• Abstract and Figures
• Public Full-text

Acta Medica Mediterranea, 2018, 34: 119 THE EFFECTS OF ALLOPURINOL ON REDOX SENSITIVE TRANSCRIPTION FACTORS, PRO- INFLAMMATORY CYTOKINES AND HEME OXYGENASE-1 IN ACETIC ACID-INDUCED COLITIS IN RATS ULVI DEMIREL1, MEHMET YALNIZ1, CEMAL ORHAN2, NURETTIN TUNC1, ABDURRAHMAN SAHIN1, MEHMET TUZCU3, KAZIM SAHIN2, IBRAHIM HANIFI OZERCAN4, IBRAHIM HALIL BAHÇECIOGLU1 1Department of Gastroenterology, School of Medicine, Firat University, Elazig, Turkey - 2Department of Animal Nutrition, Faculty of Veterinary Science, Firat University, Elazig, Turkey - 3Department of Biology, Faculty of Science, Fırat University, Elazig, Turkey - 4Department of Pathology, School of Medicine, Firat University, Elazig, Turkey ABSTRACT Introduction: Oxidative stress plays an important role in pathophysiology of inflammatory bowel diseases. We aimed to inve- stigate the effects of antioxidant allopurinol (AP) and the potential mechanisms leading amelioration upon acetic acid induced coli- tis. Materials and methods: Twenty-eight rats were divided in four groups. Control rats received 1 mL of NaCl (0.9%). In the AA group rats received 1 mL of 5% (v/v) acetic acid. The rats in the AAAP group received 1 mL of 5% (v/v) acetic acid. Additionally these rats received 100 mg/kg AP. AP group rats received only 100 mg/kg AP. Histopathological, biochemical and western blot analy- sis were done. Results: In the AA group, colonic injury scores, malondialdehyde (MDA), NF-κB p65, TNFα, COX2, AP-1 and IL-6 levels were significantly higher than in the control group (P<0.05). In the AAAP treatment group the colonic injury scores, MDA, NF-κB p65, TNFα, COX2, AP-1 and IL-6 levels significantly decreased when compared to the AA treated rats (P<0.05). The expression of HO-1 in the AAAP group significantly increased when compared to the AA treated rats (P<0.05). Conclusion: Intra colonic acetic acid instillation causes severe intestinal injury accompanied to increased oxidative stress. AP treatment ameliorates this injury by overcoming oxidative stress and regulating cellular redox balance in favor of antioxidant defense mechanisms probably via manipulation of redox sensitive transcription factors. Keywords: Oxidative Stress, Allopurinol, Acetic Acid, Colitis, Redox Sensitive Transcription Factors. DOI: 10.19193/0393-6384_2018_1_20 Received November 30, 2017; Accepted January 20, 2018 Introduction pathways for excessive reactive oxygen species (ROS) generation, and this enzyme is present at the Ulcerative colitis (UC) and Crohn's disease highest level in the intestine. XO-derived ROS (CD) are chronic and idiopathic intestinal inflam- causes tissue injury in various ways such as direct matory disorders. Although the pathophysiology of damaging cellular components, activation of these disorders is not completely understood, accu- nuclear factor-kappa B (NF-қB) signaling pathway, mulated experimental and clinical data suggests and upregulation of inducible nitric oxide synthase that increased oxidative stress plays important role (iNOS)(2, 3). Allopurinol (AP) is a competitive in these diseases initiation and progression(1). inhibitor of XO enzyme and has dose dependent Xanthine oxidase (XO) enzyme is one of the major free radical scavenging ability(4). 120 Ulvi Demirel, Mehmet Yalniz et Al Additionally, AP decreases tumor necrosis fac- deprived of not water for 24 h prior to the induction tor-alpha (TNF-α) and the expression of intercellu- of chemical colitis. The study protocol was in lar adhesion molecules-1 (ICAM-1) production. It accordance with the guidelines for animal research has been also reported that AP can down-regulate and approved by the Ethics Committee of Firat ROS dependent activation of NF-қB which is a University Medical Faculty. very important mediator of tissue inflammation. Useful effects of XO inhibition have been reported Experimental Design and Induction of with AP and amflutizole treatment in different coli- Colitis tis models(5-7). Twenty eight rats were divided four group: Heme oxygenase-1 (HO-1) is considered as a Control group (C), acetic acid group (AA), acetic major cellular defense system against oxidative acid+100 mg/kg allopurinol (AAAP) group, and insults. In increased oxidative stress conditions HO- only 100 mg/kg allopurinol (AP) group. Each group 1 production is upregulated by redox sensitive tran- consisted of seven rats. Fasted animals were lightly scription factors, including NF-қB, activator pro- anesthetized with ether. Control rats received 1 mL tein-1 (AP-1), and nuclear factor E2-related factor- of NaCl (0.9%) by intracolonic with a flexible plas- 2 (Nrf2)(8). Cyclooxygenase-2 (COX-2) is also tic rubber catheter with an outside diameter of 2 induced mainly at sites of inflammation by above mm which was inserted 8 cm into the colon via the factors like HO-1 enzyme. It has been demonstrated anus. In the AA group rats received 1 mL of 5% that intestinal inflammation can be prevented by (v/v) acetic acid in 0.9% NaCl by intracolonic with inhibition of COX-2 expression(9). At the present the same tip of cannula in the control group (10). day, it is well known that synthesis pro-inflammato- The rats in the AAAP group were subjected to the ry mediators and antioxidant defense enzymes are same procedure in the AA group rats. Additionally, strictly controlled at the nuclear level by aforemen- these rats received 100 mg/kg AP (Ürikoliz® 300 tioned redox-sensitive transcription factors. mg tablet, obtained from Sandoz, Turkey) by Therefore, development of the new treatments tar- intraperitoneal (ip) route at the same day of the col- geting the manipulation of these redox sensitive itis induction and AP treatment was continued for signaling is considered as ideal strategies to prevent seven consecutive days. To avoid chemical, infec- or treat oxidative stress-related diseases. tious peritonitis and achieve optimal dissolution, In contrast to drugs that have been already the AP was dissolved in sterile water and then titrat- used in inflammatory bowel diseases (IBD) treat- ed to pH 11 with NaOH under strict sterile condi- ment including 5-ASA, corticosteroids and biologi- tions(7). cal agents, allopurinol is cheap, and has been gener- Allopurinol treatment solutions were prepared ally regarded as a safe drug. All these data about AP and used daily. In the AP group rats were subjected suggest that AP may be a considerably proper can- to the same procedure in the control group rats. didate for prevention of intestinal inflammation. In Additionally, in this group rats received AP treat- this study we aimed to investigate the effects of AP ment at the same dose, route and duration in the treatment on redox sensitive transcription factors AAAP group rats. At the end of the seventh day all (AP-1, NF-қB, Nrf2, and TNF-α) and these factors rats were decapitated. The colon was removed at mediated synthesis of pro-inflammatory COX-2 the level of 8 cm from the anus. The colon were interleukin-6 (IL-6) and antioxidant HO-1 enzyme opened longitudinally, and rinsed with saline solu- in acetic acid-induced colitis in rats. tion. Then, colon samples were cut in two parts. One part fixed in 10% neutral buffered formalin for Material and methods histopathological examination. Other tissue samples were stored at -70°C for subsequent measurement Animals of tissue malondialdehyde (MDA), NF-қB, AP-1, Healthy, male, Albino, Wistar strain rats, Nrf2, and TNF-α, HO-1, COX2 and IL-6 levels. weighing 250-300g, were purchased from the Firat University Laboratory Animal Research Center Histopathological Examination of Colon (Elazig, Turkey). The animals were housed at tem- Distal colon segments which were fixed in perature-controlled (24±1 °С), humidity of 55 ±5%, 10% phosphate-buffered formaldehyde, embedded alternating 12 h light/12 h dark cycles, with normal in paraffin blocks and sliced in 5 µm thickness sec- rat chow and fresh water ad libitum. Rats were tions. These sections were stained with hematoxylin The effects of allopurinol on redox sensitive transcription factors, pro-inflammatory cytokines... 121 and eosin for light microscopic examination. The was added to the supernatant. Equal amounts of tissue slices were scanned and scored by two expert protein (50 μg) were electrophoresed and subse- pathologists who were unaware of sample assign- quently transferred to nitrocellulose membrane ment to the experimental groups. Histological scor- (Schleicher and Schuell Inc., Keene, NH, USA). ing was determined by examining each specimen Blots on nitrocellulose membrane were washed for following features of damage: submucosal twice for 5 min each in PBS and blocked with 1% edema, vasodilation, inflammatory cell infiltration bovine serum albumin in PBS for 1 h prior to appli- and necrosis. The degree of intestinal injury was cation of the primary antibody. The antibody scored to above features using a scale of 0-3 (nor- against Nrf-2, TNFα, COX2, AP-1, and IL-6 was mal: 0, mild: 1, moderate: 2, severe: 3) like previ- the purchased from Santa Cruz Biotechnology, Inc. ously described(11). (Santa Cruz, CA, USA). Antibodies against HO-1 and NF-κB p65 were purchased from Abcam Determination of Colonic Malondialdehyde (Cambridge, UK). Primary antibody was diluted Levels (1:1000) in the same buffer containing 0.05% The colonic samples (100 mg) were homoge- Tween-20. The nitrocellulose membrane was incu- nized in 1 mL of 0.05M Trizma base buffer (pH bated overnight at 4 °C with protein antibody. The 7.4). The homogenates were centrifuged (16,000g, blots were washed and incubated with horseradish 4 ºC) for 30 min. The supernatant was collected and peroxidase-conjugated goat anti-mouse IgG stored at -80 ºC. The colonic MDA level was mea- (Abcam, Cambridge, UK). Specific binding was sured using the fully automatic High Performance detected using diaminobenzidine and H2O2 as sub- Liquid Chromatography (HPLC) (Shimadzu, strates. Protein loading was controlled using a mon- Kyoto, Japan) equipped with a pump (LC-20AD), oclonal mouse antibody against β-actin antibody an ultraviolet-visible detector (SPD-20A), an inert- (A5316; Sigma).

Load more

  8

Copy Link