百草枯通过激活线粒体凋亡通路诱导人肺Ⅱ型上皮样A549细胞凋亡

Mitochondria-mediated Apoptosis in Human Lung Type Ⅱ Alveolar Epithelial-like A549 Cells by Paraquat

目的探讨百草枯(PQ)诱导人肺Ⅱ型上皮样A549细胞凋亡过程中线粒体凋亡通路的发生机制。方法体外培养A549细胞,对照组加入RPMI 1640培养液,实验组加入不同浓度的PQ(50、100、150和200μmol/L),2组细胞继续培养24和48 h。MTT法检测细胞活性;Hoechst 33258染色法观察细胞核的形态;流式细胞术检测细胞凋亡率以及线粒体膜电位;分光光度法检测caspase-3和caspase-9的活化程度;Western blotting检测线粒体凋亡相关蛋白Bcl-2、Bcl-x L、Bax和Bak的表达。结果 MTT结果显示,PQ对A549细胞具有显著的生长抑制作用。Hoechst染色显示,不同浓度的PQ作用于A549细胞24和48 h后,可发现细胞凋亡的特征性改变,如细胞核固缩、核碎裂、凋亡小体形成,并且细胞的凋亡程度随着PQ浓度的增大和作用时间的延长而加重。细胞凋亡率升高也证实了这一结果。线粒体膜电位出现不同程度的降低;caspase-3和caspase-9活性不同程度的增加;抗凋亡蛋白Bcl-2、Bcl-x L的表达量降低;促凋亡蛋白Bax、Bak的表达量升高。以上结果均呈时间与浓度依赖性。结论 PQ通过激活线粒体凋亡通路诱导A549细胞凋亡。

Objective To investigate the apoptosis mechanism induced by paraquat （PQ） in human type ]I alveolar epithelial-like A549 cells. Methods A549 cells were cultured in vitro. The cells in the experimental group were exposed to various concentrations of PQ （50, 100,150,and 200 μmol/L）,while those in the control group were cultured in RPM11640 medium. After treatment for 24 and 48 h,the cell survival rate was assessed by MTT assay. Morphological changes in the nuclei were observed by Hoechst 33258 fluorescence staining. Cellular apoptosis and mitochondrial transmembrane potential were assayed by flow cytometry. The activities of caspase-3 and caspase-9 were assayed by spectrophotometry. Western blotting was used to analyze the expression of proteins in the Bcl-2 family, such as Bcl-2, Bcl-xL, Bax, and Bak. Results PQ exhibited significant anti-proliferative activity in A549 cells. PQ-treated A549 cells were subjected to Hoechst 33258 staining. The hallmarks of apoptosis were detected, and the degree of apoptosis increased. Mitochondrial membrane po- tential was decreased, the levels of active caspase-3 and caspase-9 increased, the expression of Bcl-2 and Bcl-xL was decreased, and the expression of Bax and Bak was increased. These effects occurred in concentration- and time-dependent manners. Conclusion PQ effi- ciently induced intracellular apoptosis through the mitochondrial pathway in A549 cells.