活性氧簇调控细颗粒物诱导人支气管上皮细胞凋亡的机制研究

Mechanism of ROS Regulating PM_(2.5)-induced Apoptosis in Human Bronchial Epithelial Cells

目的探讨活性氧簇(reactive oxidative species,ROS)调控细颗粒物(PM2.5)诱导人支气管上皮(human bronchial epithelial,HBE)细胞凋亡的机制研究。方法分别以0 g/mL、100 g/mL、200 g/mL、400 g/mL的PM2.5刺激HBE细胞,24 h后用CCK-8法检测各组细胞活性,用DCFH-DA荧光标记法检测细胞内ROS生成情况,用Annexin VAlexa Fluor 488/PI双标记流式细胞术检测细胞凋亡率,用Western blot法检测凋亡相关蛋白Bax,Bcl-2,Caspase-3的表达变化。结果 CCK-8法结果显示PM2.5对HBE细胞活性有明显的抑制作用,并可诱导HBE细胞内ROS生成增多、细胞凋亡及凋亡相关蛋白Bax,Caspase-3表达增高,Bcl-2表达降低。此外,用N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)抑制ROS生成可以减轻PM2.5对HBE细胞活性的抑制及对HBE细胞凋亡的诱导,提示PM2.5引起的细胞凋亡与ROS有关。结论 PM2.5可诱导HBE细胞内ROS生成增多,进而促使HBE细胞凋亡,这可能是影响呼吸系统功能的机制之一。

Objective To investigate the mechanism of reactive oxidative species （ROS） regulating the apoptosis of human bronchial epithelial cells （HBE） induced by PM2.5.Methods HBE cells were stimulated with 0,100,200 and400 μg / mL PM2.5 for 24 h respectively,then the activity was detected by CCK-8,the generation of ROS in cells was detected by DCFH-DA fluorescent labeling,the apoptosis rate was detected by Annexin V-Alexa Fluor 488 / PI double-labeled flow cytometry,the apoptosis-related proteins expression of Bax,Bcl-2 and Caspase-3 were detected by Western blot.Results The results of CCK-8 assay showed that PM25 significantly inhibited the activity of HBE,induced the generation of ROS in HBEcellsandapoptosis,it also increased the expression ofBax,Caspase-3,and decrease the expression ofBcl-2;in addition,inhibition of ROS production with N-acetyl-L-cysteine （NAC） attenuated PM2 2-induced inhibition of HBE activity and apoptosis,suggesting that PM25-induced apoptosis was associated with ROS.Conclusions PM25 can induce the generation of ROS in HBE cells and then induce the apoptosis,which may be one of the mechanisms that affect the respiratory system function.