Study On The Mechanism Of Er-miao-san Aqueous Extract In Regulating FLS NF-κB Signaling Pathway In CIA Rats

Objective:In this study,a rat model of collagen induced arthritis(Collagen induced arthritis,CIA)was established and fibroblast-like synoviocytes(FLS)were isolated,purified and cultured from the joint synovium of CIA rats.In this study we are aimed to study the effect of different concentrations aqueous extract of EMS on the proliferation,migration and expression of inflammatory factors in FLS of CIA rats and based on nuclear factor-κB(NF-κB)signaling pathway to explore its mechanism of action,which can provide certain experimental for the basis clinical treatment of rheumatoid arthritis.Methods:Twenty-four female SPF Wistar rats were randomly divided into Normal group and CIA group.Normal group was routinely raised,while CIA group was immunized with bovine type II collagen twice to establish a CIA rat model.The weight,foot volume and joint scores of the rats were recorded weekly.Rats’ joints were taken,fixed and decalcified,and Masson staining was used to observe the pathological changes of the rat joint tissues 42 weeks after the initial immunization and anesthesia with 10%chloral hydratethe.the CIA rat joint synovial tissue was stripped under aseptic conditions,then purified and cultivated CIA-FLS,added different concentrations of EMS aqueous extracts(200,400,800,1600,3200μg/m L)for intervention.MTT method and transwell chamber were used to detect the proliferation and migration ability of CIA-FLS,real-time fluorescent quantitative PCR(q RT-PCR))and Western blot to detect the m RNA and protein expression levels of NF-κB signaling pathway related molecules IL-6,TNF-α,NF-κBp65 in CIA-FLS.Results:1.Compared with Normal group,14 days after the initial immunization,the weight of the rats in the CIA group decreased significantly(P < 0.05).During the modeling process,the weight of the rats in the CIA group did not increase significantly(P > 0.05).Compared with Normal group,7 days after the initial immunization,the arthritis score and foot volume of the CIA group increased significantly(P < 0.01).7 days after the initial immunization,the arthritis score and foot volume of the CIA group increased significantly(P <0.05),the arthritis score and foot volume of rats in the Normal group did not change significantly(P > 0.05).The results of Masson staining showed that the ankle joint capsule cavity of the rats in Normal group was smooth,the tissue morphology was complete,covered with a thin layer of hyaline cartilage,and synovial cells grew well.The ankle joints of the rats in the CIA group showed destruction of joint cavity,incomplete structure,and partial damage of articular cartilage,proliferation of synovial cells,mesenchymal congestion and edema,and infiltration of inflammatory cells to varying degrees.This shows that this study successfully constructed a CIA rat model.2.CIA-FLS was isolated,purified and cultured.Long fusiform or spindle-shaped CIA-FLS were adhered to the wall,among which a few had multiple neuron-like protrusions.The nucleus was oval in the center of the cell.With CIA-FLS Cells continue to divide and proliferate,the CIA-FLS cells gradually increase,contacting each other and intertwining into a network under an inverted microscope,which indicated that this study has successfully separated and purified CIA-FLS.3.Study the effect of different concentrations of EMS aqueous extracts on the proliferation and migration ability of CIA-FLS.The results showed that after 48 h of EMS aqueous extracts with different concentrations of CIA-FLS,Compared with Control group,the EMS aqueous extracts showed a dose-dependent Inhibition on the proliferation of CIA-FLS(P < 0.05).1600 μg/m L EMS aqueous extract significantly inhibited the proliferation and migration of CIA-FLS(P < 0.01),indicating that there is abnormal proliferation and migration in CIA-FLS.EMS aqueous extract can inhibit the proliferation and migration ability of CIA-FLS to varying degrees.4.The effect of different concentrations of EMS aqueous extracts on the expression of IL-6、TNF-α,NF-κBp65 m RNA and protein levels in CIA-FLS showed that 1600 μg/m L EMS aqueous extracts significantly inhibited IL-6、TNF-α,NF-κBp65 m RNA and protein level expression in CIA-FLS(P < 0.01).EMS may inhibit the expression of IL-6、TNF-α,NF-κBp65 m RNA and protein in CIA-FLS,and negatively regulate NF-κB signaling pathway to inhibite the hyperproliferation and inflammation of FLS,which indicating that EMS aqueous extract may inhibit the activation of NF-κB signaling pathway and production of inflammatory factors to reduce the proliferation and inflammation of CIA-FLS.Conclusion:This study has separated and purified CIA-FLS successfully.The EMS aqueous extract inhibits the proliferation,migration and inflammatory factor expression of CIA-FLS,and the optimal concentration is 1600 μg/m L.EMS aqueous extract may inhibit the activation of NF-κB signaling pathway and the production of inflammatory factors,thereby reducing the proliferation and inflammation of CIA-FLS.This study provides a certain experimental basis for the later clinical application of EMS in the treatment of rheumatoid arthritis.