Effect Of Lentivirus-mediated Overexpression Or Silencing Of MnSOD On Proliferation And Apoptosis Of Resveratrol-treated Fibroblast-like Synoviocytes In Rheumatoid Arthritis

Background Rheumatoid arthritis?RA?is an autoimmune disease involving hyper-proliferation of the synovial membrane and fibroblast-like synoviocytes?FLSs?,leading to progressive joint destruction.However,its pathogenesis remains unclear.Compare to the normal joint,the pathognomonic finding of RA is the expansive synovial tissue,called pannus,which erodes cartilage and bone at the cartilage–bone interface.The pannus behaves like a locally invasive tumor,and the potential imbalance between the growth and death of RA-FLSs is considered a target in the treatment of the disease.There is increasing evidence that reactive oxygen species?ROS?as inflammatory mediators promote FLSs abnormal proliferation.Mitochondrial superoxide dismutase?MnSOD?is a nuclear-encoded mitochondrial enzyme that scavenges superoxide radicals and is thought to be an important determinant of sensitivity to ROS-induced cytotoxicity.Despite the ROS is associated with RA,the relationship between MnSOD and RA remains obscure.There are many studies indicated that SIRT3?sirtuin 3?is the primary mitochondrial acetyl-lysine deacetylase that modulates various proteins to control mitochondrial function and mtROS generation.SIRT3 primarily regulates mtROS clearance by altering the acetylation of MnSOD.More importantly,SIRT3 directly binds and deacetylates MnSOD,which increases MnSOD activity and leads to a significant effect on mtROS homeostasis and cell apoptosis.Resveratrol?Res?is a polyphenol derivatives which exhibits a pro-apoptotic effect in a variety of human cancers by triggering mitochondria apoptosis pathway.However,whether and how Res affects this pathway in RA-FLSs what we are studying.Objective FLSs in the synovial intimal lining play a key role in cartilage destruction.We previously found that Res could promote FLSs apoptosis in adjuvant arthris?AA?rats,but the underlying mechanism was not clear.According to our latest study,Res can suppress the expression of mitochondrial superoxide dismutase?MnSOD?and RA-FLSs proliferation.This suppression was associated with elevated mitochondrial reactive oxygen species?mtROS?levels.Therefore,we hypothesized that Res–mediated RA-FLSs apoptosis might occur via the MnSOD-mtROS pathway.Methods The lentiviral vectors were digested with restriction endonucleases to obtain linearized vectors.The recombinant plasmids were named GV358 and GV248.The recombinant plasmids were transformed into the competent bacteria,and the monoclonal colonies were identified by PCR.The positive clones were sequenced.293T cells were infected with recombinant lentivirus,and the expression level of MnSOD was detected by Western bloting.RA-FLSs were infected with lentiviruses and screened with puromycin at a concentration of 8?g/mL,and the infection rate exceeded 90%.We divided the RA-FLSs into four groups:a control group,a negative control?NC?group,a MnSOD overexpression group,and a MnSOD RNAi group.The four groups of RA-FLSs were tested using confocal laser scanning microscopy,CCK-8 assays,flow cytometry,and Western bloting was conducted to determine the involvement of the MnSOD-mtROS pathway.Result Compared with the NC group,the MnSOD overexpression group treated with different concentrations of Res?0,25,50,100,and 200?M?and 5?M H₂O₂ showed reduced levels of mtROS,increased B-cell-lymphoma-2?Bcl-2?,reduced Bcl-2Associated X protein?Bax?,and fewer apoptotic cells.The MnSOD RNAi group showed the opposite results.Conclusion Thus,we conclude that Res could facilitate RA-FLSs apoptosis by regulating MnSOD expression and mtROS levels.Our findings show a novel mechanism for the beneficial effects of Res,especially in relation to the MnSOD-mtROS signaling pathway in RA.