小檗碱抑制类风湿关节炎患者的成纤维样滑膜细胞的自噬并促进其凋亡:基于下调ROS/mTOR信号通路

Berberine inhibits autophagy and promotes apoptosis of fibroblast-like synovial cells from rheumatoid arthritis patients through the ROS/mTOR signaling pathway

目的探究小檗碱(BBR)对类风湿关节炎(RA)成纤维样滑膜细胞(FLSs)凋亡/自噬失衡的调控作用及机制。方法CCK-8法检测BBR对RA-FLSs的增殖抑制作用,实验设空白对照组、TNF-α(25 ng/mL)组、TNF-α+BBR(10、20、30、40、50、60、70、80μmol/L)组,Annexin V/PI双染流式法和JC-1免疫荧光染色检测BBR对RA-FLSs凋亡的影响,Western blot检测BBR对RAFLSs自噬和凋亡相关蛋白表达水平的影响。进一步增加自噬诱导剂RAPA和自噬抑制剂氯喹(CQ)作为对照,激光共聚焦检测mCherry-EGFP-LC3B观察自噬流变化;并设置活性氧(ROS)模拟物H2O2和ROS抑制剂NAC观察BBR对ROS、mTOR、p-mTOR水平的影响。结果CCK-8结果显示BBR可呈时间和浓度依赖性抑制RA-FLSs增殖活力,流式和JC-1染色结果显示BBR(30μmol/L)可显著增加RA-FLSs凋亡率(P<0.01),降低线粒体膜电位(P<0.05)。Western blot结果显示BBR处理后Bcl-2/Bax(P<0.05)和LC3B-Ⅱ/Ⅰ(P<0.01)的比值降低,p62蛋白表达升高(P<0.05)。mCherry-EGFP-LC3B自噬流检测结果也显示,BBR可阻断自噬流。免疫荧光结果证实BBR显著降低TNF-α诱导后ROS水平,上调自噬调控蛋白p-mTOR表达水平(P<0.01),且受ROS水平调控,合用RAPA可显著降低BBR对RA-FLSs的促凋亡作用(P<0.01)。结论BBR可能是通过调控ROS-mTOR抑制RA-FLSs自噬,促进其凋亡。

Objective To evaluate the regulatory effect of berberine on autophagy and apoptosis balance of fibroblast-like synoviocytes(FLSs)from patients with in rheumatoid arthritis(RA)and explore the mechanism.Methods The inhibitory effect of 10,20,30,40,50,60,70,and 80μmol/L berberine on RA-FLS proliferation was assessed using CCK-8 method.Annexin V/PI and JC-1 immunofluorescence staining was used to analyze the effect of berberine(30μmol/L)on apoptosis of 25 ng/mL TNF-α-induced RA-FLSs,and Western blotting was performed to detect the changes in the expression levels of autophagy-and apoptosis-related proteins.The cells were further treated with the autophagy inducer RAPA and the autophagy inhibitor chloroquine to observe the changes in autophagic flow by laser confocal detection of mCherry-EGFP-LC3B.RA-FLSs were treated with the reactive oxygen species(ROS)mimic H2O2 or the ROS inhibitor NAC,and the effects of berberine on ROS,mTOR and p-mTOR levels were observed.Results The results of CCK-8 assay showed that berberine significantly inhibited the proliferation of RA-FLSs in a time-and concentration-dependent manner.Flow cytometry and JC-1 staining showed that berberine(30μmol/L)significantly increased apoptosis rate(P<0.01)and reduced the mitochondrial membrane potential of RA-FLSs(P<0.05).Berberine treatment obviously decreased the ratios of Bcl-2/Bax(P<0.05)and LC3B-Ⅱ/Ⅰ(P<0.01)and increased the expression of p62 protein in the cells(P<0.05).Detection of mCherry-EGFP-LC3B autophagy flow revealed obvious autophagy flow block in berberine-treated RA-FLSs.Berberine significantly reduced the level of ROS in TNF-α-induced RA-FLSs and upregulated the expression level of autophagy-related protein p-mTOR(P<0.01);this effect was regulated by ROS level,and the combined use of RAPA significantly reduced the pro-apoptotic effect of berberine in RA-FLSs(P<0.01).Conclusion Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.