Acid-sensing Ion Channel 1a Mediates Acid-induced Pyroptosis Through Calpain-2/Calcineurin Pathway In Rat Articular Chondrocytes

The pyroptosis is a causative agent of rheumatoid arthritis(RA),a systemic autoimmune disease merged with degenerative articular cartilage.Previous results have been revealed extracellular acidosis triggered pyroptosis through activation of NLRP3inflammasome in chondrocytes.Nevertheless,the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear.Acid-sensing ion channels(ASICs)belong to an extracellular H~+-activated cation channel family.Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis up-regulate calpain and calcineurin expression in arthritis.Studies have shown that:calpain and calcineurin are closely related to pyroptosis.However,the role of acid-sensing ion channel 1a(ASIC1a)in the pyroptosis in rat articular chondrocytes under acid condition remains indefinite,and the potential role of calpain-2/calcineurin pathway in this process needs to further explore.The aim of this study is to investigate the role of acid-sensing ion channel 1a(ASIC1a)in the pyroptosis in rat articular chondrocytes under acid condition,and expore the potential role of calpain-2/calcineurin pathway in this process.1.Effect of extracellular acidosis on ASIC1a,calpain,calcineurin and NLRP3inflammasome in vitro.Articular chondrocytes were subjected to different pH conditions ranging from 7.4to 6.0 or pH 6.0 with different time(1,3,6,12 or 24 h).The research results showed that extracellular acidosis induced the protein expression of ASIC1a in a pH-and time-dependent manner,and the mRNA and protein expressions of calpain,calcineurin,NLRP3,ASC and caspase-1 were significantly increased in a time-dependent manner.2.The effects of silencing and blocking ASIC1a on calpain-2,calcineurin and pyroptosis in extracellular acid-stimulated articular chondrocytes.ASIC1a-shRNA and PcTX1(a specific inhibitor of ASIC1a)were used to explore the role of ASIC1a in the expression of calpain-2,calcineurin and pyroptosis.Results showed that the inhibition of ASIC1a could decrease the cell death accompanied with the decreased IL-1?level,and the decreased expression of ASIC1a,calpain-2,calcineurin and NLRP3 inflammasome proteins.The above results suggest that blocking ASIC1a can inhibit the extracellular acid-stimulated increase of calpain and calcineurin protein expression and the pyroptosis of primary rat articular chondrocytes induced by acidification stimulation.3.The effects of silencing and blocking calpain-2 on calcineurin and pyroptosis in extracellular acid-stimulated articular chondrocytes.Calpain-2-shRNA and calpeptin were used to explore the role of calpain-2 in the expression of calcineurin and pyroptosis.Results showed that the inhibition of calpain-2could decrease the cell death accompanied with the decreased IL-1?level,and the decreased expression of calpain-2,calcineurin and NLRP3 inflammasome proteins.The above results suggest that blocking calpain-2 can inhibit the extracellular acid-stimulated increase of calcineurin protein expression and the pyroptosis of primary rat articular chondrocytes induced by acidification stimulation.4.The effects of silencing and blocking calcineurin on pyroptosis in extracellular acid-stimulated articular chondrocytes.Calcineurin-shRNA and CsA were used to explore the role of calcineurin in the expression of calcineurin and pyroptosis.Results showed that the inhibition of calcineurin could decrease the cell death accompanied with the decreased IL-1?level,and the decreased expression of calcineurin and NLRP3 inflammasome proteins.The above results suggest that blocking calcineurin can inhibit the extracellular acid-induced pyroptosis of primary rat articular chondrocytes.Taken together,these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes,the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.