The Role Of ASIC1a In Regulating Synovial Invasion Of Rheumatoid Arthritis Via Ca²⁺/Rac1 Signaling Pathway And Its Mechanism

Rheumatoid arthritis?RA?is a chronic autoimmune-disease of unknown origin that primarily affects the joints and ultimately leads to their destruction.Rheumatoid arthritis fibroblast-like synoviocytes?RA-FLSs?located at the edge of the synovium were identified as key players in the pathophysiological process of RA and reported to have many similar properties to various tumor cells.Acid?sensing ion channels?ASICs?which emerged as key receptors for extracellular acidic PH,are differently expressed during various diseases and have been implicated in underlying pathogenesis.ASIC1a is involved in tumor cells migration and invasion.Here,we investigated the roles of ASIC1a in the migration and invasion of RA-FLSs.Objectives:In this experiment,RA fibroblast-like synovial cells?RA-FLSs?and RA synovial tissue and adjuvant arthritis?AA?rats were used to detect the expression of ASIC1a in RA-FLSs and RA synovial tissue and AA rats.In order to investigate whether ASIC1a regulates the migration and invasion of rheumatoid arthritis fibroblast-like synovial cells?RA-FLSs?and leads to the invasive destruction of articular cartilage.We also investigated whether ASIC1a activates Rac1 signal by promoting Ca²⁺influx,thereby mediating the migration and invasion of RA-FLSs.Methods:The expression of ASIC1a in synovial tissue of RA patients and normal people was detected by immunohistochemistry.Western blot,immunofluorescence and q RT-PCR were used to detect the expression of ASIC1a in RA synovial fibroblasts and normal human synovial fibroblasts.Freund's adjuvant was used to establish an AA rat model of adjuvant arthritis.The AA group of the treatment group was injected with PCTX-1 or triamcinolone acetate?1 mg/kg?or PBS on the 14th day after the successful modeling.Synovial hyperplasia,infiltration,and the invasion of cartilage at the joints of rats were observed by HE staining and toluidine blue staining.Western blot and immunohistochemistry were used to detect the expression of migration and invasion related genes and proteins in the synovium of rats.RA-FLSs were transfected with ASIC1a sh RNA lentiviral plasmid to construct ASIC1a gene silencing group and overexpression group.Use q RT-PCR,western blot,and ELISA to evaluate target genes and proteins in RA-FLSs transfected with overexpression-ASIC1a or ASIC1a-RNAi at different PH.The migration and invasion of RA-FLSs were detected by wound healing experiment and transwell experiments.The calcium ion in the RA-FLSs was labeled with Fluo-3 AM,a calcium fluorescence probe,and the fluorescence of calcium in the cells of the control group,the PH6.0 acidification group and the PCTX-1+PH6.0acidification group were detected by laser confocal microscope.The activity of Rac1 in PH7.4,PH6.0 and PH6.0+ASIC1a-RNAi or overexpression-ASIC1a were detected by Rac1 activity detection kit.We used Rac1 activity detection kit to detect changes in Rac1 activity in RA-FLSs after BAPTA-AM chelated with intracellular calcium ions.We use Rac1 specific inhibitor NSC23766 to inhibit Rac1 activity and then use q RT-PCR,western blot and ELISA to evaluate the expression of target genes and proteins in ASIC1a-RNAi or overexpression-ASIC1a at different PH.Wound healing experiment and transwell experiment were used to detect cell migration and invasion.Results:Our results showed ASIC1a overexpressed in RA synovial tissues and RA-FLSs.Inhibition of ASIC1a by PCTX-1 reduces synovial invasion and the expressions of MMP2,MMP9,p-FAK to protect articular cartilage in AA rats.Moreover,the acidity-promoted invasion and migration as well as the expressions of MMP2,MMP9,p-FAK of RA-FLSs were down-regulated by ASIC1a-RNAi and PCTX-1 while they were increased by overexpression-ASIC1a.ASIC1a mediated Ca²⁺influx and the activation of Rac1,which was decreased by the intracellular calcium chelating agent BAPTA-AM.Meanwhile,the migration and invasion as well as the expressions of MMP2,MMP9,p-FAK of RA-FLSs were decreased by Rac1 specific blocker NSC23766.in conclusion:1.ASIC1a regulates the migration and invasion of RA-FLSs.2.ASIC1a regulates the migration and invasion of RA-FLSs through the Ca²⁺/Rac1signaling pathway.