Effects of daphresin on synovial fibroblasts in rheumatoid arthritis based on PERK/ATF4/CHOP signaling pathway / 中国药理学通报

ABSTRACT

Aim To investigate the effects of DAP on human rheumatoid arthritis synovial fibroblasts(RA-FLS)and its relationship with endoplasmic reticulum stress(ERS)PERK/ATF4/CHOP signaling pathway.Methods RA-FLS cells were cultured and identified by immunofluorescence assay for Vimentin and CD68.CCK-8 detected(0,5,10,20,30,40,50,60,70)mg·L-1 DAP on the proliferation activity of RA-FLS cells.According to the proliferation activity,the experiment was divided into blank group(Blank),low dose group(L-DAP),medium dose group(M-DAP),high dose group(H-DAP)and high dose+specific inhibitor 4-PBA group(H-DAP+4-PBA).The levels of TNF-α and IL-6 were detected by ELISA.Cell apoptosis was detected by flow cytometry.The invasion and migration of cells were detected by Transwell and scratches assays,and the expressions of endoplasmic reticulum stress-related proteins GRP78,PERK,P-PERK,ATF4,CHOP,Caspase-12,C-Caspase-12 and Bcl-2 were assessed by Western blot.Results CCK-8 results showed that compared with 0 mg·L-1 DAP group,the proliferation activity of each group in 20-70 mg·L-1 DAP group was significantly different(P<0.05),and the proliferation rate corresponding to 0,20,40 and 60 mg·L-1 DAP group was significantly different(P<0.05).This concentration was used as the basis for blank,low-dose,medium-dose,high-dose groups and high-dose+4-PBA experimental grouping.DAP could inhibit the release of inflammatory cytokines IL-6 and TNF-α from RA-FLS cells in concentration,reduce the invasion ability of cells,promote apoptosis,upregulate the expression of PERK,P-PERK,ATF4,GRP78,CHOP,Caspase-12, C-caspase-12 proteins and decrease the expression level of anti-apoptotic protein Bcl-2.Another set of experiments demonstrated that high dose DAP+4-PBA could up-regulate the expression of IL-6 and TNF-α,as well as the invasion of cells,and inhibit the expression of apoptosis and ERs-related proteins.Conclusions Daphresin regulates the secretion of inflammatory factors by activating the PERK/ATF4/CHOP signaling pathway,inhibits the proliferation and invasion of RA-FLS cells,and induces apoptosis,which is expected to be a potential therapeutic pathway for RA.