Effect And Mechanism Of Danggui Niantong Decotion On Apoptosis Of Synovial Fibroblasts In Rheumatoid Arthritis

ObjectiveRheumatoid arthritis(RA)is an autoimmune disease characterized by synovial hyperplasia and progressive destruction of articular cartilage.RA has the characteristics of repeated attack,lingering disease and high disability rate.Long term use of western medicine treatment can cause toxic side effects and poor efficacy,but Traditional Chinese Medicine has the characteristics of better curative effect and less side effects.Therefore,the main purpose of this study was to investigate the effect of serum containing Danggui Niantong Decoction on the proliferation and apoptosis of synovial fibroblasts,further explore its mechanism,and provide theoretical basis for the clinical application of Danggui Niantong Decoction.Methods.1.The therapeutic effect of Danggui Niantong Decoction on adjuvant arthritis(AA)rat model.The animal model of AA rats was established by injecting M.tuberculosis(Mtb)adjuvant.After intervention by groups which contained blank control group,the model group,the DGNTD group and the Leigongteng group as the positive control,the changes of body weight,swelling degree of toes,pathological changes of synovium and inflammatory cytokines in each group were detected.2.The effect of Danggui Niantong Decoction on Fas gene and related protein in synovium of AA rats.The expression of Fas mRNA and Fas,FADD,Caspase-8,Caspase-3,Bcl-2,Bax related protein in synovium was measured.3.Effect of Danggui Niantong Decoction on survival,apoptosis and migration of RAFLS.RA FLS were identified by immunofluorescence.MTT colorimetry was used to detect the cell survival rate.Hoechst 33342 and flow cytometry were used to detect the apoptotic characteristics and apoptotic rate of RA FLS.Scratch and invasion test were used to detect migration.4.Effect of Danggui Niantong Decoction on Fas signal transduction pathway of RA FLS.The expression of apoptosis related molecules Fas mRNA,FADD mRNA,Caspase-8 mRNA,Caspase-3 mRNA,Bcl-2 mRNA,Bax mRNA and their protein were detected before and after medication administration.The apoptosis of RA FLS was detected after inhibition of Fas pathway.Results1.Vivo Experiments:Danggui Niantong Decoction could significantly increase the weight of AA rats,reduce the swelling degree of their toes,improve the pathological changes of synovium,reduce inflammatory cytokines and increase the expression of Fas mRNA,Fas protein,FADD protein,Caspase-8 protein,Caspase-3 protein,Bax protein and reduce the protein expression of Bcl-2 in synovium of AA rats.2.Vitro Experiments:Immunofluorescence staining showed that the cultured cells was RA FLS.Compared with the blank group,increasing dose and intervention time of DGNTD could inhibit the proliferation of RA FLS significantly(P<0.05).The inhibitory effect of middle and high dose on 48h was obvious,with the inhibition rates of 48.27%and 53.49%.And middle and high dose of DGNTD on 48h could promote apoptosis in RA FLS,with the apoptosis rates of(10.31±3.64)%and(16.37±4.75)%.Meanwhile,DGNTD could effectively inhibit the migration of RA FLS.Compared with the blank group,middle and high dose of medicinal serum’s expression levels of Fas mRNA,Caspas-8 mRNA、Caspase-3 mRNA、FADD mRNA and Bax mRNA were increased significantly,while the expression levels of Bcl-2 mRNA was decreased(P<0.05).And the protein expressions of Fas,FADD,Caspase-8,Caspase-3 and Bax were also increased while the Bcl-2 protein was also decreased in a dose-dependent manner(P<0.05).Fas inhibitor can effectively inhibit the expression of Fas apoptosis-related proteins and the apoptosis of RA FLS cells mediated by Fas.ConclusionsDanggui Niantong Decoction can improve RA symptoms by inhibiting the proliferation,inducing apoptosis and reducing migration of RA FLS whose mechanism is related to the regulation of Fas pathway and mitochondrial apoptosis pathway.