Study On The Mechanism Of Huayu Tongbi Recipe In Inhibiting The Inflammatory Proliferation Of Synovial Cells In The Co-culture Of Lymphocytes

Objective To investigate the effect of Huayu Tongbi prescription on the proliferation and differentiation of T cells and synovial cell inflammatory proliferation,to clarify the mechanism of the effect of Huayu Tongbi prescription,so as to provide experimental basis for the application of Qi,blood and poison theory in the prevention and treatment of rheumatoid arthritis by traditional Chinese medicine.Methods Rat synovial cells(RSC-364)and rat splenic lymphocytes were cultured in Transwell mode,with IL-1β decoction as the inflammatory stimulator,Huayu Tongbi decoction freeze-drying powder as the intervention drug,and IL-1β decoction receptor antagonist as the positive control drug.Morphological changes of RSC-364 were observed by HE staining.The apoptosis rate of RSC-364 and the proliferation and differentiation of T lymphocytes were detected by flow cytometry.Western Blot was used to detect the expression of RSC-364 NF-κB and JAK/STAT signal pathway key cytokines,as well as the expression of NF-κB pathway key cytokines in T lymphocytes and Th cells specific transcription factors T-bet,GATA-3 and ROR-γt.Results Compared with the normal group,RSC-364 cells in the model group showed significantly irregular morphological changes and cytoplasm damage at different degrees under HE staining.The apoptosis rate of RSC-364 in the model group decreased after 24 hours of intervention.In the model group,key cytokines of the RSC-364 NF-κB pathway,TRAF2,MyD88,p-IKKα/β,NF-κBp50 and JAK/STAT pathways,were significantly increased.The ratio of CD4+IFN-γ+Th1 cells and CD4+IL-17+Th17 cells increased to different degrees,while the ratio of CD4+IL-4+Th2 cells decreased significantly.T-bet and ROR-yt,the key cytokines in Th cells specific transcription factors were significantly increased and GATA-3 expression was decreased at 12h.The expression levels of key cytokines in the NF-κB pathway of lymphocytes TRAF2,p-IKKα/β and NF-κBp50 were all increased,indicating successful induction in the rat inflammatory cell model.Compared with the model group,the apoptosis rate of RSC-364 increased in each drug intervention group.Key cytokines of the RSC-364 NF-κB pathway,TRAF2,MyD88,p-IKKa/p,NF-κBp50 and JAK/STAT pathways,JAK1,p-STAT3 were all decreased.The ratio of lymphocyte CD4+IFN-γ+Th1 cells in each drug intervention group was significantly decreased.The ratio of CD4+IL-17+Th17 cells in each drug group decreased significantly after 24h of intervention.The proportion of CD4+IL-4+Th2 cells after 12h intervention in each drug intervention group was significantly increased.The ratio of CD4+CD25+Treg cells increased most significantly in the 250g/ml group of Huayu Tongbi decoction.T-bet and ROR-yt,the key cytokines in Th cell proliferation and differentiation,were significantly decreased,while GATA-3 expression was significantly increased.The expression levels of key cytokines in the NF-κB pathway of lymphocytes were decreased,including TRAF2,p-IKKα/β,and NF-KBp50,with the most significant reduction at 24h after intervention.Conclusion IL-1β was used to induce and successfully establish the rat synovial cell inflammatory proliferation model by inducing the inflammatory proliferation of synovial cells,inducing the proliferation and differentiation of T lymphocytes,and activating the NF-κB signaling pathway of synovial cells and T lymphocytes and the JAK/STAT signaling pathway of synovial cells.The treatment of rheumatoid arthritis may be achieved by restoring the immune balance of T lymphocytes,regulating the NF-κB and JAK/STAT pathways of synovial cells,and thereby inhibiting the inflammatory proliferation and immune damage of synovial cells.