Effect Of Danggui Niantong Decoction On Mitochondrial Apoptosis In Synovial Tissue Of Wind-damp-heat Arthralgia RA Model Rats

Background and ObjectiveRheumatoid arthritis(RA)is a chronic autoimmune disease characterized by excessive proliferation of synovium and infiltration of inflammatory cells,leading to progressive destruction of bone and cartilage.The imbalance of proliferation and apoptosis of RA fibroblast like synoviocytes(FLS)is the main reason for the excessive proliferation of RA synoviocytes.Therefore,this study intends to confirm that Danggui Niantong Decoction may promote the mitochondrial apoptosisby regulating Akt-Bax/Bcl-2 pathway in vitro and in vivo,so as to play an important role in the prevention and treatment of wind-damp-heat arthralgia of RA.MethodsThe animal model of wind-damp-heat arthralgia RA rats(FSR rats)was established by artificial climate chamber on the basis of injecting M.tuberculosis.The general state,body mass,arthritis index and toe swelling of rats in each group were detected regularly.HE staining results of ankle joint and synovial tissue,micro CT scanning,and expression levels of IL-1β and TNF-α in synovium and serum were used to judge the construction of the model.The general state,body weight growth,toe volume,AI and spleen index of FSR rats were observed after administration.The pathological changes of synovium and spleen were observed by HE staining.4D-LFQ quantitative proteomics was used to detect the differentially expressed proteins,in synovial tissue of FSR rats after Danggui Niantong Decoction intervention,and the differentially expressed proteins were analyzed by bioinformatics.The mRNA and protein expressions of Akt Bax/Bcl-2 pathway related genes were detected by immunohistochemistry,RT qPCR and WB.Primary cultured RA-FLS were identified by immunofluorescence:After intervention with serum containing Danggui Niantong Decoction,apoptosis level and mitochondrial membrane potential level were detected by Hoechst 33342/PI double staining and JC-1 probe.The mRNA and protein levels of Akt-Bax/Bcl-2 pathway related genes were detected by RT-qPCR and WB.Results1.The establishmen of wind-damp-heat arthralgia RA model rats.Compared with the normal group and AA model group,FSR group had worse general condition,slower body weight growth,severe foot and ankle joint deformity,structural damage and bone damage,obvious proliferation and inflammatory infiltration of synovial tissue,and significantly increased expression levels of IL-1β and TNF-α in synovial tissue and serum.2.Effect of Danggui Niantong Decoction on wind-damp-heat arthralgia RA model rats.(1)Compared with FSR group,Danggui Niantong Decoction and MTX group can improve the general state,body weight,toe volume,arthritis index,synovial hyperplasia and immune level of rats.(2)814 differentially expressed proteins were identified and analyzed by bioinformatics.There are 13 differentially expressed proteins related to apoptosis.And the interaction between these proteins and Bax/Bcl-2,key regulator of mitochondrial apoptosis was analyzed by STRING database.(3)Compared with FSR group,Akt phosphorylation decreased,Bax/Bcl-2 ratio increased,cytochrome c release and Caspase-9 activation increased in Danggui Niantong Decoction groups and MTX groups.3.The effect of Danggui Niantong Decoction on mitochondrial apoptosis of RA fibroblast like synoviocytes.Compared with the blank serum group,the apoptotic level of ra-fls in Danggui Niantong decoction containing serum group increased,and the mitochondrial membrane potential decreased,p-Akt/Akt ratio decreased,Bax/Bcl-2 ratio increased,cytochrome c release and Caspase-9 activation increased.ConclusionsDanggui Niantong Decoction can promote the mitochondrial apoptosis level of RA-FLS by affecting the Akt-Bax/Bcl-2 signaling pathway,and then play a preventive and therapeutic role on wind-damp-heat arthralgia rats.