Effect Of RNF43 On The Synovial Proliferation And Inflammation In Rheumatoid Arthritis

Background:Rheumatoid Arthritis(RA)is a chronic,inflammatory disease,which can manifest as irreversible cartilage erosion and bone destruction,synovitis and pannus formation,fibroblast like synoviocytes secreted by synovium The over proliferation of synoviocytes(FLS)can secrete a variety of cytokines,leading to infiltration of various inflammatory cells,so as to cause synovial thickening and chronic inflammation,which is considered to play an important role in the pathogenesis of RA.The activation of ra-fls is related to a variety of signaling pathways.Wnt signaling pathway is the hot spot of current research.Researchers believe that there is an over activation of Wnt signaling pathway in the process of RA occurrence and development.Ring finger Protein43,RNF434 is a relatively new Wnt signal pathway inhibitor,which is a E3 ubiquitination ligase.It can bind with Wnt receptor protein and induce its ubiquitination and degradation,so as to play a role in inhibiting Wnt signal pathway.The current research on RNF43 focuses on tumor,and the role of rnf43 in RA has not been opened Therefore,we took the lead in detecting the expression level of rnf43 in RA patients,the regulatory function of rnf43 in ra-fls,and further exploring the effect of rnf43 on collagen induced arthritis mice in vivo,and speculated the possible regulatory mode.ObjectivePart 1: To observe the expression of RNF4343 in synovium of patients with RA?OA and Trauma.Part 2:To investigate the effect of RNF43 on proliferation,apoptosis and inflammatory response of fibroblast like synoviocytes(FLS)in vitro,and to explore the effect of RNF43 on joint swelling,synovitis and bone destruction of CIA model in vivo.MethodsPart 1: Firstly,the synovium of patients with RA,OA and trauma was collected in cooperation with the Department of osteoarthritis,ra-fls was extracted,and the authenticity of RA-FLS was identified;secondly,the synovium of the three patients was observed on pathological sections,and statistical analysis was done;finally,the expression level of RNF43 in the synovium of the three patients was observed.Part 2: Firstly,construct RNF43 lentivirus vector to infect RA-FLS;secondly,investigating the proliferation,cycle,migration and expression level of inflammatory factors of RA-FLS after infection;finally,construct CIA model,carry out in vivo experiments of lentivirus,and study the level of inflammatory factors in joints,synovium and body of CIA model.ResultsPart 1:FLS was successfully obtained by enzyme digestion and its source was identified.RNF43 was expressed in RA,OA and Trauma patients.However,there was no significant difference in RNF43 expression between RA and OA.We speculate that the reason of this phenomenon may be the over activation of Wnt signaling pathway in RA and OA patients,which may lead to the increase of RNF43 feedback expression.Part 2:(1)when the number of infection(MOI)I = 30,the infection efficiency can meet the experimental requirements.(2)Compared with lv-ctr group,LV-RNF43 can reduce the activity of RA-FLS cells.Compared with Sh-ctr group,Sh-RNF43 can enhance the activity of RA-FLS cells.After TNF-? stimulation,the activity of both groups decreased,but the trend was the same.(3)Compared with LV-ctr,LV-RNF43 can significantly promote the apoptosis of RA-FLS.Compared with Sh-ctr,Sh-RNF43 can inhibit the apoptosis of RA-FLS cells.(4)Compared with LV-ctr,over expression of LV-RNF43 can reduce the levels of IL-1 ?,IL-6,IL-8 m RNA and expression.Compared with Sh-ctr,Sh-RNF43 can promote the levels of IL-1 ?,IL-6,IL-8 m RNA and expression.(5)The migration rate of rafls in LV-ctr group was lower than that in LV-RNF43 group,while the migration effect of RA-FLS in Sh-RNF43 group had no significant change compared with that in Sh CTR group.(6)In the construction of CIA model,the arthritis scores of Sh CTR and LV CTR groups were similar;The score of CIA arthritis in LV-RNF43 group reached the peak around the 40 th day,and gradually decreased with time,which was significantly lower than LV CTR score;the score of CIA arthritis in Sh-RNF43 group increased rapidly around the 32 nd day,and reached the peak on the 49 th day,and the score was higher than that of Sh CTR.(7)the dosage of 10 UL lentivirus in one knee could reach the dose of successful infection of CIA.(8)Western blot Sh owed that the expression of LV-RNF43 was higher than that of LV CTR group,and that of Sh CTR group was higher than that of Sh-rnf43 group.(9)LV-RNF43 and LV-ctr were compared.It was found that the degree of pathological changes in the knee joint of mice in LV-RNF43 group was lighter than that in the control group.LV-RNF43 arthritis score was 3.2 ± 0.45,LV-ctr score was 4.6 ± 0.54,indicating that promoting the expression of RNF43 can reduce the joint inflammation.(10)In vivo experiment of CIA model rats: compared with LV-ctr group,the content of TNF-?,IL-1 ?,IL-6 in LV-RNF43 group decreased,compared with Sh-ctr group,the content of TNF-?,IL-1 ?,IL-6 in Sh-RNF43 RNF43 group increased,which indicated that promoting the expression of RNF43 can inhibit the expression of inflammatory factors in CIA model rats,and inhibiting the expression of RNF43 can promote the expression of inflammatory factors in CIA model rats.Conclusion:Through human tissue samples,in vitro experiments and animal model construction,the research group completed the determination of tissue samples,discussed the effect of RNF43 on the biological activity of RA-FLS cells,and verified it again in the CIA model.We found that RNF43 can inhibit the proliferation of RA-FLS and the expression of related inflammatory factors in vivo and in vitro,because RNF43 is an inhibitor of Wnt signaling pathway,and Wnt signaling pathway is related to the occurrence and development of RA.Therefore,we conclude that RNF43 may be a potential target for RA diagnosis and treatment.