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目的研究活性氧/c-Jun氨基末端激酶(ROS/JNK)途径在四川新康温石棉诱导A549人肺上皮细胞凋亡过程的作用。方法采用(12.5、25、50、100、200)μg/m L温石棉作用A549细胞24 h,MTT法检测细胞存活率;设置ROS抑制组,异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双染色结合流式细胞术检测细胞凋亡、JC-1标记检测线粒体膜电位、二氯二氢荧光黄乙酰乙酯(DCFH-DA)负载检测ROS水平,Western blot法检测磷酸化的c-JNK1(p-JNK1)、p-JNK2、裂解型胱天蛋白酶3(c-caspase-3)蛋白水平。结果随着温石棉剂量增大,细胞存活率下降,温石棉半数抑制剂量为223.43μg/m L;细胞凋亡率呈现剂量-效应关系,胞内ROS水平升高,p-JNK1、p-JNK2、c-caspase-3蛋白水平明显上调,线粒体膜电位降低;与相同剂量温石棉组比较,ROS抑制剂能显著抑制细胞凋亡,降低ROS水平,减少线粒体膜电位的降低程度,并下调p-JNK1、p-JNK2和c-caspase-3蛋白的表达。结论四川新康温石棉可能通过ROS/JNK途径诱导A549细胞发生凋亡。
Objective To investigate the role of reactive oxygen species / c-Jun N-terminal kinase (ROS / JNK) pathway in the apoptosis of A549 human lung epithelial cells induced by Asconsin in Sichuan. Methods A549 cells were treated with chrysotile (12.5, 25, 50, 100, 200) μg / m L for 24 h and the cell viability was determined by MTT assay. ROS inhibition group, fluorescein isothiocyanate labeled Ⅴ / Apoptosis was detected by double staining of annexinⅤ-FITC / PI and flow cytometry. The mitochondrial membrane potential was measured by JC-1 labeling, and ROS level was detected by DCFH-DA loading The protein levels of phosphorylated c-JNK1 (p-JNK1), p-JNK2 and c-caspase-3 were detected by Western blot. Results As the dosage of chrysotile asbestos increased, the cell viability decreased, and the half dose of chrysotile asbestos was 223.43μg / m L; the rate of apoptosis was dose-dependent and the level of intracellular ROS was increased. The expressions of p-JNK1, p-JNK2 , The level of c-caspase-3 protein was significantly increased and the mitochondrial membrane potential was decreased. Compared with the same dose of chrysotile, ROS inhibitor significantly inhibited cell apoptosis, decreased ROS level, decreased the mitochondrial membrane potential and decreased the expression of p- JNK1, p-JNK2 and c-caspase-3 protein expression. Conclusions Asbestos may induce apoptosis in A549 cells via ROS / JNK pathway.