Berberine Promotes The Apoptosis Of Fibroblast-like Synoviocytes In Rheumatoid Arthritis By Inhibiting Autophagy Through ROS-STAT3

Background: Rheumatoid arthritis(RA)is a chronic,inflammatory,autoimmune disease characterized by synovial inflammation and cartilage and bone destruction.Activation and excessive proliferation of fibroblasts(FLS)are thought to be important factors in promoting the pathological progression of RA,and our previous studies have found that berberine(BBR)can exert good joint protection in rats with adjuvant arthritis(AA).The regulation of autophagy apoptosis imbalance plays an important role in synovial injury and mediation of chronic inflammatory response in RA joints.Therefore,indepth study of whether BBR affects synovial inflammatory damage by regulating FLS autophagy apoptosis imbalance may provide a new theoretical basis and experimental basis for elucidating the network regulation role of BBR in the treatment of RA.Objective: 1.To investigate the role of signal transducer and activator of transcription 3(Stat3)in regulating autophagy apoptosis in rheumatoid arthritis fibroblast-like synovial cells(RA-FLSs),and its molecular mechanism.2.In vitro to explore the molecular mechanism of BBR regulating the autophagy apoptosis balance of RA-FLSs.Methods: Synovial cases of RA and OA patients were screened and collected,pathological tissue wax blocks were selected before hospitalization,and TUNEL staining was performed to detect apoptosis of synovial histiocytes.Immunohistochemical analysis detected the expression levels of p-STAT3 and autophagy apoptosis-related proteins BCL-2 and Beclin1,and analyzed the correlation with the level of disease activity related indicators.The Freund’s complete adjuvant was fully emulsified and injected into the plantar of rats to establish a rat AA model.Normal control group,model group,MTX positive control group,berberine(BBR)administration group were established.Hematoxylin-eosin(HE)staining to observe joint pathological changes;Tomato Osolid green staining observed the pathological changes of articular cartilage.Immunohistochemical analysis detected p-STAT3,Beclin-1,Bcl-2;The CCK-8 method detected the proliferation inhibition effect of BBR on RA-FLSs,and the Annexin VFITC/PI apoptosis kit detected the effect of BBR on the apoptosis of RA-FLSs.Western blot detects Bcl-2,Bax,p62,Beclin-1,LC3 protein levels;m Cherry-EGFP-LC3 B detects autophagy flow;Laser confocal microscopy to observe JC-1,ROS and immunofluorescence;Results: 1.Selected clinical data of RA and OA patients 6 patients with RA and 6 patients with OA were non-selectively included in the study.The clinical data of the two groups were analyzed,and the ESR and CRP levels of disease activity related indicators in the RA group were significantly higher than those in the OA group.2.The expression levels of Beclin-1 and p-STAT3 in synovial tissue of RA patients were significantly higher than those in OA patients.In order to clarify the differences in the expression levels of autophagy-related protein and p-STAT3 in synovial tissue of RA and OA,the expression of Beclin1 and p-STAT3 in synovial tissue of RA and OA patients was detected by immunohistochemistry.Compared with the synovial tissue of the OA group,the expression level of Beclin1 protein in the synovial tissue of the RA group was significantly regulated,and the expression level of p-STAT3 was also significantly upregulated.The positive staining immunohistochemical scores of Beclin1 in the synovial tissue of the RA group were significantly higher than those in the OA group (P<0.001).3.The apoptosis level of fibroblasts in the synovial tissue of the OA group was significantly higher than that in the RA group.In order to clarify the difference in apoptosis levels of fibroblasts in synovial tissues of RA and OA patients,the expression of fibroblast Bcl-2 in synovial tissues of RA and OA patients was detected by immunohistochemistry.Compared with synovial tissue in the OA group,the apoptosis level of fibroblasts in synovial tissue in patients in the RA group was significantly down-regulated(P<0.001).The results of TUNEL staining were further verified,and the scores of fibroblast apoptosis in the synovial tissue of the joint synovial tissue of patients in the OA group were significantly higher than those in the RA group according to the percentage of positive stained cells.4.Correlation between autophagy apoptosis-related protein and p-STAT3 expression levels in joint synovial tissue in RA patients with disease activity.Pearson bivariate correlation analysis was used to analyze the correlation between the immunohistochemical scores of Beclin-1,Bcl-2 protein and p-STAT3 in the synovial tissue of RA patients and the level of disease activity indicators reflected in the serum of the same patient.The immunohistochemical scores of Beclin-1 and pSTAT3 in the synovial tissue of the lesions of RA patients were positively correlated with the ESR,RF antibody and CRP levels of the same patient(P < 0.05).The immunohistochemical score of Apoptosis negatively correlated protein Bcl-2 was strongly positively correlated with ESR,RF and CRP levels in the same patient(all P < 0.05).5.BBR can significantly reduce the expression of Beclin-1 and p-STAT3 in joint synovial tissue in AA rats The expression of fibroblast-like synovial cells Bcl-2,Beclin-1 and p-STAT3 in joint synovial tissues was detected by HE,Saffred O-solid green staining and immunohistochemical staining,BBR could significantly inhibit joint injury and cartilage erosion in AA rats,and significantly reduce the expression of Bcl-2,Beclin-1 and p-STAT3.6.BBR can significantly inhibit autophagy of RA-FLSs and promote apoptosis The results of CCK-8 detection of RA-FLSs showed that BBR could significantly inhibit the proliferation of RA-FLSs,and the flow cytometry results showed that BBR could promote the apoptosis ratio of RA-FLSs,which further verified the results of Western Blot,and BBR could inhibit the autophagy of RA-FLSs and block the autophagy flow.7.BBR regulates the autophagy apoptosis of RA-FLSs and is related to the levels of ROS and p-STAT3 The immunofluorescence results showed that BBR had a significant effect on the levels of intracellular ROS and p-STAT3 in RA-FLSs,and further by introducing the ROS mimic H2O2 and the inhibitor NAC,it was found that BBR regulated the intracellular p-STAT3 levels of RA-FLSs by the level of ROS.Overexpression of STAT3 can significantly inhibit the regulation of autophagy apoptosis level of RA-FLSs by BBR.Conclusions: 1.RA-FLSs upregulated the expression of p-STAT3 and Beclin1 in RA patients,and the levels of these proteins were positively correlated with the levels of serum markers of RA disease activity;The apoptosis of RA-FLSs was significantly reduced,and the negative correlation protein Bcl-2 of RA-FLSs was positively correlated with the level of clinical serum disease activity index.The imbalance of autophagy/apoptosis in synovial tissue in patients with RA promotes the progression of the disease;p-STAT3 mediates autophagy/apoptosis imbalance in lesion synovial tissue.2.BBR can significantly inhibit the expression of p-STAT3,Bcl-2 and Beclin-1 in the synovial tissue of AA rats,and alleviate articular cartilage damage in AA rats.3.In vitro studies have shown that BBR can significantly inhibit the proliferation of RA-FLSs,inhibit RA-FLSs autophagy and promote apoptosis,and this process is related to the regulation of ROS and p-STAT3 by BBR.