Discussion on therapeutic mechanism of total flavonoids of Pterocarya Hupehensis Skan on rheumatoid arthritis based on Fas/FasL mediated mitochondrial apoptosis
Objective:To explore the possible mechanism of total flavonoids of Pterocarya Hupehensis Skan(PHSTF)in the treatment of rheumatoid arthritis(RA)through Fas/FasL mediated mitochondrial apoptosis.Methods:Collagen induced arthritis(CIA)-fibroblast synovial cells(FLS)cells were cultured in vitro,and the effect of PHSTF on cell survival rate was detected by CCK-8;The effect of PHSTF on cell apoptosis was detected by flow cytometry;Fas,FasL,Bax,Bcl-2,t-Bid,C-Caspase8,C-Caspase9,C-Caspase3 protein expression was detected by Western Blot.The effect of PHSTF on the expression of related proteins was further tested after adding the Fas inhibitor KR-33493.In vivo experiment using type Ⅱ collagen induction method to construct CIA rat replicated RA model.Establish control group,model group,PHSTF group,and TG group with 8 rats in each group.The AI index and toe swelling degree of each group were evaluated;and the TNF-α,IL-1β,IL-6 in serum was detected by ELISA.The pathological changes of the knee joint were observed through HE staining.The expression of mitochondrial apoptosis related proteins mediated by Fas/FasL in synovial tissue was detected through Western Blot.Results:In vitro experiments had shown that PHSTF could significantly induce apoptosis in CIA-FLS cells(P＜0.0l).Compared with the control group,the expression of Fas,FasL,Bax,t-Bid,C-Caspase8,C-Caspase9,and C-Caspase3 proteins was increased in all concentration groups of PHSTF,while the expression of anti apoptotic protein Bcl-2 was significantly reduced(P＜0.05,P＜0.01).After adding the Fas inhibitor KR-33493 to interfere with the Fas signaling pathway,it reversed the expression of Fas signaling pathway related proteins.In vivo experiments showed that compared with the model group,the AI score and toe swelling of the PHSTF and TG groups were significantly reduced(P＜0.05),the pathological changes of the knee joint were improved,and the the level of TNF-α,IL-1β,IL-6 in serum was significantly reduced(P＜0.01).The expression of Fas,FasL,Bax,t-Bid,C-Caspase8,C-Caspase9,and C-Caspase3 proteins was increased,while the expression of Bcl-2 protein was significantly reduced(P＜0.01).Conclusion:PHSTF can activate Fas/FasL mediated mitochondrial apoptosis,inhibit CIA-FLS cell proliferation,promote apoptosis,improve CIA rat arthritis phenotype,and play a therapeutic role in RA.
Total flavonoids of Pterocarya Hupehensis Skan(PHSTF)Rheumatoid arthritis(RA)Fibroblast synovial cells(FLS)Collagen induced arthritis(CIA)Fas/FasL
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