ASlC1a介导的自噬对佐剂性关节炎（AA）大鼠关节软骨细胞的作用及其机制

Acid-sensing ion channels (ASICs) belonging to the degenerin/epithelial sodium channel superfamily, are transiently activated by extracellular acidification. It has been reported that ASIC1a plays an important role in inflammation, cerebral ischemia and tumour, in which tissue acidosis is a common feature. Our previous studies have been demonstrated that ASIC1a subunit is expressed in articular chondrocytes of rats with adjuvant arthritis. In addition, activation of ASIC1a could be responsible for autophagy of articular chondrocytes by increaseing the intracellular calcium ([Ca2+]i). Therefore, the aim of this study is to investigate the role of ASIC1a in acid-induced autophagy of articular chondrocytes and effects of chondrocyte autophagy on rheumatoid arthritis. So we carry out the following researches: (1) The chondrocytes were incubated in different pH extracelluar solution and different expression of ASIC1a state models were established in chondrocytes. To investigate the role of ASIC1a in acid-induced autophagy of articular chondrocytes and its possible mechanisms, the current of ASIC1a induced by H+ was recorded using the whole-cell patch clamp and the Ca2+ concentration was detected by laser scanning confocal microscopy. (2) Chondrocytes were isolated and cultured from AA rats in different time points. To analysis the connection of cell autophagy and apoptosis, chondrocytes were transfected with small interfering RNA specifically to down-regulate the expression of ASIC1a or treated with 3-MA. (3) Amiloride combined with chloroquineon were uesd in vivo study to determine the effects of chondrocyte autophagy on rheumatoid arthritis.

酸敏感离子通道（ASICs）属于DEG/ENaC超家族一员，H+ 是最早发现的ASICs通道的激动剂，参与某些病理反应过程。我们前期实验研究证实在AA大鼠关节软骨细胞上有ASIC1a表达，激活可导致胞外钙内流并与关节软骨细胞自噬有关。在此基础上试图（1）建立不同胞外酸化环境及不同ASIC1a的表达状态模型，建立全细胞膜片钳记录H+激活ASIC1a电流，激光共聚焦显微镜检测细胞钙离子浓度，分析细胞内钙离子的超载对细胞自噬水平的影响及其分子机制。（2）分别取AA大鼠不同时间点的软骨细胞，通过转染特异性小干扰RNA技术使Beclin1表达沉默和3-MA来抑制自噬，分析其在AA发展不同时期自噬和凋亡之间的内在联系。（3）给予自噬的抑制剂，分析ASIC1a对自噬发生的影响及自噬水平的改变对佐剂性关节炎病变的影响，初步阐明ASIC1a介导的自噬在AA大鼠关节软骨细胞病变中的作用。

ASICs是一类由细胞外H＋激活的阳离子通道，属于上皮钠通道(ENaC)/退化蛋白(DEG)超家族，具有七个亚基，即ASIC1a、ASIC1b1、ASIC1b2、ASIC2a、ASIC2b、ASIC3、ASIC4。我们课题组之前研究发现，ASIC1a介导细胞外酸化诱导的大鼠软骨细胞凋亡，从而促进关节软骨的破坏。另一方面，我们的前期研究发现，细胞外酸化通过活化ASIC1a诱导软骨细胞自噬，这与其诱导的细胞凋亡密切相关。为此我们进行以下研究：（1）ASIC1a在酸诱导的软骨细胞自噬中的作用，结果显示：①与 pH 7.4 组相比，pH 6.0组软骨细胞自噬蛋白 LC3 表达量明显增加，且软骨细胞生存率随酸化时间的延长而降低。②给予ASICs非特异性阻断剂阿米洛利（Amiloride）和 ASIC1a特异性阻断剂PcTx-1处理后，自噬基因Beclin-1 mRNA 和蛋白表达水平明显降低。③与pH 6.0 酸化刺激组比较，ASIC1a 特异性阻滞剂 PcTx-1 组胞质中自噬小体的数量明显减少。④与pH 6.0 酸化刺激组比较，转染特异性siRNA沉默ASIC1a 的表达后，软骨细胞中LC3Ⅱ蛋白以及自噬基因Beclin1 、ULK1 mRNA 表达量明显下调，酸性自噬溶酶体数目明显减少。⑤应用钙离子螯合剂BAPTA-AM 及特异性siRNA ASIC1a 后，软骨细胞Beclin1 、ULK1 mRNA和 GFP-LC3 融合蛋白表达量显著减少，且两者联合应用组降低水平更明显。综上，提示细胞外酸化通过ASIC1a诱导大鼠软骨细胞自噬。（2）自噬在ASIC1a介导的大鼠关节软骨细胞凋亡中的作用，结果显示：在体内和体外实验中，使用自噬阻断剂3-MA和沉默自噬基因Beclin1抑制自噬后，软骨细胞中凋亡标志性蛋白剪切性PARP、Caspase-3/9的表达水平明显增加，细胞凋亡率明显升高；使用雷帕霉素诱导自噬，骨细胞中剪切性PARP、Caspase-3/9的表达水平明显减少，细胞凋亡率明显降低。（3）ASIC1a在酸诱导的软骨细胞自噬中的作用机制，结果显示：ASIC1a通过MAPK/ERK和AMPK/FoxO3通路介导酸诱导的软骨细胞自噬。综上所述，ASIC1a在酸诱导的软骨细胞自噬中发挥重要作用，为针对ASIC1a为靶点开发治疗RA的药物提供理论依据。