Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MA
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Int. J. Biol. Sci. 2016, Vol. 12 72 Ivyspring International Publisher International Journal of Biological Sciences 2016; 12(1): 72-86. doi: 10.7150/ijbs.13188 Research Paper Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways Hongming Lv1#, Zhenxiang Yu2#, Yuwei Zheng1, Lidong Wang1, Xiaofeng Qin1, Genhong Cheng1, Xinxin Ci1 1. Institute of Translational Medicine, The First Hospital of Jilin University, College of Veterinary Medicine, Jilin University, Changchun, China 2. Department of Respiratory Medicine, The First Hospital of Jilin University, Changchun, China. # These authors contributed equally to this work. Corresponding author: X.-X.Ci, Institute of Translational Medicine, The First Hospital, Jilin University, Changchun, 130001, China. Tel.: +86 431 88783044; E-mail addresses: [email protected] (X.-X.Ci.) © Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions. Received: 2015.07.08; Accepted: 2015.11.02; Published: 2016.01.01 Abstract Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H2O2-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expres- sion of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways. Key words: Isovitexin; LPS; inflammation; oxidative stress; ALI Introduction Oxidative damage and inflammation are associ- patients themselves, plays an essential role in regu- ated with the pathogenesis of various diseases, in- lating lung injury. For example, the antioxidant cluding acute lung injury (ALI) [1, 2]. Both ALI and its N-acetylcysteine attenuates pulmonary function in more serious form, acute respiratory distress syn- ARDS patients [5, 6]. Lipopolysaccharide (LPS), a drome (ARDS), are severe and acute respiratory dys- pathogenic endotoxin present in the outer membranes functions. Both conditions present with overwhelm- of Gram-negative bacteria, enhances the formation of ing lung inflammation and cause high mortality rates reactive oxygen species (ROS) and inflammatory me- worldwide [3, 4]. Furthermore, oxidative stress, which diators and is commonly used to induce pharmaco- is present in many animal models of ALI and in ALI logical research models of ALI [7, 8]. http://www.ijbs.com Int. J. Biol. Sci. 2016, Vol. 12 73 Accumulating evidence suggests that LPS can tivities, including anti-inflammatory and antioxidant activate nuclear factor-kappa B (NF-κB) and three activities, which may play a key role in the prevention mitogen-activated protein kinase (MAPK) pathways, of various diseases, including AMD, cancer and dia- inculding the c-Jun NH2-terminal kinase (JNK), ex- betes [22, 23]. The glycosylflavonoid isovitexin (IV) tracellular signal-regulated kinase (ERK), and p38 (Fig. 1A), extracted from rice hulls of Oryza sativa, pathways, which result in the overexpression of in- possesses various biological activities, including an- flammatory mediators such as tumor necrosis factor-α ti-inflammatory and antioxidant activities [24]. (TNF-α), interleukin-6 (IL-6), NO and prostaglandin However, the protective effect of IV against (PG) E2. Cyclooxygenase-2 (COX-2) and inducible LPS-induced damage has not yet been demonstrated nitric oxide synthase (iNOS) are responsible for the in vitro or in vivo. In the present study, we evaluated production of PGE2 and NO, respectively [9, 10]. On the protective effect of IV and the mechanisms related one hand, activation of inflammatory signaling re- to inflammation and oxidative stress in RAW 264.7 sulted in increasing adhesion molecules expressions, cells exposed to LPS and in an LPS-induced ALI including intercellular adhesion molecule (ICAM)-1 mouse model. and vascular cell adhesion molecule (VCAM)-1, which play significant roles in cell adhesion and mi- Materials and Methods gration [11, 12]. On the other hand, these Reagents and chemicals pro-inflammatory cytokines lead to further damage of epithelial and endothelial cells in the lung. This Isovitexin (IV) with a purity >98% was pur- damage leads to the activation of peripheral poly- chased from the National Institute for the Control of morphonuclear neutrophils (PMNs). Subsequently, Pharmaceutical and Biological Products (Jilin, China). over-activation of PMNs can regulate inflammatory LPS (Escherichia coli 055:B5) and dimethyl sulfoxide responses and induce tissue damage through oxida- (DMSO) were purchased from Sigma-Aldrich (St. tive stress [13], the primary source of which is respir- Louis, MO, USA). Antibodies against p-(ERK1/2), atory burst and degranulation. Oxidative stress can ERK1/2, p-(JNK1/2), JNK1/2, p-p38, p38, p65, IκB, increase the production of ROS, myeloperoxidase p-IκB, Nrf2, HO-1, Lamin B and β-actin were pur- (MPO) and malondialdehyde (MDA) and decrease chased from Cell Signaling (Boston, MA, USA). the production of anti-oxidative enzymes, including SP600125, SB203580, and U0126 (specific inhibitors of reduced glutathione (GSH), superoxide dismutase the JNK1/2, p38, and ERK1/2, respectively) were (SOD), and heme oxygenase (HO)-1, which collec- purchased from Sigma-Aldrich (St. Louis, MO). tively protect lung tissues against oxidative damage ICAM-1 and VCAM-1 polyclonal antibody were in vivo [14]. These anti-oxidative enzymes are medi- purchased from ABclonal (MA, USA). The tin proto- ated by nuclear factor-erythroid 2-related factor 2 porphyrin IX (SnPP IX, HO-1 inhibitor) was pur- (Nrf2) [15, 16]. Nrf2 plays an essential role in protect- chased from Calbiochem (La Jolla, CA, USA). Control ing cells against various inflammatory- and oxidative siRNA and Nrf2 siRNA were purchased from Santa stress-induced diseases, including age-related macu- Cruz Biotechnology (Santa Cruz, CA, USA). A lar degeneration (AMD), chronic obstructive pulmo- Prime-Script RT-PCR kit was purchased from Takara nary disease (COPD) and asthma [17, 18]. Under un- (Dalian, China). In addition, GSH, MDA, SOD and stressed conditions, Nrf2 is located in the cytoplasm MPO test kits were obtained from Nanjing Jiancheng in association with its repressor protein, Kelch-like Bioengineering Institute (Nanjing, China). Total ECH-associated protein 1 (Keap1). Under stress con- ROS/Superoxide Detection Kit was purchased from ditions, Nrf2 dissociates from Keap1, translocates into Enzo Life Sciences Inc (Farmingdale, NY, USA). All the nucleus and binds to the antioxidant response other chemicals, unless specifically stated elsewhere, element (ARE). This interaction leads to the induction were purchased from Sigma-Aldrich (St. Louis, MO, of phase II detoxifying enzymes and cytoprotective USA). genes, such as HO-1 [19]. HO-1 is the major an- Animals ti-inflammatory and anti-oxidative enzyme among Male BALB/c mice, 6-8 weeks old and weighing the genes that are mediated by Nrf2 activation [20]. approximately 18-20 g, were obtained from Liaoning Several reports have suggested that the activation of Changsheng Technology Industrial Co., LTD (Certif- HO-1 counteracts the inflammatory and oxidative icate SCXK2010-0001; Liaoning, China). All animals damage that occurs in LPS-induced ALI [21]. There- were housed in a room kept at a temperature of 24 ± 1 fore, suppressing inflammation and oxidative stress °C under a 12 h light-dark cycle with a relative hu- may be an effective method of preventing and treating midity of approximately 40-80%. The animals were ALI. allowed free access to tap water and normal food after Flavonoids exhibit a series of pharmaceutical ac- feeding for several days. All studies were performed http://www.ijbs.com Int. J. Biol. Sci. 2016, Vol. 12 74 in accordance with the International Guiding Princi- 1 h, after which LPS (2 μg/ml) was added to the ples for Biomedical Research Involving Animals, treatment group of plates for an additional 24 h. Next, which was published by the Council for the Interna- the cells were stained with 100 μl of ROS/Superoxide tional Organizations of Medical Sciences. detection reagent for 1 h to detect ROS generation. Absorbance was measured in a multi-detection reader Cell culture and MTT assay (Bio-Tek Instruments Inc.)