The Vital Role Of Acid-sensing Ion Channel1a In The Pyroptosis Of Chondrocytes In AA Rats And The Potential Mechanisms

Rheumatoid arthritis（RA）is a chronic systemic disease with a high incidence and poor prognosis.Increasing evidence have shown that accumulation of inflammatory metabolite intra-articular results to the decreasing of pH of joint fluid,which is one of the main pathogenesis of RA that lead to excessive apoptosis of cartilage cells,eventually cause the pathological changes of joints.Pyroptosis is a kind of proinflammatory programmed cell death,which is featured by the activation of Caspase-1 and the secretion of proinflammatory cytokines IL-1β/18.Recent studies have found that pyroptosis also participated in some autoimmune diseases,such as RA.Acid-sensitive ion channels（ASICs）are classes of extracellular acidosis activated cation channels.It has been reported that ASICs mainly transparent sodium and calcium permeability.Results of our previous studies indicated the presence of ASIC1 a in the articular chondrocytes of rats,and the expression of ASIC1 a was upregulated in the chondrocytes from rat with adjuvant arthritis.What’s more,ASIC1 a could mediate chondrocytes apoptosis induced by extracellular acidosis via adjusting [Ca²⁺]i.In summary,the results of these preliminary studies indicated that apoptosis of articular chondrocytes occured during RA accompanied by large amounts of inflammatory cytokines released into synovial fluid,while extracellular acidosis（pH6.0）can significantly induce apoptosis of articular chondrocytes and is a certain pH-dependent style.This phenomenon is consistent with the features of pyroptosis,but whether articular chondrocytes pyroptosis happens in the course of RA and the role of ASIC1 a in that process is still unclear.Reactive oxygen species（ROS）is an important molecule inthe cell,which plays an important role in the development of many diseases.Previous studies have shown that ROS is involved in [Ca²⁺]i and play an important role in the process of pyroptosis.However,the role of ROS in ASIC1 a mediated apoptosis of articular chondrocytes is unknown.The aim of this study is to investigate the role of ASIC1 a in chondrocytes pyroptosis of AA rats.1.The role of ASIC1 a in the pyroptosis of articular chondrocytes in vivo.The AA rat model was established by intradermal injection of Freund’s complete adjuvant（20mg/ml）100μl at the left hind paw.The AA rats were randomly divided into3 groups including the model group,the aspirin group（50mg/kg）,and the amiloride group（100mg/kg）.All the rats were sacrificed at day 28.The mRNA and protein expression of ASC,NLRP3,Caspase-1,ASIC1 a,IL-1β and IL-18 were upregulated in the joints of AA rats as compared with the normal rats,while the expression of Col 2a in cartilage was decreased.However,these changes were reversed by aspirin,as well as amiloride,which is an inhibitor of ASIC1 a.2.Extracellular acidosis induced pyroptosis of primary articular chondrocytes in vitro.Primary articular chondrocytes were stimulated by several pH as well as pH 6.0 for different times with or without AC-YVAD-CHO to observe the pyroptosis of primary articular chondrocytes.Compared with the normal cells,extracellular acidosis could increase the mRNA and protein expression of ASIC1 a,IL-1β,IL-18,ASC,NLRP3 and Caspase-1 in primary articular chondrocytes in a time-and dose-dependent manner,accompanied with the promoting release of LDH.Moreover,extracellular acidosis also induced the synthesis of ROS intracellular.3.The role of ASIC1 a in extracellular acidosis induced pyroptosis of articular chondrocytes in vitro.Primary articular chondrocytes were pre-incubated with Pctx-1（a specific inhibitor of ASIC1a）or transfected with ASIC1 a shRNA to investigate the role of ASIC1 a in extracellular acidosis induced pyroptosis of primary articular chondrocytes.Results showed that Pctx-1 and ASIC1 a shRNA attenuated the extracellular acidosis induced pyroptosis of primary articular chondrocytes by detecting for ASC,NLRP3,Caspase-1,L-1β and IL-18,as well as suppressed [Ca²⁺]i and the expression of ROS intracellular.4.The role of Ca²⁺ and ROS in ASIC1 a mediated extracellular acidosis induced pyroptosis of primary articular chondrocytes in vitro.Chelating agents of Ca²⁺ BAPTA-AM could attenuate the extracellular acidosis induced pyroptosis of primary articular chondrocytes by detecting for the expression of ASC,NLRP3 and Caspase-1.BAPTA-AM also had a negative effect on the concentration of IL-1β and IL-18 in the culture medium as well as suppressed the expression of ROS intracellular.Interestingly,NAC,the inhibitor of ROS intracellular could significantly suppress the pyroptosis of primary articular chondrocytes.These results indicated that ASIC1 a mediated the pyroptosis of chondrocytes of AA rats,the mechanism of which might be associated with its ability of promoting [Ca²⁺]i,accelerating the synthesis of ROS,and activating the NLRP3 inflammasome.