氧化应激对肺泡Ⅱ型上皮细胞的损伤作用及JNK信号转导机制

Injury of alveolar type Ⅱ cell and JNK signal transduction induced by hydrogen peroxide

目的:探讨氧化应激状态下肺泡II型上皮细胞(Alveolar type Ⅱ epithelial cell,ATⅡ)存活、凋亡和JNK(c-jun NH2-terminal kinase)的调控机制。方法:采用过氧化氢(Hydrogen peroxide,H2O2)刺激原代大鼠ATⅡ细胞,复制活性氧(Reactive oxygen species,ROS)攻击ATⅡ细胞损伤模型。蛋白质免疫印迹法(Western blot)检测ATⅡ细胞受500μmol/LH2O2刺激后磷酸化JNK的动态变化,四甲基偶氮唑盐反应比色法(MTT法)检测细胞存活率,流式细胞术检测细胞凋亡率,并观察JNK抑制剂SP600125干预前后存活率和凋亡率的变化。结果:ATⅡ细胞在受H2O2刺激后随作用时间的延长细胞存活率下降,凋亡率增加;H2O2刺激可导致JNK的磷酸化激活,使用SP600125后,细胞存活率增加,凋亡率降低。结论:H2O2以时间依赖的方式诱导ATⅡ细胞凋亡,抑制JNK信号激活对氧化应激状态下的ATⅡ细胞可能起到保护作用。

Objective：To investigate survival and apoptotic responses of alveolar epithelial type Ⅱ cells （AT Ⅱ cell） under oxidative stress and to discuss the regulation mechanism mediated by c-jun NH2-terminal kinase （JNK）. Methods：Primary rat alveolar epithelial type Ⅱ cells were attacked by Hydrogen peroxide （H2O2） and cells were pretreated with SP600125 in some groups. Cell viability,apoptotic rate and the expression of phosphorylated JNK （p-JNK） were measured by 3- （4,5-dimethylthiazol-2-yl）-2,5-diphenyhetrazolium bromide（MTT） assay,flow cytometry and western blot analysis, respectively. Results：Compared with control group, decreased cell viability and increased apoptotic rate in AT Ⅱ cells occurred in time-dependent manner when treated with 500μmol/L H2O2 in different time. H2O2 induces phosphorylation of JNK. SP600125 enhanced cell viability and decreased apoptotic rate after H2O2-exposure. Conclusion：Apoptosis could be induced by H2O2 in ATⅡ cells in time-dependent manner. JNK signaling pathway may play a proapoptotic role in the regulation of apoptosis induced by H2O2.