The Role Of Acid-sensing Ion Channels In Acid-induced Apoptosis Of Rat Atricular Chondrocyte And Its Mitochondria Mechanisms

Background: Rheumatoid arthritis （RA） is a chronic, systemic autoimmune diseasecharacterized by systemic inflammation, persistent synovitis, andprogressive joint destruction. It has long been recognized that tissueacidosis is an important feature of RA marked by intra-articulardecreases in pH. Acid sensing ion channels，a member-belong to theepithelial sodium channel/degenerin （ENaC/DEG） family ofamiloride-sensitive transmembrane ion channel, could be activated byextracellular acidification. It has been demonstrated in our previousstudy that ASICs were expressed abundantly in rat articularchondrocytes and involved in the articular chondrocyte injury ofadjuvant arthritis.Objective: The aim of the present study is to investigate the role of acid-sensing ionchannels in acid-induced apoptosis of rat articular chondrocyte and itsmitochodria mechanisms, with the apoptosis model of rat chondrocytesinduced by extracellular acidosis.Methods:1. Articular chondrocytes were obtained from rats using type IIcollagenase digestion method. The chondrocytes were divided into fourgroups and incubated in different pH extracelluar solution （pH7.4,pH6.5, pH6.0, pH5.5） for three hours. MTT assay was employed todetermine the proliferation of articular chondrocytes in acidic solutions.Effects of extracellular acidosis on chondrocyte apoptosis were observed by Hoechst33258and Annexin V/PI staining. The mRNAexpression of Cathepsin K were detected by real time reversetranscription PCR.2. Cells were pre-treated with nimodipine （5μM）, x-conotoxin MVIIC（3μM）, or thapsigargin （1μM）, which were added to eliminate theeffect of voltage-gated Ca2+channels, following acid incubation （pH6.0）for3h alone or with various final concentrations of amiloride （12.5,25,50,100, or200μM）. After treatment, the cells were washed twice withphosphate-buffered saline （PBS） and re-cultured in complete Dulbecco’smodified Eagle’s medium （DMEM） containing10%FBS for4h.Chondrocytes cultured in the pH7.4solution without amiloride wereused as controls. MTT assay was employed to determine theproliferation of articular chondrocytes in various groups; cellularmorphologic changes were determined using AO/EB staining; theannexin V/propidium iodide （PI） dual-staining assay was performedaccording to the manufacturer’s instructions; the mitochondrialmembrane potential was assessed using Rhodamine123staining, mRNAexpression of Bcl-2family genes by RT-PCR analysis. Assay ofcaspase-3,caspase-9in articular chondrocytes were determined usingcaspase kits and immunocytochemisty.Results:1. Proliferation of articular chondrocytes were inhibited observably byextracellular acidosis.The MTT result of the present study showed that the proliferation of the ratarticular chondrocytes was inhibited by extracellular acidosis with bothpH6.0and pH5.5. Combined with flow cytometry experiment, It showedthat decreasing in articular chondrocytes was due to acid-induced apoptosis.Furthermore, chondrocytes apoptosis was related to the degree ofacidification.2. Inhibition of acid-sensing ion channels by amiloride protects ratarticular chondrocytes from acid-induced apoptosis. Survival rate of groups treated by amiloride was superior thanacidification model group. We used AO/EB staining to observe changesin cellular morphologies. After drug treatment, there was an increase inthe number of viable cells, while apoptotic and necrotic cells werereduced. Acid-induced apoptosis in articular chondrocytes wasevaluated by annexin V-FITC and PI staining. the apoptotic ratio was32.8%in acid-induced chondrocytes, while amiloride significantlyinhibited acid-induced apoptosis ratio. Incubation with the necessarycofactors significantly decreased the fluorescent intensity inchondrocytes. Amiloride could significantly suppress the loss ofmitochondrial membrane potential induced by acidic extracellularsolution in a dose-dependent manner.3. Effect of amiloride on mitochondrial signal transduction pathwayduring acid-induced articular chondrocytes apoptosis in ratsTo investigate Bad, Bcl-xl, Bcl-2, and Bax gene expression by RT-PCR, weanalyzed the expression levels of genes in different groups. As we expected,expression of the anti-apoptosis gene, Bcl-2, was increased, while themRNA levels of the pro-apoptotic genes, Bax/Bad, decreased.Chondrocytes treated with100μM of amiloride showed a significantdecrease in caspase-3/9activity compared with acid-treated cells.Furthermore, chondrocytes treated with100μM of amiloride showed asignificant decrease in protein expression of cyt-c、caspase-3/9comparedwith acid-induced cells.Conclusion:1. Extracellular acidosis can restrain the proliferation and promote theapoptosis of articular chondrocytes in vitro, in addition, apoptosis wasrelated to the degree of acidification.2. Inhibition of acid-sensing ion channels by amiloride protects ratarticular chondrocytes from acid-induced apoptosis.3. Effect of amiloride on acid-induced apoptosis of rat articularchondrocytes could be achieved by blocking ASICs, inhibiting [Ca²⁺]i in acid-induced articular chondrocytes, protecting the mitochondrialfunction of rat articular chondrocytes, regulating the expression ofBcl-2family genes and controling cyt-c releasing and caspase activity.