Effect Of Resveratrol On Oxidative Stress And Apoptosis In H₂O₂-treated Rheumatoid Arthritis Fibroblast-like Synoviocytes Via Nrf2-Keap1 Pathway

Background:Rheumatoid arthritis?RA?is an autoimmune disease caused by a combination of genetic,hormonal,and environmental factors that cause slow development and constant damage to joints.RA can cause inflammation of joint tissues and abnormal bone morphology,which prevents patients from moving normally.In recent clinical trials,it has been shown that there is a correlation between selective RA-specific rheumatoid factor and anti-cyclic citrullinated peptide antibody,and the presence of the two affects each other to make clinical symptoms of RA patients continue to worsen.In China,the main target tissues of established RA models are cartilage and synovial tissue,which are characterized by intimal hyperplasia and accumulation of subsynovial inflammatory cells.At present,RA patients are mainly used to control symptoms and tissue inflammation,maintain joint function,and avoid joint deformities.Oxidative stress is common in the initial stages of chronic disease.In the normal human body,there is a balance between the generation and resistance of the body's oxidation,and the body's oxidation and anti-oxidation balance appears disordered in patients with RA.Persistent inflammatory injury,long-term drug treatment,pain,and limited functional activities cause the patient's body to be in an oxidative stress state,manifested by the excessive production of reactive oxygen species?ROS?and insufficient ability to resist oxidation,which can damage joints The proteins,lipids,nucleic acids,and other matrix components produced by synovial tissue break the original balance of oxidation and oxidation,which triggers an oxidative stress response and directly leads to hypoxia in local tissues.Under normal physiological conditions,when ROS is present at low concentrations,it can be used as a"redox transporter"in cells to perform its functions of cell signal transduction and functional regulation.The production of superoxide dismutase?SOD?,glutathione peroxidase?GSH-Px?,etc.is conducive to oxidative resistance in cells.Nrf2?NF-E2-related factor 2?,a key transcription factor in oxidative stress,can activate phase II detoxification enzyme,promote Nrf2 and Keap1 decoupling,and transport active Nrf2 to the nucleus,which is formed after binding with Maf protein Dimers composed of different types of subunits are then combined with AREs to activate target gene expression and inhibit the synthesis of mitochondria and NADPH,thereby inhibiting the production of ROS.Fibroblast-like synoviocytes in rheumatoid arthritis?RA-FLSs?have the characteristics of tumor-like cells,that is,abnormal proliferation causes inflammation to disrupt the body's original steady state.When the ROS level exceeds a certain standard,it may induce the opening of the transient pore of mitochondrial permeability,damage the mitochondria,affect the synthesis of adenosine triphophate?ATP?,and induce apoptosis.Resveratrol?Res?is a plant antitoxin and has many biologically active polyphenolic compounds.Res naturally exists in cis and trans isomers.The trans isomers are dominant in plants,and its physiological function is significantly better than that of cis structural function in responding to damage caused by UV and fungal infection.Objective:To explore the effects of different concentrations of Res on oxidative stress and apoptosis in RA-FLSs treated with hydrogen peroxide?H₂O₂?through the Nrf2-Keap1 pathway.Methods:The CCK-8 method was used to detect the proliferation of RA-FLSs cells treated with 5?M H₂O₂ and different concentrations of Res?0,1,10,20,40?M?,and the RA-FLSs migration ability was measured by scratch test.After the above treatment,the contents of malonaldehyde?MDA?and reactive oxygen species?ROS?in the supernatant of RA-FLSs were detected;Hoechst33342 and flow cytometry were used to detect the effects of RA-FLSs apoptosis;Western Bloting The expression of Nrf2,Keap1,HO-1,NF-?B,Bcl-2 and Bax proteins was detected by immunofluorescence.At the same time,after using Nrf2 to interfere with Nrf2,it was verified whether Res had an effect through Nrf2-Keap1 pathway.5?M H₂O₂ and 40?M Res were used to treat RA-FLSs after Nrf2si RNA infection,and the mitochondrial ROS content was detected by mitochondrial staining?red fluorescence?;apoptosis was detected by flow cytometry;Nrf2,Keap1,HO-1,Bcl-2 and Bax protein expression.Result:?1?RA-FLSs treated with 5?M H₂O₂ showed abnormal proliferation.Res inhibited the abnormal proliferation and migration of RA-FLSs in a dose-dependent manner.?2?RA-FLSs treated with 5?M H₂O₂increased intracellular MDA and ROS levels.Res activated Nrf2 nuclear expression and Keap1 in a dose-dependent manner,increased HO-1 expression,and increased the production of antioxidant enzymes.Reduce MDA and ROS levels in RA-FLSs.?3?Res prevents I?B phosphorylation by activating Nrf2,thereby preventing the activation of NF-?B p65,reducing the expression of Bcl-2,increasing the expression of Bax,and inducing apoptosis in RA-FLSs.This is consistent with the analysis results of flow cytometry,which indicate that the cells obviously tend to apoptosis.?4?After RA-FLSs knocked down Nrf2 protein by lentivirus infection,compared with NC group,although Res can activate the Nrf2-Keap1 pathway,the activation was not obvious,and Res'ability to reduce mt ROS was significantly lower than before lentiviral infection It is shown that the loss of Nrf2 protein affects the antioxidant level of Res.?5?After RA-FLSs knocked down Nrf2 protein by lentivirus infection,Bcl2protein expression and Bcl2/Bax ratio in the 40?M Res treatment group were lower than those in the NC group and the 5?M H₂O₂ group,indicating that the loss of Nrf2protein affected the induction of Res.The ability to apoptotic,and consistent with the results of flow cytometry analysis.Conclusion:?1?Res inhibits the production of ROS and MDA in RA-FLSs through the Nrf2-Keap1 pathway and reduces the damage caused by oxidative stress.?2?Res inhibits the activity of NF-?B p65 by activating the Nrf2-Keap1 pathway,inhibits the abnormal proliferation and migration of RA-FLSs,and induces apoptosis of RA-FLSs.?3?Because RA-FLSs was infected by lentivirus,it reduced Nrf2 expression,so Res'ability to exert anti-oxidation and induce apoptosis through Nrf2-Keap1 pathway was weakened.