Genistein And Daidzein Protect Collagen Induced Arthritis By Increasing IgG Glycosylation And Reducing Osteoclast Activation

Backgroud:Rheumatoid arthritis(RA)is an autoimmune disease characterized by joint swelling and pain.Pathology is characterized by arthritic cell infiltration and bone and joint destruction.It is a widely affected autoimmune disease,affecting about 0.5-1%of the world’s population.Estrogen can improve the glycosylation of autoantibodies and inhibit the activity of osteoclasts,thus protecting arthritis.Phytoestrogens are similar in structure with estrogen and can bind to estrogen receptor.Studies have shown that they have protective effects on arthritis,but the mechanism is not clear.The phytoestrogens effects on the glycosylation of IgG and the effects on osteoclasts have not been reported previously.In this study,collagen-induced arthritis(CIA)mice were administered phytoestrogens,polyclonal IgG from peripheral blood and monoclonal IgG antibody screened by hybridoma cells to examine the effect of glycosylation level;bone marrow-derived macrophages(BMMs)from mice were extracted and induced by ligand of nuclear factor NF-kappa B receptor(RANKL)to simulate the differentiation of osteoclasts in vivo environment,to investigate the phytoestrogens effects on IgG glycosylation and osteoclast activity and their protective effects on bone and joints.Methods:We gavaged female DBA1/J mice with 20 mg/kg phytoestrogens(Genistein and Daidzein)in CIA modeling,and collected specimens on days 42 and 81,respectively.The effects of phytoestrogens on joint inflammation and bone destruction in mice were investigated by clinical scores,HE staining and micro-CT.The effects of phytoestrogens on peripheral blood inflammatory cells such as neutrophils,B cells,macrophages and CD4+T cells(Th1 and Th17)were examined by flow cytometry.Mouse peritoneal macrophages were taken to phagocytose E.coli labeled with FITC to evaluate their phagocytic function.ELISA was used to screen monoclonal IgG-producing hybridoma cells were and to detect the avidity and specificity of IgG antibodies.IgG subtypes,polyclonal and monoclonal IgG glycosylation types and levels were detected by Lectin-ELISA.Antibody glycosylation(B4galt1,St6gal1)and related genes of osteoclasts(TRAP,CTSK,MMP9,NFATc1 and c-Fos)were analyzed by qRT-PCR.Establish osteoclasts differentiated and matured by RANKL-induced BMMs,and detected the effect of phytoestrogens on osteoclastogenesis by TRAP staining;detected the effect of phytoestrogens on the NF-κB(IκB-α and p65)and AKT(Akt,pAkt)signaling pathways and downstream transcription factors NFATc1 and c-Fos of osteoclasts by Western blot,and studied the effect of phytoestrogens on osteoclast-related activation pathways.Results:Phytoestrogens(Genistein and Daidzein)can reduce the inflammatory cells infiltrated in peripheral blood and joints of CIA mice,such as neutrophils,macrophages,B cells and T cells,inhibit the phagocytic function of macrophages,and reduce the inflammatory response.Phytoestrogens(Genistein and Daidzein)can increase the levels of galactosylation and sialylation of polyclonal and monoclonal antibody IgG.Phytoestrogens inhibited the osteoclast-related genes,NF-κB signaling pathway and downstream transcription factors NFATc1 and c-Fos.Conclusion:In CIA mice,phytoestrogens(Genistein and Daidzein)protect bone and joints by alleviating immune inflammation,increasing the level of antibody IgG galactosylation and sialylation,inhibiting NF-kappa B signaling pathway and NFATc1 and c-Fos transcription factors.