Effects Of Acid-sensing Ion Channella On Acid-induced Autophagy Of Articular Chondrocytes And Its Possible Mechanisms

Acid-sensing ion channels(ASICs), a member-belong to the epithelial sodiumchannel/degenerin(ENaC/DEG) family of amiloride-sensitive transmembrane ionchannel, could be activated by extracellular acidification. It has long been recognizedthat ASIC1a played an important role in inflammation, cerebral ischemia and tumour,in which tissue acidosis is a common feature. It has been demonstrated in our previousstudy that ASIC1a was expressed strongly in rat articular chondrocytes and involvedin the articular chondrocytes injury of adjuvant arthritis. Recently, it has been reportedthat autophagy is activated in articular cartilage from patients with RA and involed inthe progress of cartilage damage, however, the mechanisms remains unknown.Primary rat articular chondrocytes was choosed in the present study to furtherinvestigate the effect of acid-sensing ion channel1a on acid-induced autophagy of ratarticular chondrocyte and explore the possible mechanisms.OBJECTIVE To establish the acidosis model by observing the effect ofextracelluar acidosis on articular chondrocytes autophagy, and investigate the effect ofinhibition of acid-sensing ion channel1a(ASIC1a) on the level of autophagy and therole of MAPK pathways in ASIC1a regulates autophagy from acid-induced articular cartilage cells, through which discovering the effect of ASIC1a on acid-inducedautophagy of articular chondrocytes and its possible mechanisms.METHODS1. Primary rat articular chondrocytes were obtained using typeⅡcollagenasedigestion method. The chondrocytes were incubated in different pH extracelluarsolution (pH7.4, pH7.0, pH6.5, pH6.0, pH5.5) for three hours. The expression ofautophagy markers light chain3(LC3) in articular chondrocytes after acidation wasdetected through western blot. Effects of extracelluar acid solutions (pH6.0) atdifferent time points (0min,15min,30min,1h,2h,3h,4h,5h) on autophagy andviability of articular chondrocytes were detected by western blot and MTT assay,respectively.2. The acidulated chondrocytes were cultured with the ASIC1a antagonist Amilorideor PcTX1, The mRNA expression of Beclin-1and protein expression of pERK,pP38MAPK and LC3were detected using reverse transcription-polymerase chainreaction (RT-PCR) and western blot, respectively. The autophagic vacuoles wasobserved through transmission electron microscope (TEM).3. The pERK1/2inhibitor PD98059and pP38MAPK inhibitor SB203580were usedto explore the effect of ERK1/2and P38MAPK on acid-induced autophagy, mRNAexpression of Beclin-1and protein expression of LC3were determined by RT-PCRand western blot, respectively.RESULTS1. Activation of autophagy in articular chondrocytes were promoted observably byextracellular acidosis.The western blot result of the present study showed that the activation of autophagy inarticular chondrocytes was remarkably increased as the medium pH decreased, withno significant difference between pH6.0group and pH5.5group. The survival rate ofarticular chondrocytes was significantly reduced in acidosis group with a time-dependent manner, and the expression of LC3increased within3hours, butgradually decreased after3hours. Accordingly, exposure to acidic medium (pH6.0)for3h was selected as the acidosis condition in the following experiments.2. The acid-induced autophagy of articular chondrocytes was involved in theactivation of ASIC1a.The mRNA expression of Beclin1and the protein expression of LC3, pERK1/2andpP38MAPK were both significantly increased in the pH6.0group as compared withthe normal group, which could remarkably inhibit by treatment with amiloride orPcTX1. Consistent with this result, the number of autophagosome was increased inpH6.0group, but significantly decreased in amiloride or PcTX1group.3. The effect of acid-induced autophagy of articular chondrocytes might partly resultfrom the role of ERK1/2、p38MAPK.Results of the RT-PCR analysis revealed that the inhibition of ERK1/2phosphorylation could result in a decreased amount of Beclin-1mRNA expression,but inhibition of the phosphorylation of p38MAPK could not attenuate the mRNAexpression of Beclin-1. Consistent with these results, protein expression levels of LC3in acid-induced chondrocytes was decreased in the PD98059-treated group ascompared with the pH6.0group. However, the amount of LC3present inSB203580-treated group was not significantly changed as compared with the pH6.0group.CONCLUSION1. Extracellular acidosis could promote the autophagy and restrain the survival rateof articular chondrocytes in vitro, in addition, it was related to the degree ofacidification.2. Inhibition of acid-sensing ion channel1a could attenuate acid-induced autophagyof articular chondrocytes. 3. The effect of blocking ASIC1a on acid-induced autophagy of articularchondrocytes was involved with the inhibition of ERK1/2phosphorylation, but notp38MAPK phosphorylation.