Investigating The Mechanism Of Sirolimus In The Treatment Of Rheumatoid Arthritis Based On AKT/mTORC1 Pathway

Background:Rheumatoid arthritis(RA)is an autoimmune disease characterized by abnormal proliferation of synovial membranes.The main pathological changes are synovial hyperplasia,persistent synovitis,formation of vascular opacities,and destruction of cartilage.The pathogenesis of RA has yet to be elucidated.It has been shown that the development of RA is associated with the activation of the mTOR pathway,and inhibition of the AKT/mTORC1 signalling effectively slows down the pathogenesis of RA.In this study,we verified whether sirolimus has a therapeutic effect on the CIA model in vivo.In vitro experiments were performed by extracting primary synovial fibroblasts from RA patients and culturing them to investigate the effect of sirolimus on synovial fibroblasts and the role of AKT/mTORC1 pathway in RA treatment and its molecular mechanism,providing an experimental basis for the clinical treatment of RA with sirolimus.Purpose:(1)To observe the interventional effect of sirolimus on CIA mice.(2)To investigate the effects of sirolimus on the proliferation and apoptosis of RA synovial fibroblasts(RA-FLSs).we further explored the molecular mechanisms of the signaling pathways associated with sirolimus-induced apoptosis in RA-FLSs.Methods:1.Establishment the Collagen-Induced Arthritis(CIA)mouse model: C57BL/6J mice were induced with chicken type II collagen to prepare the arthritis model.The mice were randomly divided into two groups:the CIA group without intervention(CIA control Group)and the Sirolimus intervention CIA group(CIA Sirolimus Group).The severity of arthritis in the mice was assessed by clinical scores;the genetic differences were analyzed by transcriptomics with the peripheral blood of the mice.2.The synovial tissue specimens were collected from RA patients,and primary synovial cells were isolated by the tissue block method.The proliferation of RA synovial fibroblasts with different concentrations of sirolimus by the CCK-8 method.In order to analyze the effect of sirolimus on RA-FLSs,the apoptosis rate of RA-FLSs was detected by flow cytometry.the m RNA expression of AKT,mTOR,Bcl-2,and Bax in RA-FLSs was analyzed by real-time fluorescence PCR assay after sirolimus intervention.The expression levels of apoptotic proteins Bcl-2,Bax,Caspase 3,Caspase 9 and cyclin CDK2 and CD1 proteins in the RA-FLSs were analyzed by protein immunoblotting(Western blot).The expression of AKT/mTORC1 pathway proteins and their phosphorylated proteins were analyzed.Results:1.Sirolimus relieved CIA-related symptoms and reduced arthritis scores(P <0.05).A total of 540 differential genes were observed after sirolimus treatment,in which 249 differential genes were upregulated and 291 differential genes were downregulated.2.Sirolimus inhibited the cell proliferation of RA-FLSs:The CCK-8 results showed that the different concentrations of sirolimus(1-25 nmol/L)had significant inhibitory effect on RA-FLSs compared with the PBS control(P<0.05).The cellular inhibition rate of RA-FLSs was 50.4% at 10 nmol/L concentration of sirolimus and48 h,which was close to 50%(IC50).Therefore,10 n M sirolimus and 48 h were selected as the optimal action time for the follow-up experiment.3.Sirolimus-induced apoptosis in RA-FLSs cells and mechanism study:The flow cytometry results showed that the apoptosis rate in RA-FLSs cells after sirolimus-treated increased significantly compared with the PBS control group.The q RT-PCR and Western blot results showed that the m RNA and protein expression level of pro-apoptotic protein Bax was increased and anti-apoptotic protein Bcl-2 was decreased in the sirolimus group compared with the control group.Western blot results showed that sirolimus intervention promoted the expression of apoptotic proteins Caspase 3 and Caspase 9 and inhibited the expression of cyclins CDK2 and CD1 expression.The AKT/mTORC1 signaling pathway is essential in regulating the proliferative activity of synovial fibroblasts in patients with rheumatoid arthritis.RT-PCR results showed that the expression levels of AKT m RNA and mTOR m RNA in RA-FLS were reduced by sirolimus treatment.Western blot results showed that intracellular AKT,mTOR,p70S6 and p70S6 were reduced by sirolimus treatment.mTOR,p70S6 K,and4EBP1 protein phosphorylation expression levels were decreased by sirolimus treatment,but there was no difference with total protein expression.The results indicates that sirolimus can inhibit AKT/mTORC1 signaling pathway.Conclusion:1.Sirolimus can effectively alleviate joint inflammation in CIA mice.2.Sirolimus can effectively inhibit cell proliferation and promote the apoptosis of RA-FLSs.3.Sirolimus inhibited cell proliferation and induced apoptosis of RA-FLSs through the AKT/mTORC1 pathway.