酸敏感离子通道1a介导细胞内钙离子内流调节椎间盘终板软骨细胞凋亡

Acid-sensing ion channel la mediating acid-induced increases in intracellular calcium influx induces endplate chondrocytes apoptosis

目的：观察酸敏感离子通道1a（ASIC1a）介导的钙离子内流在酸诱导的终板软骨细胞凋亡的作用。方法：SD培养大鼠终板软骨细胞，慢病毒为载体转染终板软骨细胞沉默ASIC1a，钙离子成像分析细胞内Ca2＋浓度（［Ca2＋］i）变化，采用四甲基偶氮唑蓝（MTT）实验分析酸诱导的细胞活力；ELISA测定凋亡率；荧光染料Hoechst 33342检测细胞凋亡。免疫印迹检测活化型半胱天冬酶-9（cleaved caspase-9）、活化型半胱天冬酶-3（cleaved caspase-3）等凋亡相关蛋白的表达。结果：阻断ASIC1a可以抑制酸诱导的［Ca2＋］i升高；用ASIC1asiRNA或PcTX1特异性阻断ASIC1a，明显抑制酸诱导的终板软骨细胞凋亡；胞外钙离子浓度降低抑制酸诱导的终板软骨细胞凋亡，与阻断ASIC1a结果一致，提示ASIC1a介导的钙离子内流在酸诱导凋亡中的作用。阻断ASIC1a，抑制酸诱导的cleaved caspase-9、cleaved caspase-3蛋白的表达。结论：酸激活的ASIC1a可能通过钙超载促使大鼠椎间盘终板软骨细胞凋亡。

Objective： To investigate whether acid-sensing ion channel la （ASICla） contributes to acid-induced endplate chondrocyte apoptosis by calcium influx. Methods： Rat endplate chondrocytes were cultured in vitro. Lentivirus-packing siRNA expression vector was transfected with ASIC1a small interfering RNA（siRNA）. Cell viability was determined using a 3-（4, 5-dimethylthiazol-2-yi）-2, 5-diphenyltetrazolium bromide（MTT） assay. Apoptosis was analyzed by DNA fragmentation ELISA and Hoechst 33342 for morphologic determination. Acid-induced intracellular Ca^2＋ concentration （[Ca^2＋] i） was investigated by calcium imaging. Expression of apoptosis-relevant protein including cleaved caspase-9 and -3 was determined by Western blotting. Results： Blockade of ASIC1a using ASIC1a-siRNA or PcTX1 inhibited acid-induced cell apoptosis and elevation of [Ca^2＋]i. Acid-induced [Ca^2＋]i increase via ASIC1a was involved in endplate chondrocyte apoptosis. Meanwhile, expression of apoptotic proteins including cleaved caspase-9 and -3 was inhibited by ASICla-siRNA or PcTX1. Conclusion： Acid-induced [Ca^2＋ ]i elevation via activation of ASICla is involved in acid-induced endplate chondrocyte apoptosis.