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 80 μmol/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 (30 μmol/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 (30 μmol/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.
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、80 μmol/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RA-FLSs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(30 μmol/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。
Keywords: apoptosis; autophagy; berberine; fibroblast-like synoviocytes; mTOR; reactive oxygen species; rheumatoid arthritis.