechnology and Biodiversity Center Research (BIOTEC), Federal University of Piauí.
BLAFICA – Laboratory of Pharmacology of Inflammation and Cancer, Department of Physiology and Pharmacology, Federal University of Ceará.
CLaboratory of Proteins and Carbohydrates of Marine Algae, Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, CE, Brazil.
DLaboratory of Polimers, Departament of Organic and Inorganic Chemistry, Federal University of Ceará, Fortaleza, Brazil.
INTRODUCTION: Various studies have investigated new drugs that can help to avoid or minimize excessive inflammatory process. Marine natural products have been the focus of pharmacological interest to discover new chemicals, because many have been described as having anti-inflamatória even. Knowing this, the objective of this study was to evaluate the anti-inflammatory PLS extracted from red seaweed Agardhiella ramosissima in classical models of acute inflammation in mice. METHODS: The paw oedema was induced by the injection into the right hind paw of mice (n=6) 50 μL of a suspension of carrageenan (Cg,500 μg/paw), dextran ( Dxt, 500 μg/paw), serotonin (1% w/v) or histamine (1% w/v). To verified the anti-inflammatory effect of PLS, mice were pre-treated intraperitoneally with (i.p.) PLS (1, 10 and 30 mg•kg-1) or indomethacin (10 mg/kg) before 1 hour carrageenan treatment. Paw volume was measured immediately before and at 1, 2, 3, and 4 h after carrageenan treatment or 30 min, 1, 2, 3 and 4 hrs after Dxt, histamine or serotonin injections. RESULTS: PLS prevented paw edema induced by Cg (3h: 0,025±0,003), Dxt (0.0024 ± 0.0014), histamine (0.012 ± .0055) and serotonin 0.012 ± 0.0036) when compared those groups with groups that only received the Cg (3h: 0,090±0,003), Dxt (30 min: 0.090 ± 0.007 ml), histamine (0.090 ± 0.008 ml) or serotonin (0.090 ± 0.017 ml). CONCLUSION: According our results we can infer that PLS extracted from red seaweed Agardhiella ramosissima has anti-inflammatory effect by modulating the action of several mediators involved in acute inflammatory process.
THE ANTIINFLAMMATORY MECHANISM OF 15D-PGJ2 ON RA-INDUCED TMJ HYPERALGESIA.
MARIANA DA SILVA QUINTEIRO1; FABIANA FURTADO FREITAS2; MARCELO HENRIQUE NAPIMOGA3; JULIANA TRINDADE CLEMENTE NAPIMOGA4.
1,2,4.LABORATORY OF OROFACIAL PAIN, DEPARTMENT OF PHYSIOLOGY, PIRACICABA DENTAL SCHOOL, STATE UNIVERSITY O, PIRACICABA - SP - BRASIL; 3.LABORATORY OF IMMUNOLOGY AND MOLECULAR BIOLOGY, SÃO LEOPOLDO MANDIC INSTITUTE AN RESEARCH CENTER, CAMPINAS - SP - BRASIL.
Introduction: Inflammation of the temporomandibular joint (TMJ) induced by Rheumatoid Arthritis (RA) have often resulted in persistent pain and caused distress to many patients. Considering that not all patients respond to traditional drugs therapy to RA and it has demonstrated that 15d-PGJ2 into the TMJ has a potential antinociceptive effect, the aim of this study was to evaluate the peripheral effect of 15d-PGJ2 in RA-induced TMJ inflammatory hyperalgesia in rats as well its mechanisms. Methods and Results: Antigen-induced arthritis (AIA) was generated in rats with methylated bovine serum albumin (mBSA). RA-induced TMJ hyperalgesia in rats was assessed by measuring the behavioral nociceptive responses, such as rubbing the orofacial region and flinching the head, induced by the injections of low dose of formalin (0.5%) into TMJ prior challenge with an intra-articular injection os mBSA. After behavioral experiments, animals were terminally anesthetized and periarticular tissues were removed for ELISA, leukocytes migration, plasma-protein extravasation and histopathological analysis. The intra-articular injection of mBSA, but not PBS (control), in immunized rats induced dose-and-time-dependent behavioral nociceptive responses in which the peak of behavioral nociceptive responses were obtained by using 10 ug/TMJ of mBSA after 24 h.
Pretreatment (15min) with 15d-PGJ2 (30, 100 and 300 ng/TMJ) inhibited the RA-induced release of TNF-α, IL-1β, IL- 18, IL-12 and KC (p<0,05: ANOVA, Tukey Test) as well the RA-induced leukocytes migration and plasma-protein extravasation (p<0,05: ANOVA, Tukey Test). In addition the histopathological analysis showed that 15d-PGJ2 reduced the inflammatory parameters induced by RA. Conclusion: The 15d-PGJ2 might be used as a potential anti- inflammatory drug to treat RA inflammation of the temporomandibular joint (TMJ).
Financial support: Governmental Brazilian's Financial Support - FAPESP, CNPq and CAPES.
THE ANTINOCICEPTIVE POTENTIAL OF THE SLOW RELEASING H2S DONOR GYY4137 IN A MODEL OF NEUROPATHIC PAIN
FILIPHE DE PAULA NUNES MESQUITA1; BRUNA CAETANO DOS SANTOS2; MARK WOOD3; MATTHEW WHITEMAN4; MARCELO NICOLÁS MUSCARÁ5; SORAIA KÁTIA PEREIRA COSTA6.
1,2,5,6.UNIVERSITY OF SÃO PAULO, SÃO PAULO - SP - BRASIL; 3,4.UNIVERSITY OF EXETER MEDICAL SCHOOL, EXETER - REINO UNIDO.
Introduction: Chronic neuropathic pain is a debilitating and distressing complication of neuroinflammation or chronic effects of cancer treatment, that exerts neither biological warning value nor protective function, and it is difficult to treat. Fast and slow releasing H2S donors are an intriguing novel class of compounds with potential analgesic activity; however, their roles on neuropathic pain have received remarkably little attention. The goal of this study was to explore the antinociceptive potential of the slow releasing H2S donor GYY-4137 in a model of paclitaxel-induced peripheral neuropathy, an effective anti-neoplastic agent.
Methods and Results: This study was approved by the ICB/USP Animal Ethics Committee. Adult male rats (n=10) were given four intraperitoneal (i.p.) injections on alternate days of vehicle or paclitaxel (2.0 mgkg-1). Behavioral tests for pain using mechanical and thermal stimuli applied to the hind paws, and tests for motor performance (Rota Rod) were taken before, during and after dosing on day 12th. Up to two weeks, paclitaxel did not cause mechano-allodynia (16,2±55,1 vs 45,4±35,0 AUC g x day)but had significant effect on increasing cold-allodynia (32,8±14,8 vs -27,6±23,3 AUC Nociceptive Behaviour) motor performance (-300,4± 257,6 vs. 193,7±115,7 AUC Sec x day) and body weight loss (97,1±76,2 vs. 224,6±14,2 AUC ponderal evolution) as compared to vehicle-treated (n=5) animals, respectively. On day 12th post paclitaxel injection, the therapeutic administration of GYY-4137 (50mg kg-1, i.p., n=5) significantly decreased cold allodynia latency (-10,2±9,9 vs. 2,6±9,2 AUC Nociceptive Behaviour) but failed to affect motor performance (15,33±13 vs.-10,8±39,5) when compared with paclitaxel-alone group treated with vehicle.
Conclusions: Neuropathic pain began within 4 days after paclitaxel injection and lasted for the time measured (12 days), thus suggesting this as valid model of neuropathic pain, in which the acute administration of H2S donor GYY- 4137 plays a partial, but protective, role. To the best of our knowledge, the present study is the first to demonstrate the inhibitory effect of slow releasing H2S donor in paclitaxel-induced neuropathy model in rat, and further provide the suggestion that GYY-4137 might be considered an option for preventing or ameliorates paclitaxel-induced neuropathic pain. Further studies using GYY-4137 in distinct neuropathic models and action mechanisms comprehension are necessary to a better understanding.
Financial support: CAPES, CNPq, FAPESP (2011/16645-5) and Irene Maria Gouvea
THE CYTOKINE MIF CONTROLS NEUTROPHIL RECRUITMENT IN ARTICULAR INFLAMMATION INDUCED BY URIC ACID CRYSTALS
IZABELA GALVÃO1; IRLA PAULA RODRIGUES2; LIVIA DUARTE TAVARES3; ANA CAROLINA DIAS4; LUCAS SECCHIM RIBEIRO5; DANIELLE GLORIA SOUZA6; MAURO MARTINS TEIXEIRA7; FLAVIO ALMEIDA AMARAL8.
1.DEPARTAMENT OF BIOCHEMISTRY AND IMMUNOLOGY, BELO HORIZONTE - MG - BRASIL; 2,3,6.DEPARTAMENT OF MICROBIOLOGY - ICB - UFMG, BELO HORIZONTE - MG - BRASIL; 4,5,7,8.DEPARTAMENT OF BIOCHEMISTRY AND IMMUNOLOGY - ICB - UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is expressed by a wide variety of cells and plays an important role in the pathogenesis of numerous inflammatory and autoimmune diseases. Gout is an inflammatory arthropathy caused by precipitation of monosodium urate (MSU) crystals in the joint, which is associated with the production of important inflammatory mediators, such as the cytokines IL-1β, TNF-α and CXCL1, as well as neutrophil recruitment. This study aimed to verify the importance of MIF in the gouty inflammation in mice. Methods and Results: All experiments were performed in male C57/Bl6 mice (8-12 weeks old; 6 animals per group). MSU crystals injection time-dependently increased MIF and its receptor (CD74) expression on synovial tissue (WB) and recombinant MIF injection (100 ng; 6h) caused neutrophil influx to articular cavity (V: 0.10 ± 0.09; MIF: 6.81 ± 3.4 - cells x 104). The inhibition of MIF activity (ISO-1; 50 μg; i.a.) was effective in reduction neutrophil recruitment and hypernociception both before (-1 h) and after (3 h) MSU injection ((V: 3.85 ± 1.64; MSU: 84.16 ± 8.50; ISO-1: 30.33 ± 4.98 - cells x 104)(V: 0.17 ±0.19; MSU: 12.47 ± 7.92; ISO-1: 3.85 ± 3.66- cells x 104)). This effect was dependent of CXCL1 synthesis, since pre-treatment with ISO-1 was capable to reduce the expression of CXCL1 one hour after MSU crystal injection (RT-PCR) (V: 6.57 ± 7.76; MSU: 21.43 ± 6.23; ISO-1: 12.80 ± 7.71 – fold change over 18S housekeeping gene). Furthermore, ISO-1 treatment reduced IL-1β production on inflamed tissue (V: 54.71 ± 70.92; MSU: 580.27 ± 495.57; ISO-1: 273.86 ± 335.30 - pg/100mg of tissue) compared to vehicle-treated mice. Conclusion These results suggest that MIF plays an important role in the pathogenesis of gout, which have a major contribution to neutrophil recruitment to joint cavity after MSU crystals injection, partially dependent of CXCL1 production.
Financial support: CNPq, FAPEMIG, CAPES.
THE EFFECTS OF PI3K AND SRC-FAMILY KINASE INHIBITORS ON OSTEOCLAST DEVELOPMENT
DÁNIEL CSETE1; DÁVID GYŐRI2; TIBOR VÁNTUS3; GYÖRGY KÉRI4; CSABA SZÁNTAI-KIS5; LEN STEPHENS6; PHILLIP T HAWKINS7; ATTILA MÓCSAI8.
1,2,8.SEMMELWEIS UNIVERSITY, BUDAPEST - HUNGRIA; 3,4,5.VICHEM CHEMIE RESEARCH LTD., BUDAPEST - HUNGRIA; 6,7.THE BABRAHAM INSTITUTE, CAMBRIDGE - REINO UNIDO.
Introduction: Osteoclasts are the unique bone-resorbing cells of hematopoietic origin. Their development is directed primarily by M-CSF and RANKL, as well as by integrin-mediated adhesive interactions and immunoreceptor-like activation signals. Phosphoinositide 3-kinases (PI3K) are lipid kinases involved in important biological functions. Prior studies suggested a role for PI3K activity in osteoclasts, but the importance of the specific PI3K isoforms in osteoclast biology is poorly understood. The nonreceptor tyrosine kinase Src has been identified as one of the first proteins essential for normal osteoclast function. The role of the other Src family kinases expressed in osteoclasts has been only partially explored. Here we tested the role of the different PI3K isoforms and distinct Src-family kinases in osteoclast development using a pharmacological approach.
Methods and Results: Bone marrow cells were isolated from the femurs and tibiae of C57Bl/6 mice and differentiated into osteoclasts in vitro in the presence of recombinant M-CSF and RANKL. Cultures were treated with isoform specific PI3K inhibitors TGX221 (PI3Kβ inhibitor), IC87114 (PI3Kδ inhibitor), or different Src-family kinase inhibitors from Vichem's compound library (dasatinib, bosutinib and 13 other compounds (C1-C13)). Vehicle control samples received DMSO. Osteoclast differentiation was examined after 5 days by osteoclast-specific TRAP-staining. The number of developing osteoclasts was strongly reduced, the IC50 are the following: TGX221: 50 nM, IC87114: 500 nM, dasatinib: 3 nM, bosutinib: 50 nM, C1-C13: 3 nM, 3 nM, 1 nM, 10 nM, 3 nM, 30 nM, 30 nM, 30 nM, 100 nM, 300 nM, 3 nM, 300 nM, and 30 nM. For actin ring formation assays, cells were stained with Alexa488-Phalloidin or Lifeact- EGFP expressing cells were used. The presence of TGX221 or IC87114 significantly decreased the actin ring formation of mature osteoclasts.
Conclusion: Our results indicate that PI3Kβ, PI3Kδ and Src-family kinases play a critical role in osteoclastogenesis. The different roles of the distinct PI3K isoforms/Src-family kinases in osteoclast development promises novel therapeutic strategies based on the use of selective PI3K/Src-kinase inhibitors in diseases characterized by pathological bone loss.
Financial support: The work was supported by the EU FP7 (MOLINFLAM and TARKINAID projects) and the Wellcome Trust.
THE INVOLVEMENT OF THE HO-1 PATHWAY IN THE ANTI-INFLAMMATORY EFFECT OF A LECTIN ISOLATED FROM THE GREEN SEAWEED CAULERPA CUPRESSOIDES
ISMAEL NILO LINO DE QUEIROZ1; ANA LUÍZA GOMES QUINDERÉ2; NATÁSSIA ALBUQUERQUE RIBEIRO3; RENATA LINE DA CONCEIÇÃO RIVANOR4; CHISTIANE OLIVEIRA COURA5; JOSÉ ARIÉVILO GURGEL RODRIGUES6; HELLÍADA VASCONCELOS CHAVES7; MIRNA MARQUES BEZERRA8; EDFRANCK DE SOUSA OLIVEIRA VANDERLEI9; IANNA WIVIANNE FERNANDES DE ARAÚJO10; VÍTOR HUGO POMIN11; PAULO ANTONIO DE SOUZA MOURÃO12; NORMA MARIA BARROS BENEVIDES13.
1,11,12.IBQM-UFRJ, RIO DE JANEIRO - RJ - BRASIL; 2,3,4,5,6,7,8,9,10,13.UFC, FORTALEZA - CE - BRASIL.
Introduction: The search for new compounds for controlling inflammation, with minimal side effects has focused on seaweed. The aim of this work was to investigate the effect of the isolated lectin from the green seaweed Caulerpa cupressoides in classical models of inflammation. Methods and Results: The lectin (CcL) was extracted with Tris-HCl 25 mM, pH 7.5 and then isolated by ion exchange chromatography (DEAE-cellulose). We used male Wistar rats (180- 220 g) (CEPA 80/10). Animals (n=6) received CcL (0.1, 1 or 10 mg/kg; i.v.) 30 min before carrageenan (Cg) (700 µg/paw) or dextran (400 µg/paw), administered by intraplantar injection. In addition, myeloperoxidase (MPO) activity was also determined. To confirm the lectin effect, additional groups of rats received CcL (1 mg/kg; i.v.) associated with its binding sugar mucin (8 mg/kg; i.v.), and another group received only mucin (8 mg/kg; i.v.). To analyze the involvement of HO-1 in the anti-inflammatory efficacy of CcL, the animals were pretreated with ZnPP IX (3 mg/kg; s.c.), followed by injection of CcL (1 mg/kg; i.v.). Paw volume was measured immediately before (zero time) the stimulus and at selected time intervals (0.5, 1, 2, 3 and 4 h) using a plethysmometer. The results were expressed as difference between the basal volume and the volume in the selected time interval. CcL inhibited (p<0.05) the paw edema induced by Cg (0.65±0.04 mL) in doses 0.1, 1.0 and 10 mg/kg at all tested intervals, mainly, at the third hour time-point by 48.13, 60.03 and 82.14%, respectively and confirmed by the MPO activity in the paw tissue by 43.66, 93.55 and 96.56%, respectively. Additionally, dextran induced (p<0.05) increase in vascular permeability, with a maximum effect at 30 min. (0.76±0.16 mL) after administration, decreasing over the subsequent hours. Treatment of animals with different doses of CcL inhibited (p<0.05) paw edema induced by dextran at 0.5 hr by 17.34, 44.65 and 59.26%, respectively. Mucin, administered alone, did not modify the inflammatory response induced by Cg or dextran. However, the inflammatory effect was reduced when CcL (1 mg/kg) was combined with its inhibitor mucina. The anti- inflammatory effect of CcL (1 mg/kg) on the Cg-induced rat paw edema test was reduced in the presence of ZnPP-IX. Conclusion: The results indicate that anti-inflammatory effect of CcL is related to the integrity of the HO-1/BVD/CO pathway.
Financial support: CNPq, CAPES and FUNCAP.
THE N-ACYLHYDRAZONE DERIVATIVE LASSBIO-897 SUPPRESSES LUNG INFLAMMATION CAUSED BY SILICA PARTICLES IN MICE.
ANA CAROLINA SANTOS ARANTES1; MANUELA DE JESUS LANZETTI2; TATIANA PAULA TEIXEIRA FERREIRA3; BIANCA TORRES CIAMBARELLA4; ELIEZER JESUS DE LACERDA BARREIRO5; CARLOS ALBERTO MANSOUR FRAGA6; MARCO AURÉLIO MARTINS7; PATRICIA MACHADO RODRIGUES E SILVA8.
1,2,3,4,7,8.OSWALDO CRUZ INSTITUTE/FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL; 5,6.LASSBIO / FEDERAL UNIVERSITY OF RIO DE JANEIRO, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Silicosis is an occupational disease caused by prolonged inhalation of dust containing free crystalline silica particles, which is characterized by an intense pulmonary inflammation with formation of fibrotic nodules. As there is no effective treatment for fibrotic diseases, in this study we investigated the effect of the 1,3-N-acylidrazonic benzodioxolic derivative LASSBio-897 on the experimental silicosis in mice. Effect of the compound was also evaluated in some target cells in vitro. Methods and Results: Swiss-Webster mice were intranasally instilled with silica (10 mg) and analyzes performed after 28 days. The animals received daily administration of LASSBio-897 (2 and 5 mg/kg, p.o.) for 7 days, starting 21 days post-silica. Lung function was evaluated by invasive plethysmography, in absence or presence of methacholine. Histological techniques (morphology/morphometry) and immunohistochemistry were performed. Collagen content and cytokine generation were quantified by Sircol and ELISA, respectively. Therapeutic treatment of silicotic mice with LASSBio-897 inhibited the reduction in lung function (resistance and elastance) and airways hyperreactivity to methacholine as well as collagen deposition and granuloma formation. Values of granuloma area were 42.2% ± 9.5%; 10.2% ± 3.7%; and 11.0% ± 3.6%; (mean ± SEM; n=6, p<0.05), in silica- and silica-stimulated mice treated with 2 and 5 mg/kg of LASSBio-897, respectively. Cytokine generation and the increased number of α-SMA positive cells in the lungs of silicotic mice were also sensitive to treatment with LASSBio-897. The evaluation of LASSBio-897 effect on the functionality of target cells in vitro showed that, at the concentrations varying from 0.01-10μM, the compound reduced IL-13-induced silicotic lung fibroblast proliferation and collagen production. No effect was noted on murine alveolar macrophages (AMJ2C11) and human lung epithelial cells (A549). Conclusion: Our results show that compound LASSBio-897 inhibited the inflammatory and fibrogenic response caused by silica particles in the lungs of mice, by a mechanism associated with suppression of cytokine production and fibroblast activation.
Financial support: FIOCRUZ, CNPq, FAPERJ, INCT-INOFAR, PRONEX.
THE PERIPHERAL ANTIEDEMATOGENIC EFFECT INDUCED BY INTRATHECAL MORPHINE IS MEDIATED BY POTASSIUM CHANNEL OPENING.
VANESSA RAFAELLA FOLETTO DA SILVA; CARLOS ROGERIO TONUSSI.
UFSC, FLORIANÓPOLIS - SC - BRASIL.
Introduction: It is known that dorsal root antidromic potentials running in tiny primary afferents fibers can enhance a distal inflammatory process. A previous study showed that peripheral inflammatory edema can be inhibited by intrathecal administration of morphine, which seems to involve the nitric oxide/cGMP pathway. This study extends these findings checking whether potassium channels may be also involved in the morphine's effect.
Methods and Results: Male Wistar rats (250-350g) received intrathecal (i.t.) injections of phosphate-buffered saline (PBS; 20 μL), morphine (MOR; 37 nmol), morphine either with 4-aminopyridine (MOR+4AP), or glibenclamide (MOR+GLI), or dequalinium (MOR+DQL), or nicorandil (MOR+NIC), 30 minutes before left hindpaw stimulation with carrageenan (150 μg/ 50 μL). Edema was measured as paw volume increase (in mL). Plasma leakage and neutrophil migration were indirectly evaluated by the optical density (OD) of Evans blue dye in the tissue and myeloperoxidase (MPO) assay, respectively. Vascular congestion was measured by histological score. Comparing to PBS (0.35 ± 0.03 mL), MOR inhibited edema (0.28 ± 0.07 mL), plasma leakage (MOR = 12.74 ± 0.99 OD; PBS = 16.36 ± 1.06 OD), and vascular congestion (MOR = 1.6 ± 0.36; PBS = 2.6 ± 0.19), but had no effect on MPO activity (MOR = 0.09 ± 0.003 OD; PBS=0.098 ± 0.003 OD). Co-injection with K+ channel blockers 4AP (10 nmol) (MOR = 0.15 ± 0.06 mL; MOR+4AP = 0.3 ± 0.06 mL); GLI (5 nmol) (MOR = 0.15 ± 0.06 mL ; MOR+GLI = 0.3 ± 0.06 mL) and DQL (10 pmol) (MOR = 0.1 ± 0.02 mL; MOR+DQL = 0.16 ± 0.05 mL) reversed, while the K+ channel opener NIC (0.03 nmol) enhanced the effect of morphine (MOR = 0.23 ± 0.07 mL; MOR+4AP = 0.1 ± 0.06 mL). The value of “n” in all groups was 9.
Conclusion: The peripheral antiedematogenic effect produced by intrathecal morphine is likely mediated by the three major types of potassium channels. This effect seems to be due to a reduction in capillary recruitment, but not to neutrophil migration inhibition. The present findings support the idea that clinically available K+ channel openers, as minoxidil and nicorandil, could be associated to opiates for a combined spinal therapy.
Financial Support: CNPq, CAPES.
THE ROLE OF GILZ IN THE CONTEXT OF RESOLUTION OF ACUTE INFLAMMATION
JULIANA PRISCILA VAGO1; LUCIANA PÁDUA TAVARES2; CRISTIANA COUTO GARCIA3; ÉRICA LEANDRO MARCIANO VIEIRA4; CAMILA RODRIGUES CHAVES NOGUEIRA5; FREDERICO MARIANETTI SORIANI6; STEFANO BRUSCOLI7; CARLO RICCARDI8; ERIC MORAND9; PATRÍCIA MACHADO RODRIGUES SILVA10; VANESSA PINHO11; MAURO MARTINS TEIXEIRA12; LIRLÂNDIA PIRES SOUSA13.
1,2,3,4,5,6,11,12,13.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 7,8.UNIVERSITY OF PERUGIA, PERUGIA - ITÁLIA; 9.MONASH UNIVERSITY, VICTORIA - AUSTRÁLIA; 10.INSTITUTO OSWALDO CRUZ, RIO DE JANEIRO - RJ - BRASIL.
Introduction: Glucocorticoid (GC)-induced leucine zipper (GILZ) has been shown to mediate several GC functions including apoptosis and anti-inflammatory activities. The proresolutive abilities of GILZ have not been explored. In the present study, we investigated the role of GILZ on spontaneous and GC-induced resolution of neutrophilic inflammation induced by LPS.
Methods: BALB/c or GILZ-/- mice were challenged by i.pl. (intrapleural) injection of LPS (250ηg/cavity) or PBS. The peptide TAT-GILZ (a GILZ fusion protein, 0,2mg/kg), a pan caspase inhibitor (zVAD-fmk, 1mg/kg) and dexamethasone (dexa-synthetic glucocorticoid, 2mg/kg) were administered systemically (i.p.). Cells present in the pleural cavity were harvested at different times after LPS challenge or after treatment with drugs and processed for total and differential leukocyte counts, western blot analysis, and flow cytometry.
Results: GILZ expression was strongly increased during the resolution phase of LPS-induced pleurisy (48-72h) in resolution macrophages. TAT-GILZ given preventively was able to shorten resolution intervals. Interestingly, TAT- GILZ given therapeutically at different protocols, induced resolution of neutrophilic inflammation, decreased TNF-α and IL-6 levels and promoted caspase-dependent neutrophil apoptosis. TAT-GILZ also modulated the survival- controlling proteins ERK1/2, NFkB and Mcl-1. GILZ-deficiency did not modify the course of neutrophil influx in LPS- induced pleurisy but there was decreased mononuclear cell influx at 48h post-LPS. Interestingly, there is an early increase of AnxA1 intact levels in LPS-injected GILZ-/- mice as compared to wild type mice. Induction of GILZ by dexa was dependent on AnxA1, another GC-induced protein with established anti-inflammatory and proresolutive activities. Importantly, administration of dexa to GILZ-/- and AnxA1-/- mice promoted resolution of neutrophilic inflammation associated with compensatory balance of AnxA1 and GILZ expression suggesting that in vivo these proteins replace the function of each other to promote the efficient control of acute inflammation.
Conclusion: These findings indicate that endogenous GILZ is redundant for spontaneous resolution of acute inflammation, probably because AnxA1 is over-expressed in the absence of GILZ. However, therapeutic administration of GILZ is efficient to induce a pro-apoptotic program that promotes resolution of acute inflammation.
THE ROLE OF MACROPHAGES AND MAST CELLS ON P2X7-INDUCED TMJ NOCICEPTION
HORTÊNCIA MARIA XAVIER SOUSA1; MARIA CLAUDIA GONÇALVES OLIVEIRA FUSARO2; CRISTINA GOMES MACEDO3; JULIANA MAIA TEIXEIRA4; PATRICIA OLIVEIRA LIMA5; MARCELO HENRIQUE NAPIMOGA6; JULIANA TRINDADE CLEMENTE NAPIMOGA7.
1,3,5,7.FOP UNICAMP, PIRACICABA - SP - BRASIL; 2.UNICAMP, CAMPINAS - SP - BRASIL; 4.INSTITUTO DE BIOLOGIA UNICAMP, CAMPINAS - SP - BRASIL; 6.SÃO LEOPOLDO MADIC, CAMPINAS - SP - BRASIL.
Introduction: P2X7 receptors are known to induce temporomandibular joint (TMJ) nociception by indirect mechanisms, such as the release of prostaglandins and sympathetic amines. Therefore, this study aimed to evaluate the effect of inflammatory cells (macrophages, neutrophils and mast cells) and nociceptive C-fibers on P2X7-induced nociception in rats’ TMJ. Methods and Results: Male Wistar rats (± 150 g, n=4-6/group) were pretreated with 1% Thioglycollate (30 µl/TMJ/3 days), Fucoidan (20 mg/kg/30 min), Cromolyn (600 µg/TMJ/15 min), capsaicin (50 mg/kg, i.p.), and an intra-TMJ injection of P2X7 agonist BzATP (225 µg/TMJ). The rats' nociceptive behavior induced by the intra-TMJ injection of BZATP was assessed by summing the number of seconds that the animal spent rubbing the orofacial region and flinching the head in a 30-minute period. The animals were then terminally anesthetized and their periarticular tissues removed for the analysis of P2X7 expression (Western Blot). The nociceptive behavior induced by the intra-TMJ injection of BZATP (225 µg/TMJ) was significantly inhibited by Fucoidan (p<0.05: ANOVA, Tukey’s test) and Capsaicin (p<0.05: ANOVA, Tukey’s test). Cromolyn (p>0.05: ANOVA, Tukey’s test) had no effect on BZATP- induced nociception. Western Blot analysis concerning the P2X7 expression showed no statistical difference among the groups treated with BZATP, Thioglycollate + BZATP, and Fucoidan + BZATP (p>0.05: ANOVA, Tukey’s test). Conclusion: P2X7-induced nociception in the rat’s TMJ might be mediated by neutrophils and nociceptive C-fibers. Macrophages and mast cells seem to have no effect on this mechanism of pain.
Financial Support: PIBIC/UNICAMP- Programa Institucional de Bolsas de Iniciação Científica
THE ROLE OF MELANOCORTIN RECEPTORS IN OSTEOCLASTS DIFFERENTIATION AND ACTIVITY RELATED TO PERIODONTAL DISEASE
MILA FERNANDES MOREIRA MADEIRA1; TRINIDAD MONTERO MELENDEZ2; LUCY V NORLING3; CELSO MARTINS QUEIROZ-JUNIOR4; SILVIA MARIA CORDEIRO WERNECK5; DANIELE DA GLORIA DE SOUZA6; MAURO MARTINS TEIXEIRA7; TARCILIA APARECIDA DA SILVA8; MAURO PERRETTI9.
1,4,5,6,7,8.UFMG, BELO HORIZONTE - MG - BRASIL; 2,3,9.WHRI, LONDON - REINO UNIDO.
MILA FERNANDES MOREIRA MADEIRA1,3; TRINIDAD MONTERO MELENDEZ2; LUCY V NORLING2; CELSO MARTINS QUEIROZ JUNIOR1,3; SILVIA MARIA CORDEIRO WERNECK1; DANIELE DA GLORIA DE SOUZA1; MAURO MARTINS TEIXEIRA1; TARCILIA APARECIDA DA SILVA1,3; MAURO PERRETTI2.
1Immunopharmacology, Institute of Biological Science, UFMG, BH, Brazil; 2 William Harvey Research Institute – Barts and The London School of Medicine and Dentistry London EC1M 6BQ - United Kingdom; 3Oral Pathology, School of Dentistry, UFMG, BH, Brazil
Introduction: The relationship between periodontal disease (PD) and rheumatoid arthritis (RA) has been studied for more than 40 years, receiving more attention recently. PD and RA share some features such as inflammatory cell infiltration, production of inflammatory mediators and bone loss. Inflammation-induced bone loss is characterized by activation of bone resorption and inhibition of bone formation. Melanocortins and their receptors regulate inflammation by inhibiting leukocyte recruitment and reducing pro-inflammatory mediators. Recent studies in MC3 deficient mice (Mc3r-/-) suggest a role for this receptor in the regulation of osteoclast development and function. Objective: Our aim was to assess the development of alveolar bone loss in the K/BxN-serum transfer arthritis model and the role of melanocortins in this process. Methods: Arthritis was induced by injecting wild-type (WT) mice with 100 μL of K/BxN serum on days 0 and 2 (i.p.). Arthritis was monitored by measuring paw volume and clinical score. After 7 days the maxilla were analyzed for alveolar bone loss. In vitro experiments were carried out with bone marrow cells (BMC) from C57BL/6 WT mice. To generate osteoclasts cells were incubated with M-CSF for 6 days. RANKL and D[Trp8]-γMSH
(MC3 agonist) were added at day 6 until day 10. LPS from Aggregatibacter actinomycetemcomitans (AaLPS) was added at day 8 until day 10. Osteoclasts were defined as TRAP+ve cells containing more than 3 nuclei. Resorption activity was also evaluated by resorption pits counting using Osteo Assay Plate (Corning, NY, USA). Results: Arthritis in the K/BxN model was associated with alveolar bone loss (Control: 0.347±0.02; Arthritis: 0.459±0.04; p<0.05; n=5), which was significantly correlated with clinical score. Osteoclasts generated from WT BMC presented increased osteoclastogenesis after activation with AaLPS (Control: 2.19±1.13; RANKL: 29.6±3.8; RANKL+AaLPS: 67.2±5.6; 8 p<0.01; n=3) and they express MC3. Treatment with D[Trp ]-γMSH decreased osteoclastogenesis induced by AaLPS (42±7.5; p<0.05; n=3), as well as osteoclast activity. Conclusion: Inflammatory arthritis – modeled with K/BxN serum - is associated with alveolar bone loss. Melanocortin agonists may have an inhibitory effect on osteoclast activity and differentiation on AaLPS-activated cells.
Support by: CNPq, CAPES and FAPEMIG.
THE ROLE OF PANNEXIN1/P2X7 IN CARRAGEENAN-INDUCED INFLAMMASOME ACTIVATION/IL-1Β MATURATION.
ALEXANDRE HASHIMOTO PEREIRA LOPES1; RANGEL LEAL SILVA2; JHIMMY TALBOT3; RAFAEL FREITAS OLIVEIRA FRANÇA4; FERNANDO QUEIROZ CUNHA5; DARIO SIMÕES ZAMBONI6; GOMBAULT AURÉLIE7; ISABELLE COUILLIN8; BERNHARD RYFFEL9; THIAGO MATTAR CUNHA10.
1,2,3,4,5,10.FMRP-USP, RIBEIRÃO PRETO - SP - BRASIL; 6.BIOCEL-USP, RIBEIRÃO PRETO - SP - BRASIL; 7,8,9.CNRS, ORLEANS - FRANÇA.
Introduction: Carrageenan (Cg) is an algae carbohydrate that is widely used as food additive. Interestingly, Cg can induce local inflammation and have been used in animal models of inflammatory diseases (eg. ulcerative colitis) as inductor-agent. However, the mechanism of Cg-induced inflammation is not clear. We found previously that Cg can induce Interleukin 1 beta (IL-1β) production and maturation by macrophages. The IL-1β is one of the key mediators of inflammatory response upon cell activation. It is known that IL-1β maturation is dependent of activation of the molecular protein platform known as inflammasome, namely NLRP3/ASC and caspase-1. It is also described that ATP, pannexin-1 (PAN1) and the purinergic receptor P2X7 are important to inflammasome activation induced by some agents. However, the mechanism of inflammasome activation induced by Cg is not clear. Thus, the aim of this study was to investigate whether Cg-induced IL-1 β maturation is dependent of PAN1 and P2X7 signaling and inflammasome activation.
Methods and Results: In the present study peritoneal macrophages (MF) were isolated from male C57BL/6 mice (WT) and from mice genetically deficient to P2x7-/-, Nlrp3-/-, Casp1-/- and adaptor molecule Asc-/- , ethical committee (CETEA-FMRP 149/2011). The MF were stimulated by Cg (300 µg/ml) and the supernatant was removed to measurement of IL-1β by ELISA and extracellular ATP by ATP One Step kit. To investigate the participation of PAN1and P2X7, its inhibitors (carbenoxolone [CBX] and A740003, respectively) were also used in culture. Here, at the first time we provide evidences that Cg induces the release of ATP by peritoneal MF, depending in some extent to functional P2X7 and PAN1. Moreover, we show a strong correlation between ATP released and the secretion of mature IL-1β. This secretion also was dependent of inflammasome activation as the production of IL-1β by Cg was reduced in cells from Caspase-1-/-, Nlrp3-/- and Asc-/- compared to cells from WT mice.
Conclusion: The present data demonstrates that ATP released by MF after Cg stimulation can be an important second signaling to IL-1β maturation. Mainly, the production of active IL-1β is dependent of PAN1 and P2X7 and inflammasome assembling, namely NLRP3/ ASC and Caspase-1. In conclusion, inhibition of inflammasome assembling might be a target to control inflammatory diseases.
Financial support: CAPES, FAPESP.
THE ROLE OF PKC-Β ISOFORMS IN DIABETES-INDUCED HYPONOCICEPTION INTO RATS TMJ
AUGUSTO MUZILLI1; MARIA CLAUDIA OLIVEIRA FUSARO2; FABIANA FURTADO FREITAS3; CRISTINA GOMES DE MACEDO4; JULIANA TRINDADE CLEMENTE NAPIMOGA5.
1,5.PIRACICABA DENTAL SCHOOL/UNICAMP, PIRACICABA - SP - BRASIL; 2.FACULTY APPLIED SCIENCES/UNICAMP, LIMEIRA - SP - BRASIL; 3.PIRACICABA DENTAL SCHOOL/UNICAMP L SCHOOL, PIRACICABA - SP - BRASIL; 4.PIRACICABA DENTAL SCHOOL/UNICAMP L SCHOOL/UNICAMP, PIRACICABA - SP - BRASIL.
Introduction: Diabetes in early phase is known to result in painful neuropathy as a result of neurovascular complications mediated by the activation of Diacylglycerol/Proteinoquinase C pathway (DAG/PKC). Considering that immunohistochemical studies have demonstrated the presence of PKC-β isoforms in the neural tissues. The aim of this study was to evaluate the role of PKC-β1 and β2 in diabetes-induced hypernociception. Methods and Results: Wistar rats (± 150g, n=4-6/group) were treated with an intraperitoneal injection of vehicle (normoglycemic – NG) or Streptozotocin 75 mg/kg (diabetic – DB). Diabetes-induced hypernociception was assessed by the animals’ nociceptive behavior induced by an intra-articular injection of formalin 7, 14, 21, 28, 35 and 42 days after the diabetic induction. After behavioral assays, animals were terminally anesthetized and their periarticular tissue for Western Blot analysis. Early phase of diabetes induced hyponociception into TMJ of rats 7, 14, 21, 28, 35 and 42 days after the diabetic induction (p<0.05: Two-way ANOVA, Bonferroni’s test). To confirm these results, the experiments were repeated and animals receive an intra-articular injection of capsaicin. DB animals treated with an intra-articular injection of capsaicin demonstrated no behavioral response when compared to that NG rats. Western Blot analysis demonstrated that the expression of PKC-β1 and β2 into periarticular tissue was significantly higher in the DB rats than in the NG rats (p<0.05: Two-way ANOVA, Bonferroni’s test).
Conclusion: Early phase of diabetes induce hyponociception into rats TMJ that involves the activation of PKC-β1 and β2.
THE RUTHENIUM NO DONOR, [RU(HEDTA)NO], ATTENUATES ACUTE PAIN IN MICE: EFFECT ON CYTOKINE PRODUCTION AND INVOLVEMENT OF CGMP/PKG/ATP-SENSITIVE POTASSIUM CHANNEL SIGNALING PATHWAY
SANDRA SATIE MIZOKAMI1; LARISSA STAURENGO-FERRARI2; VICOTR FATTORI3; JEAN J. SILVA4; PATRICIA G. ZANICHELLI5; SANDRA R. GEORGETTI6; MARCELA M. BARACAT7; WANDER R PAVANELLI8; RUBIA CASAGRANDE9; WALDICEU A. VERRI JR10.
1,2,3,6,7,8,9,10.UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA - PR - BRASIL; 4.UNIVERSIDADE ESTADUAL DE RORAIMA, BOA VISTA - RR - BRASIL; 5.UNIVERSIDADE DE SÃO PAULO, SÃO CARLOS - SP - BRASIL.
Introduction: Nitric oxide plays an important role in various biological processes, such as analgesic and anti- inflammatory effects. The control of its local concentration, which is crucial for obtaining the desired effect, can be achieved with exogenous NO-carriers, such Ruthenium compounds. Therefore, we evaluated the analgesic mechanism of ruthenium NO donor [Ru(HEDTA)NO] focusing on the role of cytokines and activation of the cGMP/PKG/ATP-sensitive K+ channels signaling pathway. Methods: This study was approved by the Ethics Committee of Universidade Estadual de Londrina (8624.2013.20). Male swiss mice (25-30g, n=6 per experiment, representative of two experiments) were treated with [Ru(HEDTA)NO] (0.3-10 mg/kg, sc) 40 min before inflammatory stimulus. The writhing response was evaluated during 20 min after acetic acid (0.8% v/v), or PBQ (1890 μg/kg) injection, and mechanical hyperalgesia evaluated 1-5h after carrageenan (Cg; 300 ug/paw). Cytokine levels (TNFα and IL-1β) and myeloperoxidase (MPO) activity were determined 3 and 5 h after Cg injection, respectively. The analgesic effect of [Ru(HEDTA)NO] was challenged by pre-treatment with ODQ (inhibitor of guanilate cyclase, 0.3 mg/kg, ip); KT5823 (inhibitor of PKG, 0.5ul/animal, ip) and glybenclamide (inhibitor of ATP-sensitive K+ channels, 0.3 mg/kg, po). Results: [Ru(HEDTA)NO] inhibited acetic acid (up to 56.4%, from 66.2±3.5 to 29.5±4.9) and PBQ (up to 51.5%, from 40.5±5.3 to 20.0±2.0) -induced writhing response. [Ru(HEDTA)NO] also inhibited the Cg-induced mechanical hyperalgesia (up to 47.4%, from 9.6±0.9 to 5.3±0.7 g), decrease of MPO activity (up to 96.6%, from 15.0±3.1 to 3.4±1.1 neutrophils) and the production of the cytokines TNF-α (50%) and IL-1β (33.2%) in paw skin samples. Furthermore, the inhibitory effect of [Ru(HEDTA)NO] in the Cg-induced hyperalgesia and MPO activity was prevented by ODQ (39.2% [from 5.9±0.4 to 8.9±0.8 g] and 57% [from 5.1±1.7 to 11.4±1.0 neutrophils]), KT5823 (36.2% [from 5.9±0.4 to 8.5±0.5] and 54.5% [from 5.1±1.7 to 10.7±1.2 neutrophils]) and glybenclamide (35.7% [from 5.9±0.4 to 8.5±0.6 g] and 60.9% [from 5.1±1.7 to 12.4±2.2 neutrophils]), respectively. Conclusion: These results demonstrate that [Ru(HEDTA)NO] exerts its analgesic effect by inhibiting pro-nociceptive cytokine and activating the cGMP/PKG/ATP-sensitive K+ channels signaling pathway.
Financial support: CAPES, CNPq, MCTI, SETI / Fundação Araucária, and Governo do Estado do Paraná (Brazil).
THE SULFATED POLYSACCHARIDE EXTRACTED FROM SEAWEED HYPNEA MUSCIFORMIS AMELIORATES THE TNBS-INDUCED COLITIS IN RATS.
ISABELA DE SOUZA BRAÚNA1; DANDARA MARQUES2; IAGO SANTOS VERAS3; JALLES ARRUDA BATISTA4; TARCISIO VIEIRA BRITO5; RAFAEL SILVA PRUDÊNCIO6; RENAN OLIVEIRA SILVA7; MARCELLUS HENRIQUE LOIOLA PONTE SOUZA8; LUCIANO SOUSA CHAVES9; MÁRCIA RUBIA S. MELO10; ANA LUCIA PONTE FREITAS11; JAND-VENES ROLIM MEDEIROS12; ANDRÉ LUIZ REIS BARBOSA13.
1,2,3,4,5,6,7,12,13.UNIVERSIDADE FEDERAL DO PIAUÍ-UFPI, PARNAÍBA - PI - BRASIL; 8,9,10,11.UNIVERSIDADE FEDERAL DO CEARÁ- UFC, FORTALEZA - CE - BRASIL.
ISABELA DE SOUZA BRAÚNA(A) ; DANDARA MARQUES(A); IAGO SANTOS VERAS(A); JALLES ARRUDA BATISTA(A); TARCISIO VIEIRA BRITO(A); RAFAEL SILVA PRUDÊNCIO(A); RENAN OLIVEIRA SILVA(A); MARCELLUS HENRIQUE LOIOLA PONTE SOUZA(B); LUCIANO SOUSA CHAVES(C); MÁRCIA RUBIA S. MELO(C); ANA LUCIA PONTE FREITAS(C); JAND-VENES ROLIM MEDEIROS(A) AND ANDRÉ LUIZ REIS BARBOSA(A) *.
(A)LAFFEX – Laboratory of Experimental Physiopharmacology, Biotechnology and Biodiversity Center Research (BIOTEC), Federal University of Piauí.
(B)LAFICA – Laboratory of Pharmacology of Inflammation and Cancer, Department of Physiology and Pharmacology, Federal University of Ceará.
(C)Laboratory of Proteins and Carbohydrates of Marine Algae, Department of Biochemistry and Molecular Biology, Federal University of Ceará, Fortaleza, CE, Brazil.
INTRODUCTION: Substrates from plants (seaweed) characterized as bioactive compounds have become a very important in pharmaceutical research. Such compounds have been reported to exhibit a wide spectrum of pharmacological activity, including anti-inflammatory effects. This study aimed to evaluate the anti-inflammatory potential of a polysaccharide (PLS) extracted from seaweed Hypnea musciformis in the colitis induced by TNBS. METHODS: TNBS colitis was induced by intracolonic instillation of a solution of 20 mg of TNBS in 50% EtOH (ethanol) in rats (n=6). Control groups received an equivalent volume of saline. All assays were performed 3 days after colitis induction. In the experiments involving TNBS-induced colitis, rats were pre-treated once a day for three days with polysaccharide extracted from Hypnea musciformis (10, 30 and 60 mg.kg-1,500μl p.o.). Three days after colitis-induction the rats were killed the abdomens were opened, and after identification of the intestine, fragments of distal colon was excised for the evaluation of macroscopic scores of lesions, wet weight of colon, MDA and MPO assays. RESULTS: The PLS (60 mg.kg-1) significantly decreased the macroscopic scores of lesion ( macroscopic scores; PLS+TNBS GROUP: 5.25±0.75), wet weight (PLS+TNBS GROUP:0.45±0.089), MPO concentration (PLS+TNBS GROUP:0.83±0.65) and MDA (PLS+TNBS GROUP:76.27±16.30) level when compared this group with TNBS group (macroscopic scores:18.0±1.73; wet weight: 1.03±0.03; MPO:10.03±2.55; MDA:162.5±25.63). CONCLUSION: We suggest that PLS extracted from red seaweeds Hypnea musciformis has prevents intestinal inflammation by reduced macroscopic scores of lesion wet weight, MPO and MDA concentrations and consequently the intestinal damage in the colon.
THE TREATMENT WITH HIGH DOSE INTRAVENOUS IMMUNOGLOBULIN (IVIG) THERAPY PREVENTS THE DEVELOPMENT OF SEVERE DENGUE BY THE INDUCTION OF PRODUCTION OF IL-33.
REBECA DE DE PAIVA FROES ROCHA1; VIVIAN VASCONCELOS COSTA2; CAIO TAVARES FAGUNDES3; THIAGO VINICIUS ÁVILA4; DANIEL CISALPINO5; PATRICIA REGINA SOUZA6; LUCAS SECCHIN RIBEIRO7; CELSO MARTINS QUEIROZ JUNIOR8; DEBORAH FERNANDES VALADAO9; ANA CAROLINA FIALHO DIAS10; WALDICEU APARECIDO VERRI JUNIOR11; MAURO MARTINS TEIXEIRA12; DANIELLE DA GLORIA DE SOUZA13.
1,2,3,4,5,6,7,8,9,10,12,13.UFMG, BH - MG - BRASIL; 11.UNIVERSIDADE ESTADUAL DE LONDRINA, LONDRINA - PR - BRASIL.
Introduction:Dengue is caused byone of the four serotypes of Dengue virus (Denv-1-4). The hallmarks of severe dengue mayinclude thrombocytopenia, increased vascular permeability, cytokine storm associated with an exacerbated inflammatory response and shock.High dose intravenous immunoglobulin (IVIG) therapy has been used in the treatment of a large range of autoimmune and inflammatory diseases. Here we evaluated the potential therapeutic effects of the administration of high dose IVIG therapy in a model of DENV infection in mice. Methods: BALB/c mice (8 to10 weeks) were inoculated with an adapted strain of DENV-3 via thei.p route. Mice were given vehicle or IVIG daily, via i.v route, at dose of 1g/kg from day 0 to 6 of infection. Seven days after infection, mice were euthanized and blood and tissues (spleen and liver) collected for several analysis described below. Mice were accompanied for lethality until day 14th of infection. All experimental procedures were approved by CETEA/UFMG access number 113/2009. Results:Seven days after DENV-3 infection vehicle-treated mice presented severe disease manifestation characterized by markedly thrombocytopenia, increased hemoconcentration, elevated levels of hepatic transaminases in serum and massive liver damage. Higher levels of TNF-aand IFN-gand elevated viral loads were found in serum and tissues of infected mice. Conversely, high dose IVIG administration to mice clearly inhibited the major manifestations of the disease, including lethality. Mechanistically, IVIG treatment resulted in massive releaseof the immunomodulatory cytokines IL-33 and IL-10 in comparison to the vehicle-treated mice. Of note, the protection afforded by the IVIG administration during DENV infection occurred without loss of control of viral replication and IFN- γ production andwas completely dependent on IL-33 production. IVIG administration to mice deficient for IL-33 receptor (ST2-/- mice) failed to mediate protection after DENV-3 inoculation. Conclusion:IL-33 production induced by IVIG treatment is pivotal for disease amelioration during DENV infection. Interfering in the exacerbated inflammatory response induced by DENV through high dose IVIG administration can be used as an alternative therapeutic to the severe manifestations of dengue disease.
Financial support:CNPq, FAPEMIG, CAPES, INCT em dengue and PRONEX (Ministério da Saúde).
TNF-A MEDIATES THE ANTI-INFLAMMATORY EFFICAY OF PHYTOMEDICATION APLAUSE® (CIMICIFUGA RACEMOSA) IN EXPERIMENTAL PERIODONTITIS
LORENA VASCONCELOS VIEIRA; JORDÂNIA MARQUES DE OLIVEIRA; ALICE RAMOS DE FREITAS; SAMUEL MATEUS PEREIRA FILHO; LÍVIA CUNHA RIOS; DÉBORA DA SILVA FREITAS; MIRNA MARQUES BEZERRA; ANTONIO ALFREDO RODRIGUES E SILVA; LISSIANA MAGNA AGUIAR VASCONCELOS; KARUZA MARIA ALVES PEREIRA; GERARDO CRISTINO FILHO; VICENTE DE PAULO TEIXEIRA PINTO; HELLÍADA VASCONCELOS CHAVES.
FEDERAL UNIVERSITY OF CEARÁ CAMPUS SOBRAL, SOBRAL - CE - BRASIL.
Introduction: Periodontitis is a chronic inflammatory process that affects the supporting tissues of teeth characterized by extensive alveolar bone resorption. Cimicifuga racemosa is an herbaceous plant belonging to the family Ranunculaceae, native to Canada and the Atlantic coast of the United States, and is commonly used in traditional medicine as an analgesic, anti-inflammatory and for the treatment of menopause symptom. This study aimed to evaluate the effectiveness of Cimicifuga racemosa on periodontitis in rats, investigating the involvement of tumor necrosis factor (TNF-α), and the safety of this treatment. Methods: Periodontitis was induced by placing a nylon thread (3.0) in the upper molars of female Wistar rats (180 - 200 g). Rats (6 per group) were weighed and treated (per os) daily with Cimicifuga racemosa (0.01, 0.1 or 1 mg/kg) or vehicle (saline) for 11 days. Following the treatment course, alveolar bone resorption was measured using the ImageJ® software; the periodontal tissues were analyzed by histopathology (H&E) and by ELISA assay to determine the levels of TNF-α; the gastric mucosa was examined macro and microscopically (H&E); peripheral blood was collected for biochemical analysis (alanine amino transferase - AST, aspartate amino transferase – ALT, creatinine, and total alkaline phosphatase - tAP). Results: Cimicifuga racemosa (0.01, 0.1 or 1 mg/kg) reduced (P < 0.05) alveolar bone resorption (1.99 ± 0.46, 1.55 ± 0.31 and 1.64 ± 0.57, respectively), compared to the vehicle group (3.60 ± 0.31). These data were confirmed by histopathology analysis of Cimicifuga racemosa (1 mg/kg) that showed discrete cell influx, reduction in osteoclast number, cementum and alveolar process well preserved [1(0-1)], compared to the vehicle group [2(1-3)]. Further, Cimicifuga racemosa (1 mg/kg) decreased (P < 0.05) TNF-α levels in gingival tissues (17.99 ± 1.67), compared to the vehicle group (42.57 ± 2.8). Cimicifuga racemosa treatment did not change the macroscopic and histopathological analysis of gastric mucosa. Serum levels of ALT/AST, creatinine and tAP did not differ from respective controls. Conclusion: These findings suggest that Cimicifuga racemosa is safe and it reduces the alveolar bone resorption in periodontitis, at least in part, by reducing TNF-α levels.
Financial Support: CAPES, CNPq, FUNCAP, UFC.
TRANSIENT RECEPTOR POTENTIAL VANILLOID 4 (TRPV4) AS A VASCULAR REGULATOR IN SEPSIS
CLAIRE A. SAND; ANDREW D. GRANT; SUSAN D. BRAIN; MANASI NANDI.
KING'S COLLEGE LONDON, LONDON - REINO UNIDO.
Background. Sepsis is an inflammatory response to infection that can progress to septic shock, characterised by cardiovascular dysfunction and hypoperfusion. Mortality rates can reach 90% and treatment options are highly inadequate. Early preclinical models have focussed on measuring macrocirculatory changes, but there is now a growing realisation that sepsis is a disease of the microcirculation. Thus, there is a strong drive to develop more relevant preclinical models in order to better understand the vascular pathology and identify novel drug targets.
Transient receptor potential vanilloid 4 (TRPV4) is co-expressed in vascular endothelial cells with the endotoxin sensor Toll-like receptor 4. It is sensitised by inflammatory stimuli, and excessive activation leads to profound hypotension and circulatory collapse in mice. We hypothesise that TRPV4 may be an important vascular regulator in sepsis.
Methods and Results.We assessed TRPV4 activity in cultured endothelial cells (ECs) by Ca2+ fluorometry. The 2+ selective TRPV4 agonist GSK1016790A caused a robust Ca influx (EC50 24.38 ± 2.6 nM), which was diminished by the selective antagonist HC-067047 (n=5). Pre-treatement of ECs with bacterial lipopolysaccharide (LPS; 1-100 ng/ml, 24h) dose-dependently enhanced responsiveness to 100 nM GSK10190A. Interestingly, TRPV4 activity was found to correlate with cell death; pre-incubation with HC-067047 was found to preserve cell viability in the presence of LPS (as assessed by tetrazolium dye reduction; n=5).
In order to monitor microcirculatory function in vivo, we have developed a novel method of measuring murine mesenteric blood flow by laser speckle flowmetry. In two different models of sepsis (LPS, 12.5 mg/kg, iv; and cecal ligation and puncture), mesenteric blood flow was found to be significantly decreased in a time-dependent manner (p<0.01, 1-way ANOVA, n=6), consistent with impaired organ perfusion. Low mesenteric flow was found to correlate with plasma markers of organ dysfunction, validating the use of this model for assessing the impact of pharmacogenetic manipulation on sepsis outcome.
Discussion. Our data suggest that excessive TRPV4 activity in septic conditions correlates with decreased endothelial viability. We have developed and validated a novel model of microcirculatory blood flow in order to determine the importance of this pathway in the circulatory collapse that underlies sepsis mortality.
This work was funded by the British Heart Foundation
TREATMENT WITH THALIDOMIDE ANALOGUES MODULATES TNF-A, IL-6 AND IL-10 PRODUCTION AND CONTROL HISTOPATHOLOGY IN LUNGS OF MICE STIMULATED WITH LPS
VICTOR SOARES CAVALCANTE; RODOLFO TOLEDO FILGUEIRAS; TAYNÁ RODRIGUES COELHO; BARBARA MUNIZ FIGUEIREDO; SILVIA HELENA CARDOSO; GIOVANNI WILSON AMARANTE; MAURO VIEIRA ALMEIDA; NADIA RAPOSO; HENRIQUE COUTO TEIXEIRA.
UNIVERSIDADE FEDERAL DE JUIZ DE FORA, JUIZ DE FORA - MG - BRASIL.
Introduction: Thalidomide is an immunomodulatory agent with anti-inflammatory activity. Because of its serious teratogenic effects, thalidomide is only prescribed through a controlled distribution program. Thus, it becomes necessary to develop and validate new compounds derived from thalidomide with proven efficacy in modulating inflammatory responses with low toxicity. A previous study from our laboratory demonstrates that thalidomide analogues containing diamines and open phthalimide structure present high in vitro anti-inflammatory activity on key cytokines such as TNF-a and IL-6 (Biomed. Pharmacother. 66:323-329. 2012). In this study, the anti-inflammatory activity of two thalidomide analogues (GI-16 and SC-15) was evaluated in vivo after LPS-induced mouse lung inflammation. Methods and Results: C57Bl/6 mice (n=5/group) were treated (i.p.) with one dose (20mg/kg or 50mg/kg in 0.5 ml) of thalidomide, SC-15, GI-16 or dexamethasone (positive control, 20mg/kg or 50mg/kg). One hour later each mice was injected intratracheally with LPS (200ug/ml). After 24 hours, the left lung of the mice was extracted to obtain homogenate supernatants used for measurement of TNF-a, IL-6 and IL-10 production by indirect standard ELISA. The right lung was used for histological examination of paraffin sections stained with hematoxylin and eosin. Treatment with both GI-16 (20mg/kg and 50mg/kg) and SC-15 (50mg/kg) greatly inhibited LPS-induced TNF-a (around 34%) and IL-6 (66-89%) in lung homogenate (p<0,05). In contrast, thalidomide (50mg/kg) and SC-15 (50mg/kg) enhanced IL-10 (p<0,05). Histopathological analysis showed reduction in LPS–induced lung inflammation after GI-16 treatment, characterized by discrete capilary congestion. In contrast, animals treated with SC-15 showed moderate to intense inflammatory infiltrate, thickening of alveolar wall and vascular congestion at 20mg/kg, and reduced lung inflammation at 50mg/kg. Conclusions: These results indicate that the compounds SC-15 and GI-16 have marked anti-inflammatory activity, which makes them very attractive compounds and promising drug candidates to treat inflammatory conditions.
Financial support: CNPq, FAPEMIG and CAPES.
TRIMETAZIDINE AND NEUROPROTECTION: ANTIOXIDANT EFFECTS AND MODULATION OF GABAERGIC AND GLUTAMATERGIC SYSTEMS
THALES FONTENELE MORAES PINHEIRO1; LISSIANA MAGNA VASCONCELOS AGUIAR2; JONATAS CAVALCANTE LEMOS3; LUZIANA MARA FROTA SOUZA4; EMANUEL SAMPAIO ARAUJO5; MIRNA MARQUES BEZERRA6; GERARDO CRISTINO FILHO7; VICENTE TEIXEIRA PINTO8; RAYANE SIQUEIRA DE SOUZA9.
1,2,3,4,5,6,7,8.UNIVERSIDADE FEDERAL DO CEARÁ, SOBRAL - CE - BRASIL; 9.UNIVERSIDADE VALE DO ACARAÚ, SOBRAL - CE - BRASIL.
Introduction
Epilepsy is a complex neurological disorder that affects approximately 1% of the population worldwide. Currently available conventional antiepileptic drugs provide control in only 75–80% of epileptic patients. Adverse effects associated with these agents are the main limitation for their long-term use.Therefore, the development of new antiepileptic agent with improved efficacy and minimal adverse effects is very important.Trimetazidine (TMZ) is a lipophilic piperazine derivative, a drug of interest in ischemic diseases. It has demonstrated anti-oxidant, anti- inflammatory, antinociceptiveand gastroprotective properties in various experimental animal models. The aim of this study was to investigate the antioxidant effect of trimetazidineonpilocarpine-induced seizures in mice.
Methods and Results
Male Swiss mice, 25-30g received TMZ(5, 10 and 20 mg/kg, i.p.), fenobarbital (FEN 10 and 30 mg/Kg, i.p.), association (TMZ 5 + FEN 10 mg/kg, i.p.) or saline - NaCl 0,9%, i.p. during seven days.60 minutes after the last injection, pilocarpine (P320 mg/Kg, s.c.) was administered and behavioral tests were performed. After behavioral tests animals were euthanized and cerebral areas were removed for neurochemical analysis.Our results showed an increase in lipid peroxidation in the hippocampus of animals that received pilocarpine (Control = 98.8 ± 5.5 (8); P320 = 336.5 ± 25.2 (8)), and this effect was reversed with TMZ pretreatment at the doses of 10 and 20 mg/kg (TMZ5 = 284.6 ± 19.7 (8); TMZ10 = 171.3 ± 20.1 (8); TMZ20 = 175.3 ± 20.1 (8)). Co-administration of per se ineffective dose of TMZ (5 mg/kg) with subanticonvulsantdose of FEN (10 mg/kg) offered significantprotection, decreasing the MDA levels in48% (TMZ5+FEN10 = 174.6 ± 23.0 (8)) compared to P320 group.Furthermore TMZ was able to reverse the changes produced by pilocarpine in the levels of GABA and glutamate.
Conclusion
This study suggests that antioxidant properties and modulatory action exerted by TMZ on the functioning of gabaergic and glutamatergic systems promoted a neuroprotection against seizure in the pilocarpine model.
This work was supported byCNPq.
TRPV1 ANTAGONISM BY CAPSAZEPINE REDUCES SPLEEN LYMPHOCYTE AND NKT CELL ACTIVATION IN MICE INFECTED WITH PLASMODIUM BERGHEI ANKA
ELIZABETH SOARES FERNANDES1; CAROLINA XAVIER LIMA BRITO2; ARAMYS SILVA DOS REIS3; RENATO BARBOZA4; CLAUDIO ROMERO FARIAS MARINHO5; MARCOS AUGUSTO GREGOLIN GRISOTTO6.
1,2,6.UNICEUMA, SAO LUIS - MA - BRASIL; 3,4,5.UNIVERSIDADE DE SAO PAULO, SAO PAULO - SP - BRASIL.
Introduction: Malaria is a major cause of death affecting millions of new patients with thousands dying from severe malaria every year. In this context, the spleen plays a determinant role in antigen presentation, immunoglobulin production and parasite clearance (Cell Microbiol.14:343-55,2012). TRPV1 has a protective role in sepsis by modulating the immune response to bacterial infection (J Immunol.188:5741-51,2012). Herein, we investigated if TRPV1 is involved in the immune response to malaria.
Methods and Results: Malaria was induced in male C57BL/6 mice (8 week-old, n=5) by intraperitoneal (i.p.) injection of 105 red blood cells infected with P. berghei ANKA (PLoS One.7:e44004,2012). Non-infected mice were used as control (n=5). Parasitemia was accessed daily from day 5 to 7 following infection by analysis of red blood cells by microscopy. Capsazepine (n=5) or vehicle (10% DMSO in saline; n=5) was given i.p. from day 2 after infection for 6 days (2 x day, 50 mg/animal). Mice were culled on day 7 post-infection and spleen cell population phenotype and activation was evaluated by FACS. P.berghei ANKA infection similarly increased total cell population, with F4/80+(2- fold increase), F4/80+Ly6G+ (5.5-fold increase), CD3+CD4+(1.5-fold increase), CD3+CD8+ (1.5-fold increase), CD19+ (2-fold increase), CD3+NK1.1+ (4.5-fold increase) cells significantly raised (P<0.05; One-way ANOVA followed by Bonferroni) in both capsazepine- and vehicle-treated mice when compared with their control littermates. IL-2 receptor expression (denoted by CD25 expression in CD3+CD4+ and CD3+CD8+ cells) was raised in P. berghei infected mice but not changed by capsazepine treatment. Interestingly, capsazepine markedly decreased (P<0.05; mean + SD; One-way ANOVA followed by Bonferroni) the number of CD19+CD69+ (30+10%), CD3+CD4+CD69+ (46+7%) and CD3+NK1.1+CD69+ (49+17%) cells whilst CD3+CD4+CD69+ remained unaffected. CD3-NK1.1+, CD3+CD4+CD25+FOXP3+ and Ly6G+ cell populations showed no differences between groups (One-way ANOVA followed by Bonferroni). Capsazepine treatment only slightly diminished parasitemia (17+10%; mean + SD), an effect not statistically significant.
Conclusion: Our data show TRPV1 long-term blockade by capsazepine affects spleen B and T helper lymphocyte as well as NKT activation in malaria. Further investigation is needed in order to establish the impact capsazepine may have on cytokine generation and malaria severity/sequel.
Financial support: CNPq-Brazil
UNCARIA TOMENTOSA REDUCED INFLAMMATION AND OSTEOCLASTIC BONE RESORPTION IN EXPERIMENTAL PERIODONTITIS.
IRACEMA MATOS MELO1; ANA PATRÍCIA SOUZA LIMA2; MARIANA VASCONCELOS GUIMARÃES3; VILANA MARIA ADRIANO ARAÚJO4; ANA CARLA SILVA SANTOS5; THAYANNE BRASIL6; LUCIANA CARVALHO CÂNDIDO7; GERLY ANNE CASTRO BRITO8; RENATA CARVALHO LEITÃO9; RONALDO ALBUQUERQUE RIBEIRO10; VILMA LIMA11.
1,2,3,4,5,7.FACULTY OF PHARMACY, DENTISTRY AND NURSING, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 6,10,11.DEPARTMENT OF PHYSIOLOGY AND PHARMACOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL; 8,9.DEPARTAMENT OF MORPHOLOGY, FEDERAL UNIVERSITY OF CEARÁ, FORTALEZA - CE - BRASIL.
Introduction: Uncaria tomentosa (UNT) has demonstrated different biological activities, as NF-kB suppression, anti- inflammatory and antioxidant profiles. Periodontitis is an immunoinflammatory disease that results in alveolar bone loss (ABL). We evaluated if UNT affects both the inflammation and, consequently, the bone resorption in the periodontitis model. Methods and Results: Periodontitis was induced in rats by a nylon-3.0 around the left upper 2nd molar, and contralateral as control. Groups of 6 rats each received (sc) saline 0.9% (SAL) or UNT (27 and 81 mg/kg) daily until 11thd, when they were killed. Periodontitis was analyzed through macroscopy, histology, tartrate resistant acid phosphatase (TRAP) immunohistochemical staining, myeloperoxidase activity gingival (MPO) at 6thh and 11thd, and serum bone-specific alkaline phosphatase (BALP) levels. Body mass variation and serum dosages and indexes of kidney and liver were also analyzed. The data were presented as mean±standard error of the mean or median (and range). Ligature caused intense ABL (SAL=4.2±0.3), which was reduced (P<0.05) by the highest dose of UNT (UNT 81=2.7±0.3), and corroborated by increased of BALP (Baseline=136.5±11.9; SAL=93.6±24.2; UNT81=246.0±16.2). Periodontitis also caused an important (P<0.05) leukocyte infiltration, followed by destruction of alveolar bone, cementum and periodontal ligament and low level of the junctional epithelium [Unchallenged=0 (0-1); SAL=3 (2-3)], which was prevented (P <0.05) by UNT [UNT27=1.5 (0-3); UNT81=1 (1-2)]. In line with this, periodontitis induced intense immunostaining for TRAP, while UNT 81 showed less stain. Moreover, the ligature significantly increased MPO at 6thh (Unchallenged=1.04±0.13; SAL=38.3±2.4), what was reduced (P<0.05) by UNT (UNT 81=23.7±4.5). On the 11thd MPO (SAL=2.53±0.26) was reduced (p<0.05), although not back to the levels of unchallenged periodontium (1.14±0.2), while UNT did it (UNT 81= 1.4±0.5; p>0.05). Systemically, UNT did not alter the corporal mass variation and nor caused alteration on serum dosages and indexes of kidney and liver. Conclusions: UNT reduced the local inflammatory response by reducing MPO activity and leukocyte infiltration, resulting in preserving the periodontium and less ABL, without affecting the systemic parameters. Thus, UNT could be quite attractive for the treatment of inflammation underlying periodontal diseases or for other oral inflammatory conditions.
Financial support: Capes; CNPq.
VALIDATION OF A PRECLINICAL MODEL OF DIABETIC ISCHEMIC FOOT ULCERS IN MICE FED HIGH FAT OR HIGH CARBOHYDRATE DIET
LEANDRO CEOTTO FREITAS LIMA (PG)1; LUIZA DIAS DA CUNHA LIMA (PG)2; CAMILA PEREIRA ALMEIDA (IC)3; KATIA MICHELLE FREITAS (PG)4; ROBSON AUGUSTO SOUZA DOS SANTOS5; ADALIENE VERSIANI FERREIRA6; MAURO MARTINS TEIXEIRA7; SÉRGIO HENRIQUE SOUSA SANTOS8; LUCIOLA DA SILVA BARCELOS9.
1,2,3,9.1IMMUNOPHARMACOLOGY GROUP, DEPARTMENT OF 2PHYSIOLOGY AND BIOPHYSICS, ICB-UFMG, BELO HORIZONTE - MG - BRASIL; 4,8.4DEPARTMENT OF PHARMACOLOGY, ICB-UFMG, BELO HORIZONTE - MG - BRASIL; 5.2DEPARTMENT OF PHYSIOLOGY AND BIOPHYSICS, ICB-UFMG, BELO HORIZONTE - MG - BRASIL; 6.1IMMUNOPHARMACOLOGY GROUP, 5BASIC NURSING, NURSING SCHOOL-UFMG, BELO HORIZONTE - MG - BRASIL; 7.1IMMUNOPHARMACOLOGY GROUP, 3 DEPARTMENT OF BIOCHEMISTRY AND IMMUNOLOGY, ICB-UFMG, BELO HORIZONTE - MG - BRASIL.
Introduction: Diabetic foot ulcer (DFU), a severe complication of Diabetes Mellitus (DM), is the leading cause of amputations in diabetic patients and is related to high mortality, especially when associated with peripheral arterial disease. We recently established a preclinical model of ischemic DFU in streptozotocin (STZ)-induced type 1 DM in mice; however, there is no data available to date on type 2 DM (T2DM). The aim of this study is to validate a preclinical model of diabetic ischemic foot ulcers in T2DM mice by comparing the effects of high fat (HFD) and high refined carbohydrate (HCD) diets on ischemic wound healing.
Methods and Results: 8 weeks old C57BL/6 male mice were divided into five groups that received three differents diabetogenic diets for 12 weeks. HC group received a HCD and HF40 and HF60 groups received HFD with 40% and 60% of lipids, respectively. Control and STZ groups received regular diet. Bilateral hindlimb ischemia was induced by permanent occlusion of femoral arteries. At the same occasion, full-thickness wounds were created in the thigh dorsal skin of both legs using a 5-mm-wide biopsy punch. Diet feeding last until two weeks after surgery. Clinical outcome was established by determining the rate of wound closure. Hindlimbs and wound blood flow were evaluated by laser Doppler perfusion imaging. Animals were euthanized at varied time-points and wounds collected for analysis (n=5-7 animals/group). Myeloperoxidase activity was used to assess accumulation of neutrophils. Diabetic mice exhibited delayed wound closure when compared to control group (3d: C: 52±3.6% vs HF60: 39±4.304%*; 7d: C: 61.95±4% vs STZ: 47.2±4.07%**, HC: 50.9±2.4%*) as well as a reduced blood supply in wounded area (7d: C 310± 22 vs STZ 245±21*, HC 241±18*, HF40 227±27**, HF60 232±36**). Interestingly, there was a delayed infiltration of neutrophils in wounds of diabetic mice (1d: C 0.03±0.004 vs STZ 0.01±0.004*, HC 0.03±0.003, HF40 0.02±0.002, HF60 0.01±0.003*; 3d: C 0.07±0.004 vs STZ 0.05±0.007, HC 0.04±0.006***, HF40 0.04±0.007**, HF60 0.04±0.009) which was instead higher in HF40 group at day 7 post surgery (C 0.05±0.005 vs HF40 0.08±0.006*).
Conclusion: Our data suggest that healing of ischemic wounds is impaired in diet-induced T2DM and is associated with poorer wound blood flow and delayed inflammatory cell accumulation.
Financial support: FAPEMIG, CNPq, CAPES, INCT-Nanobiofar.
XYLAZINE INJECTION INDUCES PERIPHERAL ANTINOCICEPTION BY ADRENERGIC, OPIOIDERGIC AND CANNABINOIDERGIC SYSTEM INTERACTION
AMANDA CRISTINA REIS GONZAGA.
UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.
AMANDA CRISTINA REIS GONZAGA(PG)1, PATRÍCIA PAIVA-LIMA1, CELSIO QUEIROZ-JUNIOR2, MARCELO CALIARI2, VINCENZO DI MARZO3, THIAGO ROBERTO LIMA ROMERO1 AND IGOR DIMITRI GAMA DUARTE1
1 Laboratório de Dor e Analgesia, Instituto de ciências Biológicas, 2 Laboratório de Patologia Odontológica,UFMG, Belo Horizonte, MG.
3 Institute of Biomolecular Chemistry, Napoli, Italy.
Introduction: Knowing the importance of xylazine in the veterinary medicine and animal experimentation, the aim of this study was verify the adrenergic, opioid and cannabinoid system participation in the peripheral antinociceptive effect of xylazine. Methods and Results: The rat paw pressure test was used and hyperalgesia was induced by intraplantar injection of PGE2 (2 µg/paw). All drugs were administered intraplantar of Wistar rats (n=4). Xylazine (100
µg/paw) elicited a local inhibition of hyperalgesia. The α2 adrenoceptor antagonist yohimbine (20 µg/paw; 91.3±3.0), the α1 adrenoceptor antagonist prazosin (2 µg/paw; 100.0±0.0), the β adrenoceptor antagonist propranolol (600 ng/paw; 102.4±0.8), the µ-opioid receptor antagonist clocinnamox (40 µg/paw; 103.3±7.8), the δ-opioid receptor antagonist naltrindole (60 µg/paw; 98.3±2.1) and the CB1 cannabinoid antagonist AM251 (80 µg/paw; 99.1±5.9) antagonized the antinociceptive effect induced by xylazine (p<0.05), but not by the κ-opioid receptor antagonist NOR
BNI (100 µg/paw; 8.3±3.9) or the CB2 cannabinoid antagonist AM630 (100 µg/paw; 23.3±5.7) (p<0.05). The anandamide amidase inhibitor MAFP (2 µg/paw; 10.8±1.6) and the anandamide reuptake inhibitor VDM11 (20 µg/paw; 30.4±2.1) increased the peripheral antinociceptive effect of xylazine low dose (p<0.05). In addition, the imunohistochimic technique evidenced an increase of β-endorphin in the keratinocyte area after the xylazine-injection and the dosage of endocannabinoids indicated that xylazine induces a selective release of anandamide in the peripheral site. Conclusion: The results provide evidences that xylazine probably induces peripheral antinociceptive effect by activation of adrenoceptors in the resident immune cells to release β-endorphine or anandamide that activates selectively µ, δ-opioid receptors or CB1 cannabinoid receptor in peripheral.
Financial support: CNPq, CAPES and FAPEMIG.