Journal of Southern Medical University ›› 2023, Vol. 43 ›› Issue (4): 552-559.doi: 10.12122/j.issn.1673-4254.2023.04.07

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

ZONG Shiye, ZHOU Jing, CAI Weiwei, YU Yun, WANG Ying, SONG Yining, CHENG Jingwen, LI Yuhui, GAO Yi, WU Baihai, XIAN Hao, WEI Fang   

  1. School of Pharmacy, Bengbu Medical College, Bengbu 233030, China; Department of Pharmacy, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou 310007, China
  • Online:2023-04-20 Published:2023-05-16

Abstract: 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.

Key words: rheumatoid arthritis; berberine; fibroblast-like synoviocytes; autophagy; apoptosis; reactive oxygen species; mTOR