Effect Of MnSOD-SIRT3 Expression On Resveratrol In Adjuvant Arthritis Of Rat Synovialjoints

Background: In the pathogenesis of rheumatoid arthritis(RA),oxidative stress reaction is an important factor of pathogenic factors,such as joint pain and function limited in RA patients.Because patients need long-term treatment,change the way of life,which make the body in a state of stress and lipid peroxidation,increase the oxygen free radicals(ROS)generation,accumulation of ROS in the body,cause the body damage.At present,in the preparation of animal models of RA,using Freund's complete adjuvant(FCA)preparation of adjuvant arthritis(AA)rats model is relatively common,because the morphology change is similar to RA,and the model establishment is relatively simple,therefore,the AA model is often used to simulate the RA.Manganese superoxide dismutase(Mn SOD)is an important antioxidant enzymes,metal dismutase exist in eukaryotic cells,mitochondria can be super oxygen anion free radicals(O.-)2,specificity into H2O2 and O2,because mitochondrial oxidative phosphorylation is not only the body cells metabolism and generate energy,but also is the important place to produce free radicals,therefore,mitochondrial function significantly in the oxidation damage.At the same time,in the body of the mitochondria,acetylation enzyme 3(SIRT3),exist in the family of acetylation enzyme sirtuins(SIRTs),which has to acetylation enzyme activity,in the role of mitochondrial energy metabolism and regulate the body's resistance to oxidative stress.Resveratrol(Res)is multi-phenolic compounds,belong to the phytoalexin,Res has anti-inflammatory,antioxidation,antitumor,etc,this study discussed whether Res have antioxidant effect in vitro and the relationship between mitochondrial Mn SOD-SIRT3 signaling pathways in AA rats.Objective To investigate the relationship between the changes of manganese superoxide dismutase(Mn SOD),sirtuin 3(SIRT3)with adjuvant arthritis(AA)rats.Culturing synovial cells in vitro,to test the effect of Res treated fibroblast-like synoviocytes(FLS)proliferation induced by low hydrogen peroxide(H2O2),to explore the effect of the role of Res inhibited AA rats FLS on mitochondria Mn SOD,SIRT3 signaling pathways change.Methods: 10 SD rats subcutaneous injection of 0.1 ml Freund's complete adjuvant(FCA)in pelma induced by AA,another 10 rats subcutaneous injection amount of phosphate buffer solution(PBS)as control group.After 28 days,femoral artery bleeding to execute the rats and collect serum,application of spectrophotometer,two groups of rats body oxidative stress indicators were detected,glucosinolates barbituric acid method to detect malondialdehyde(MDA)content,superoxide dismutase(SOD)hydroxylamine method activity,colorimetric method for the determination of glutathione peroxidase(GSH-px)activity.Two groups of rats ankle structure change were observed by HE staining.SIRT3,Mn SOD proteins expression in synovial tissues were observed by immune histochemical methods SP method,and the average optical density(OD)values were determined by computer image analysis system.Synovial cell of rats were cultivated in vitro with tissue block cultivation method,which identified by rat vascular cell adhesion molecule-1(VCAM-1))polyclonal antibody,effect of Res with low concentration of H2O2(5 ?mol/L)on proliferation effect FLS was observed by CCK-8 method,oxidative stress related proteins of SIRT3,Mn SOD expression were detected by Western blot in AA rats FLS.Result(1)Compared with normal group rats,AA model rats serum MDA content and GSH-px oxidative stress index content and SOD activity were significantly increased(P< 0.01);HE staining showed that AA model rat articular synovial hyperplasia,inflammatory cells infiltration;Brown or yellow particles were presented in synovial tissue in rat AA model by immuno-histochemical staining,the model group mitochondrial oxidatie stress SIRT3,Mn SOD protein expression increased with computer image analysis results.(2)In vitro experiments showed that VCAM 1 polyclonal antibody identification of FLS by immunofluorescence staining;With the increase of the concentration of the Res,low concentration of H2O2 treatment of AA rats proliferation inhibition of FLS,SIRT3 and Mn SOD proteins expression decreased.Conclusion: AA model group rats exist oxidatie stress,synovial tissue inflammatory performance,mitochondrial oxidative stress protein expression in rat AA model increases;Res can suppress FLS proliferation which treated by low concentration of H2O2 in AA rats,and mitochondria oxidative stress protein SIRT3,Mn SOD protein expression.