The Role Of ASIC1a In Regulating The Proliferation Of Synovial Fibroblasts Through ERK/MAPK Signaling And Its Possible Mechanism

Rheumatoid arthritis（RA）is a long-term autoimmune disease with complex pathogenesis of arthropathy.The main changes of synovium are hyperplasia,persistent synovitis and pannus.Synovial hyperplasia is one of the significant changes in synovial tissue structure and is an important cause of joint destruction in rheumatoid arthritis.RA synovial fibroblasts（RASFs）,known as tumor-like cells,play a vital role in the pathology of RA.Their uncontrolled proliferation and invasion of surrounding tissues directly lead to joints damage.It is generally considered that controlling synovial hyperplasia and looking for the cause of excessive synovial cell proliferation are the focus of treatment of the disease.Another typical feature of RA is that the articular microenvironment is accompanied by extracellular acidification,and the p H is usually below 6.0.Acid-sensitive ion channel1a（ASIC1a）is one of the most widely studied subunits of ASICs.It can be activated by extracellular H⁺and participates in various physiological and pathological conditions due to its unique activity in mediating Ca²⁺influx.We known that ASIC1a plays an important role in RA,but its role in synovial hyperplasia and its regulatory mechanisms are poorly understood.In addition,whether ERK/MAPK signaling,as a classic proliferation pathway,is involved in the proliferation of RASFs and whether it is regulated by ASIC1a remains to be further studied.Objectives:1.This study aims to explore whether the extracellular acidification can activate ASIC1a.2.To explore whether activated ASIC1a can promote the proliferation of RASFs by regulating the activation of ERK/MAPK signaling pathway.3.Explore whether ASIC1a can conduce to RASFs proliferation in vitro and in vivo.4.Understanding the effect of ASIC1a on the proliferation of RASFs may provide insights for ASIC1a as a potential target for the treatment of RA.Understanding the effect of ASIC1a on the proliferation of RA synovial fibroblasts may provide a basis for ASIC1a as a promising target for RA.Method:1.The pathological changes of human synovial tissue and synovial hyperplasia were observed by immunohistochemistry.Collected human synovial tissue（the number of normal patients is n=3,the number of RA patients is n=25）.The pathology of synovium was observed by immunohistochemical staining.And the differential expression of PCNA and Ki67 in normal and RA synovium was to show the changes of synovial hyperplasia.2.The differential expression of ASIC1a in human synovium was observed by immunohistochemistryImmunohistochemistry and immunofluorescence was performed to observe the expression of ASIC1a in normal and RA synovium.3.To observe the expression of ASIC1a in human NSFs and RASFsFirstly,normal human synovial fibroblasts and RASFs were obtained by the method of adherents.Secondly,synovial fibroblasts were identified by the expression of CD55,detected by FCM.Finally,ASIC1a in NSFs and RASFs was detected by WB,PCR and immunofluorescence staining.4.To observe the effect of ASIC1a activated by acidification on the proliferation of RASFsRASFs were stimulated by acidification（p H 6.0）for 0 h,1.5 h,3 h,6 h,12 h and 24 h.The expression of PCNA and Ki67 in each group was detected by immunofluorescence and FCM,and the proliferation rate was observed by CCK8.5.To explore the role of silencing or overexpression of ASIC1a in the proliferation of RASFsSilencing or overexpressed ASIC1a cell lines were constructed and ASIC1a specific inhibitor PCTX-1 was used.The experimental groups were divided into control,acidification（p H 6.0）,ASIC1a-sh RNA,Pc TX-1 and ASIC1a overexpression group.The cell proliferation of each group was detected by Western blot,colony formation and others.6.To investigate the effect of acidification activated ASIC1a on the proliferation of synovial fibroblasts through the ERK/MAPK signaling pathwayThe experimental groups were divided into control,acidification,ASIC1a-sh RNA,Pc TX-1 and ASIC1a overexpression group.The action of activated ASIC1a on c Raf and ERK1/2 was detected by western blot.Then,the experiment group was divided into control,acidification,and U0126（the inhibitor of ERK1/2）group.The growth ability of RASFs in each group was observed by westrn blot,colony and CCK8.7.To investigate the effect of ASIC1a on synovial hyperplasia in vivoAdjuvant arthritis（AA）rat model was used to investigate the effect of ASIC1a on synovial hyperplasia.The animals were divided into normal,AA model,ASIC1a specific inhibitor PCTX-1（0.5μg/kg,1μg/kg,2μg/kg,starting administration after the first immunization,each time for 3 days,a total of 8 times）,and positive triamcinolone acetonide group（1 mg/kg,starting administration after the first immunization,each time for 3 days,for 8 times）.HE staining was used for observing inflammation of each animal group.and the protein of PCNA and Ki67 was observed by immunohistochemistry.Results:The expression of ASIC1a was significantly increased in human synovium and primary RASFs and the synovium of AA rats comepared with the normal group.In addition,the cell extracellular acidification can significantly increase the expression of PCNA and Ki67.And this phenomenon can be abrogated by Pc TX-1 or ASIC1a-sh RNA,indicating that extracellular acidification promotes RASFs proliferation by activating ASIC1a.The activation of p-c Raf and p-ERK can also be blocked by Pc TX-1 or ASIC1a-sh RNA,and the use of ERK inhibitors（U0126）can further inhibit the proliferation of RASFs,suggesting that suggesting that the ERK/MAPK signaling involved in the proliferation of RASFs promoted by ASIC1a.Conclusion:1.ASIC1a promotes the proliferation of rheumatoid arthritis synovial fibroblasts.2.ASIC1a promotes the proliferation of rheumatoid arthritis synovial fibroblasts by regulating the ERK/MAPK signaling pathway.