Effect Of ASIC1a Gene Sliencing On Extracellular Acid-induced Autophagy Of Rat Articular Chondrocyte And Its Possible Mechanisms

Acid-sensing ion channels?acid-sensing ion channels,ASICs?are a H+-gated cation channel that widely present in the cell membrane,they are Na⁺-selective and Ca²⁺-permeant.ASICs are involved in a variety of diseases,such as inflammation,cerebral ischemia,epilepsy and cancer.Autophagy is a lysosomal-dependent cellular degradation processes with a variety of physiological and pathological functions.Previous studies of our works found ASICs was expressed in rat articular chondrocytes,extracellular acidification stimulate chondrocytes induced the activation of autophagy,but the mechanism was not clear.This study is to investigate the effects of ASIC1 a on acid induced activation of chondrocytes autophagy and its possible mechanisms.Objective: To observe the effects of ASIC1 a on acid induced activation of chondrocytes autophagy and its possible mechanisms in vitro.Content: 1.To investigate the levels of rat articular chondrocytes autophagy in the different extracellular acidification stimulation conditions;2.Using the small interfering RNA?si RNA?technology to establish the expression of ASIC1 a silencing model and verify its silencing efficiency in vitro;3.To observe the changes in cartilage chondrocytes autophagy after the treatment with ASIC1 a si RNA or Pc TX1;4.To investigate the possible mechanisms of ASIC1 a in acid-induced activation of chondrocytes autophagy.Methods: 1.Different extracellular acidification conditions?pH7.4,7.0,6.5,6.0,5.5 and 5.0?or?pH6.0 15 min,30min,1h,2h,3h,4h,5h?,the expression LC3 II in chondrocytes were tested by Western-blot assay.2.A synthesis of of short-chain-specific fluorescence ASIC1 a si RNA-FAM was transfected into chondrocytes using Lipofectamine 2000 transfection kit,the silencing efficiency of ASIC1 a si RNA were verified by fluorescence microscope,Q-RT-PCR and Western-Blot assay.3.Rats articular cartilage chondrocytes were divided into normal group,pH6.0 group,pH6.0+Pc TX1 group,pH6.0+ASIC1a si RNA group,pH6.0+NC-RNAi group.The intracellular calcium([Ca²⁺]i)was analyzed using laser scanning confocal microscopy.Quantitative RT-PCR and Western blot were used to detect the m RNA and protein expression of autophagy markers LC3 II,Beclin1 and ULK1.4.Rats articular cartilage chondrocytes were divided into normal group,pH6.0 group,pH6.0+BAPTA-AM group,pH6.0+ASIC1a si RNA group,pH6.0+NC-RNAi group,pH6.0+BAPTA-AM+ASIC1a si RNA group.GFP-LC3 plasmid was transfected into chondrocytes to verify the levels of LC3;Quantitative RT-PCR and Western blot were used to detect the m RNA and protein expression of autophagy-related markers.5.The expression of Ca MKK?,phosphorylation levels of AMPK and m TOR were detected by Western blotting assay.6.Rats articular cartilage chondrocytes were divided into normal group,pH6.0 group,pH6.0+3-MA group,pH6.0+ASIC1a si RNA group,pH6.0+NC-RNAi group,pH6.0+3-MA+ASIC1a si RNA group.Chondrocytes apoptosis were valued by Hoechst33342 staining,flow cytometry and Quantitative RT-PCR.Results: 1.Extracellular acidification-induced the activation of autophagy on rat articular chondrocytes,the expression of LC3?exhibited a time-and pH-dependent behavior,when the expressions were the maximum at time pH6.0,3h;2.The specific ASIC1 a si RNA could be successfully transfected into rat articular chondrocytes,the expression of ASIC1 a m RNA and protein levels were significantly decreased in transfected chondrocytes compared with normal group;3.The intracellular Ca²⁺ concentration was decreased in chondrocytes treated with Pc TX1 or ASIC1 a si RNA.Moreover,inhibition or silencing of ASIC1 a was able to inhibit acid-induced autophagy in rat articular chondrocytes,showing as: expression of LC3?,Beclin1 and ULK1 were significantly reduced;results of AO staining showed that the numbers of acidic lysosomal autophagy was reduced;4.ASIC1 a si RNA or BAPTA-AM could suppress acid-induced chondrocytes autophagy activation,manifested as: levels of LC3?,Beclin1 and ULK1 were significantly reduced;the numbers of intracellular GFP-LC3 puncta was decresead.5.The expression of Ca MKK? and AMPK phosphorylation were significant reduced,and expression of m TOR was significant increased in ASIC1 a si RNA group or?and?BAPTA-AM group.6.The m RNA expression of Bax was significant decreased,but the expression of Bcl-2 was significant increased in ASIC1 a si RNA group or?and?3-MA group,chondrocytes apoptosis was significant decreased.Conclusion: 1.ASIC1 a was involved in extracellular acidification-induced activation of rat articular chondrocytes autophagy;2.The silence cells model of ASIC1 a by si RNA techonology in rat articular chondrocytes was successfully constructed.3.Inhibition or silencing of ASIC1 a was able to inhibit acid-induced the overload of Ca²⁺ and the activation of autophagy in rat articular chondrocytes.4.The effect of ASIC1 a on the activion of chondrocytes autophagy induced by extracellular acidification might be associated with the Ca MKK?/AMPK/m TOR signaling pathway.