雌激素及其受体对ASIC1a介导的关节软骨细胞凋亡的保护作用及其机制

Acid-sensitive ion channel 1a (ASIC1a) is a member of the DEG / ENaC superfamily, and it can be activated by extracellular H+ and then leads to influx of extracellular Ca2+, which participating in certain pathological processes. Our previous study showed that ASIC1a mediates the apoptosis of articular chondrocytes in adjuvant arthritis (AA) rats and found that estrogen has a protective effect on acid-induced chondrocyte apoptosis. Therefore, we carry out the following researches: (1) The different concentrations of estradiol (E2), ASIC1a specific blockers PcTX1 or specific small interfering RNA, whole cell patch clamp recording H+-activated ASIC1a current, and confocal laser scanning microscopy intracellular calcium concentration were used to investigate the role of E2 on acid-induced chondrocyte apoptosis and the relationship of E2 with ASIC1a expression and activity. (2) The estrogen receptor (ER), autophagy and ubiquitin proteasome degradation pathway were specific activated and inhibited by specific activators or blockers to analysis the role of ER and protein degradation pathway in regulating ASIC1a expression. (3) E2 and PcTX1 were given to male, female and ovariectomized AA rats, and the relationship between estrogen and ASIC1a was analyzed to elucidate the protective effect and mechanism of estrogen on ASIC1a-induced apoptosis in AA rat chondrocytes.

酸敏感离子通道1a（ASIC1a）属于DEG/ENaC超家族一员，H+激活可导致胞外Ca2+内流，参与某些病理反应过程。我们前期研究表明ASIC1a可介导佐剂型关节炎（AA）大鼠关节软骨细胞凋亡，并发现雌激素对其具有保护作用，在此基础上试图（1）使用不同浓度雌二醇（E2）及ASIC1a特异性阻断剂PcTX1或特异性小干扰RNA，全细胞膜片钳记录H+激活ASIC1a电流，激光共聚焦显微镜检测胞内钙离子浓度，分析E2抑制酸诱导的软骨细胞凋亡与ASIC1a表达和活性之间的关系。（2）通过对雌激素受体（ER）、自噬和泛素蛋白酶体降解途径的特异性激活与抑制，分析ER以及蛋白降解途径在E2调控ASIC1a表达中的作用。（3）在雌雄及卵巢摘除大鼠AA模型中给予E2、PcTX1，分析雌激素与ASIC1a之间的内在联系，初步阐明雌激素对ASIC1a介导的AA大鼠软骨细胞凋亡的保护作用与机制。

酸敏感离子通道1a（ASIC1a）属于DEG/ENaC超家族一员，H+激活可导致胞外Ca2+内流，参与某些病理反应过程。我们前期研究表明ASIC1a可介导佐剂型关节炎（AA）大鼠关节软骨细胞凋亡，并发现雌激素对其具有保护作用。为了阐明雌激素通过抑制ASIC1a表达发挥保护软骨细胞凋亡的作用与机制，设计了3部分研究内容：（1）使用不同浓度雌二醇（E2）及ASIC1a特异性阻断剂PcTx-1或特异性小干扰RNA，全细胞膜片钳记录H+激活ASIC1a电流，激光共聚焦显微镜检测胞内Ca2+浓度，分析E2抑制酸诱导的软骨细胞凋亡与ASIC1a表达和活性之间的关系；（2）通过对雌激素受体（ER）、自噬和泛素蛋白酶体降解途径的特异性激活与抑制，分析ER以及蛋白降解途径在E2调控ASIC1a表达中的作用；（3）在雌雄及卵巢摘除大鼠AA模型中给予E2、PcTx-1，分析雌激素与ASIC1a之间的内在联系。结果发现：（1）E2可以以时间和剂量依赖性方式降低酸诱导的大鼠关节软骨细胞的ASIC1a的表达以及自噬相关水平（Beclin1，LC3表达下降，p62表达升高），进而抑制软骨细胞的凋亡；（2）E2通过与ERα结合，激活AMPK/ULK1信号通路促进自噬溶酶体途径依赖的ASIC1a蛋白降解，从而保护酸诱导的关节软骨细胞损伤；（3）E2可以减少卵巢摘除AA大鼠关节病理变化，减少软骨基质破坏，降低软骨损伤，并抑制关节软骨细胞中ASIC1a蛋白的表达水平，增加自噬相关蛋白Beclin-1和LC3的表达水平。以上结果初步探究了雌激素与ASIC1a介导的AA大鼠关节软骨细胞凋亡的发生之间的关系，为以ASIC1a为靶点治疗RA的提供理论基础。