Acid Sensor ASIC1a Regulating Synovial Fibroblast Proliferation Via The Wnt/β-catenin Pathway In Rheumatoid Arthritis

Rheumatoid arthritis（RA）is a chronic autoimmune disease characterized by synovial hyperplasia and progressive joint destruction in the middle and late stages.The proliferation of activated rheumatoid arthritis synovial fibroblasts（RASF）is a direct cause of synovial hyperplasia and bone destruction.Our previous found that acid-sensing ion channel 1a（ASIC1a）induces RASF activation in response to the extracellular acidic microenvironment.However,the role of ASIC1 a on the proliferation of RASF remains unknown.Here,the effect of ASIC1 a on RASF proliferation and its molecular mechanism were investigated in vivo and in vitro.We found that ASIC1 a promotes the proliferation of RASF in an acidic p H6.0 environment and reduced synovial hyperplasia and bone damage when blocked in adjuvant arthritis（AA）rats.The canonical Wnt/β-catenin signaling was activated by ASIC1 a in an acidic p H6.0 environment,which upregulated the expression of proto-oncogene c-Myc and mediated RASF proliferation.In summary,this study reveals ASIC1 a as an acid sensor to promote RASF proliferation by the Wnt/β-catenin/c-Myc pathway,and suggests that ASIC1 a may be a potential therapeutic target for RA.Objective:The purpose of this study was to investigate the effect of acid-activated ASIC1 a on acid-induced RA synovial tissue hyperplasia and its pathophysiological significance.Methods:1.Effect of acid stimulation on RASF proliferation in vitro.RASF was stimulated with p H 6.0 acid at different time points in vitro.Ed U cell proliferation assay,CCK-8 and cell growth curve were used to explore the changes of RASF proliferation.The expression of PCNA protein was detected by Western blotting.2.Expression of ASIC1 a in RA synovial tissues.Western blotting and q-PCR were used to investigate the expression of ASIC1 a in human rheumatoid arthritis synovial tissue and normal synovial tissue.The isolated cells were identified by flow cytometry.3.The effect of acid-activated ASIC1 a on RASF proliferation.ASIC1a gene in RASF was silenced or overexpressed by ASIC1A-Si RNA or lentivirus,and the expression level of ASIC1 a was analyzed by Western blotting and qPCR to detect the silencing or overexpression effect.Western blotting was used to detect the protein expression levels of ASIC1 a and PCNA in control group,acidified group,silenced ASIC1 a group,overexpressed ASIC1 a group,and ASIC1 a specific inhibitor（Pc Tx-1）group.The proliferation of RASF under different treatments was investigated by Ed U cell proliferation experiment.4.Role of Wnt/β-catenin pathway in acid-induced RASF proliferation.Western blotting,q-PCR and immunofluorescence were used to detect the expression of β-catenin and c-Myc protein in different treatment groups.Western blotting and immunofluorescence experiments were used to investigate the effects of acid stimulation on β-catenin nuclear accumulation.RASF was treated with Wnt/β-catenin pathway inhibitor（xav939）,and the expression levels of β-catenin,c-Myc and PCNA were investigated by WB,q-PCR and IF.The proliferation of RASF under different treatments was investigated by Ed U experiment.5.Effect of blocking ASIC1 a channel on synovial tissue proliferation in AA rats.The AA rats with adjuvant arthritis were constructed.After Pc Tx-1 was injected,the changes in body weight and foot swelling of the rats were detected,and the arthritic inflammation of AA rats was scored by arthritis scoring rules.HE staining,toluidine blue staining and immunohistochemistry were used to investigate the proliferation of synovial tissue in AA rats after ASIC1 a channel blocking.Results:1.pH 6.0 acid stimulation promoted RASF proliferation in vitro;2.ASIC1 a was highly expressed in RA synovial tissue;3.Acid stimulation induced RASF proliferation by activating ASIC1 a,silencing or overexpressing ASIC1 a,the expression level of PCNA was down-regulated or upregulated,and the growth ability of RASF was also decreased or increased.4.The expression of β-catenin and c-Myc,the target genes of Wnt/β-catenin pathway,were decreased or increased accordingly after silencing or overexpression of ASIC1 a.The proliferation of acidification-induced RASF was reduced with Wnt/β-catenin pathway inhibitors.5.Inhibition of ASIC1 a channel inhibited synovial tissue proliferation in AA rats.Conclusion:1.RASF proliferation was induced by acidizing microenvironment2.Acid sensor ASIC1 a promoted RASF proliferation by regulating Wnt/β-catenin/c-Myc pathway.