摘要
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.
作者
宗世烨
周静
蔡伟伟
余芸
王颖
宋宜宁
程静文
李宇会
高艺
吴百海
咸郝
魏芳
ZONG Shiye;ZHOU Jing;CAI Weiwei;YU Yun;WANG Ying;SONG Yining;CHENG Jingwen;LI Yuhui;GAO Yi;WU Baihai;XIAN Hao;WEI Fang(School of Pharmacy,Bengbu Medical College,Bengbu 233030,China;Department of Pharmacy,Hangzhou Hospital of Traditional Chinese Medicine,Hangzhou 310007,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2023年第4期552-559,共8页
Journal of Southern Medical University
基金
国家自然科学基金(81703529)
安徽省教育厅自然科学重大项目(KJ2021ZD0086)
蚌埠医学院512人才项目(by51202203)
国家大学生创新创业训练计划项目(201910367036,202010367037)
蚌埠医学院创新药物创新团队(BYKC201904).
关键词
类风湿关节炎
小檗碱
成纤维样滑膜细胞
自噬
凋亡
ROS
mTOR
rheumatoid arthritis
berberine
fibroblast-like synoviocytes
autophagy
apoptosis
reactive oxygen species
mTOR