Zong S, Zhou J, Cai W, Yu Y, Wang Y, Song Y, Cheng J, Li Y, Gao Y, Wu B, Xian H, Wei F
School of Pharmacy, Bengbu Medical College, Bengbu 233030, China.
Department of Pharmacy, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou 310007, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2023 Apr 20;43(4):552-559. doi: 10.12122/j.issn.1673-4254.2023.04.07.
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.
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 HO or the ROS inhibitor NAC, and the effects of berberine on ROS, mTOR and p-mTOR levels were observed.
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 ( < 0.01) and reduced the mitochondrial membrane potential of RA-FLSs ( < 0.05). Berberine treatment obviously decreased the ratios of Bcl-2/Bax ( < 0.05) and LC3B-II/I ( < 0.01) and increased the expression of p62 protein in the cells ( < 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 ( < 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 ( < 0.01).
Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.
评估黄连素对类风湿关节炎（RA）患者成纤维样滑膜细胞（FLSs）自噬与凋亡平衡的调节作用并探讨其机制。
采用CCK-8法评估10、20、30、40、50、60、70和80 μmol/L黄连素对RA-FLS增殖的抑制作用。采用Annexin V/PI和JC-1免疫荧光染色分析黄连素（30 μmol/L）对25 ng/mL TNF-α诱导的RA-FLS凋亡的影响，并用蛋白质免疫印迹法检测自噬和凋亡相关蛋白表达水平的变化。用自噬诱导剂雷帕霉素（RAPA）和自噬抑制剂氯喹进一步处理细胞，通过激光共聚焦检测mCherry-EGFP-LC3B观察自噬流的变化。用活性氧（ROS）模拟物HO或ROS抑制剂NAC处理RA-FLS，观察黄连素对ROS、mTOR和p-mTOR水平的影响。
CCK-8检测结果显示，黄连素能以时间和浓度依赖性方式显著抑制RA-FLS的增殖。流式细胞术和JC-1染色显示，黄连素（30 μmol/L）显著提高凋亡率（<0.01）并降低RA-FLS的线粒体膜电位（<0.05）。黄连素处理明显降低细胞中Bcl-2/Bax比值（<0.05）和LC3B-II/I比值（<0.01），并增加p62蛋白表达（<0.05）。检测mCherry-EGFP-LC3B自噬流发现，黄连素处理的RA-FLS存在明显的自噬流阻断。黄连素显著降低TNF-α诱导的RA-FLS中ROS水平，并上调自噬相关蛋白p-mTOR的表达水平（<0.01）；这种作用受ROS水平调节，联合使用RAPA可显著降低黄连素对RA-FLS的促凋亡作用（<0.01）。
黄连素可通过调节ROS-mTOR通路抑制RA-FLS的自噬并促进其凋亡。
