Role And Potential Mechanisms Of ASIC1a-mediated NLRP3 Inflammasome Activation In AA Rat Articular Cartilage

Objective Rheumatoid arthritis(RA)is a chronic autoimmune disease,and cartilage destruction is one of the fundamental reasons for RA patients' deformity.Acid-sensing ion channel 1a(ASIC1a)is a kind of cation channel activated by extracellular acidification that exists widely on the cell membrane.Our previous study indicated that ASIC1 a was involved in the expression of NLRP3 inflammasome in AA rat articular cartilage.However,the underlying mechanism remains unclear.This study confirmed the effect of ASIC1 a on the expression of NLRP3 inflammasome in rat articular cartilage,and discussed its possible molecular mechanism and the effect of activation of NLRP3 inflammasome on articular cartilage.Methods1.In vivo,the AA rat model was established to observe the dynamic changes in the expression of NLRP3 inflammasome at different stages.The rats were sacrificed at 0,7,14,21,28 and 35 days after modeling,and the pathological changes of joints were observed by HE staining.Immunohistochemistry and Western blot were used to detect the expression of nod-likereceptorprotein3(NLRP3),apoptosis-associatedspecklikeprotein(ASC)and caspase-1 in articular cartilage tissues.2.In vitro,to observe the effect of extracellular acidification on the expression of NLRP3 inflammasome in rat articular chondrocytes.Primary rat articularchondrocytes were treated for 24 h at different pH(pH 7.4,pH 7.0,pH 6.5,pH 6.0,and pH 5.5)and acidified for different time(0,3,6,12,and 24 h)under pH 6.0.Western blot was used to detect the protein expression of ASIC1a?NLRP3,ASC,caspase-1.The expressions of NLRP3,ASC and caspase-1 mRNA were detected by qRT-PCR.3.In vitro,to observe the effect of blocking and silencing ASIC1 a on the expression of NLRP3 inflammasome in rat articular chondrocytes.Rats primary articular chondrocytes were divided into the pH 7.4 normal group,pH 6.0 group,pH 6.0+NC group,pH 6.0+ASIC1a shRNA group,and pH 6.0+ASIC1a specific inhibitor pctx-1(100ng/ml)group.Western blot was used to detect the expression levels of ASIC1 a,NLRP3,ASC,caspase-1,NF-?B p65,NF-?B pp65.4.In vitro,to observation the role of NF-?B signaling pathway in the expression of NLRP3 inflammasome mediated by ASIC1 a.Primary rat articular chondrocytes were divided into three groups: pH 7.4 normal group,pH 6.0 group and pH 6.0+NF-?B specific inhibitor BAY 11-7082(10uMl)group.Western blot,qRT-PCR and ELISA were used to detect the expressions of NLRP3,ASC,caspase-1 and IL-1?.5.In vitro,to observation the role of activation of NLRP3 inflammasome in the articular cartilage of rats.Primary rat articular chondrocytes were divided into the following three groups: pH 7.4 normal group,pH 6.0 group and pH 6.0+caspae-1specific inhibitor AC-YVAD-CHO(10uMl)group.The expression of caspase-1 was detected by Western blot,qRT-PCR and caspase-1 detection kits,the activity of articular chondrocytes in rats was detected by AO-EB staining,LDH and MTT methods,and the expression of apoptosis-related proteins bcl-2,Bax,caspase-3 and cleaved caspase-3 was detected by Western blot,and the apoptosis rate was detected by flow cytometry.The levels of IL-1??IL-6 and TNF-? were detected by qRT-PCR and ELISA.Results1.In vivo experiments showed that NLRP3 inflammasome were expressed in AA rats.The results of HE staining showed that,compared with the normal group,thejoints of the rats in the model group were significantly damaged,which was manifested as the uneven joint surface of the rats,with obvious synovial tissue dysplasia and inflammatory cell infiltration.The damage increased from the first day to the 28 th day and improved from the 35 th day.Immunohistochemistry and Western blot results showed that compared with normal rats,the expressions of NLRP3,ASC and caspase-1 proteins in the articular cartilage of the rats in the model group were significantly increased and time-dependent,and the protein expression was significantly increased at 28 or 35 days.In vitro experiments showed that extracellular acidification could up-regulate the expression level of NLRP3 inflammasome in rat articular chondrocytes,and the protein and mRNA expression was significantly increased after the treatment with pH 6.0 for 24 h.2.ASIC1 a promotes the expression of extracellular acid-induced NLRP3 inflammasome in rat articular chondrocytes.Western blot results showed that the expression levels of ASIC1 a and NLRP3 inflammasome related proteins were significantly increased under acidic conditions(pH 6.0).However,silencing and blocking of ASIC1 a significantly reduced the levels of acid-induced NLRP3 inflammasome associated proteins(NLRP3,ASC,caspase-1).The results showed that activated ASIC1 a could be involved in the expression of NLRP3 inflammasome in rat articular chondrocytes.3.NF-?B signaling pathway plays a key role in the expression of NLRP3 inflammasome in rat articular chondrocytes mediated by ASIC1 a.Western blot results showed that the NF-?B signaling pathway was significantly activated in chondrocytes after 24 h of extracellular acidification(pH6.0)compared with the control group.However,the expression of NF-?B signaling pathway was significantly reduced after silencing ASIC1 a with siRNA and the pretreatment with PcTx-1,a specific inhibitor of ASIC1 a.The acid-induced expression of NLRP3,ASC,caspase-1 and IL-1?genes and proteins was significantly reduced by the use of NF-?B signaling pathway specific inhibitor BAY 11-7082.These results suggest that ASIC1 a promotes the activation of the NF-?B signaling pathway,and the activation of the NF-?B signaling pathway plays a key role in the expression of NLRP3 inflammasome in the articularchondrocytes mediated by ASIC1 a.4.Activation of NLRP3 inflammasome promotes acid-induced apoptosis of articular chondrocytes in rats and induces expression of inflammatory factor IL-1??IL-6 and TNF-?.The results of AO-EB,MTT and LDH show that the extracellular acidification can significantly reduce the articular cartilage cell activity,however,caspase-1 inhibitor AC-YVAD-CHO can dramatically improve articular cartilage cell activity,and Western blot results show that AC-YVAD-CHO can obviously reduce extracellular acidification-induced cartilage cell apoptosis by the decrease of the apoptosis-related protein expression,and the cell apoptosis rates detected by Annexin V-FITC Apoptosis Detection Kit is consistent with the results.In addition,according to the results of qRT-PCR and ELISA,AC-YVAD-CHO can significantly reduce the expression of IL-1??IL-6 and TNF-?.These results suggest that activation of NLRP3 inflammasome can promote apoptosis and induce the expression of IL-1??IL-6 and TNF-? in rats articular chondrocytes.Conclusions1.The activation of ASIC1 a promoted the expression of acid-induced NLRP3 inflammasome in rat articular chondrocytes and was associated with the NF-?B signaling pathway.2.Activation of NLRP3 inflammasome can induce articular chondrocytes apoptosis and the expression of IL-1??IL-6 and TNF-? in rats.3.The study of NLRP3 inflammasome and related signaling pathways will expand our understanding of the function of ASIC1 a and provide a new idea for the treatment of RA with articular chondrocyte injury.