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[目的]探讨氯乙酸对人支气管上皮细胞16HBE氧化应激以及线粒体凋亡通路的影响。[方法]以16HBE细胞为研究对象,将实验分为对照组和0.5、1.0、1.5、2.0、2.5 mmol/L的氯乙酸染毒组,染毒24 h后检测细胞存活率、凋亡率及超氧化物歧化酶(SOD)活力;染毒0.25、0.5、1、8、24 h后检测细胞内活性氧(ROS)水平;染毒8、24 h后检测线粒体膜电位以及Bcl-2和Bax m RNA表达水平。用1.5、2.5 mmol/L氯乙酸染毒24 h后,检测Bcl-2、Bax、细胞色素C、Caspase-9、Caspase-3、PARP-1凋亡相关蛋白的表达水平。[结果]随氯乙酸染毒浓度升高,染毒24 h后细胞存活率、SOD活力和线粒体膜电位呈剂量依赖性下降(r=-0.902、-0.732和-0.863,P<0.05),而细胞凋亡率呈剂量依赖性上升(r=0.914,P<0.05)。与对照组相比,1.5、2.0、2.5 mmol/L剂量组细胞存活率分别降低了12%、20%和30%,SOD活力分别降低了9%、21%和30%,线粒体膜电位分别降低了11%、18%和24%,而细胞凋亡率分别是对照组的3、4和7倍。各剂量组细胞内ROS水平呈先升高后降低的趋势,在染毒0.5 h达到峰值,与对照组相比分别增加了15%、35%、48%和55%(P<0.05),染毒1 h后反而降低。染毒各时间点染毒浓度与ROS水平均存在剂量-效应关系(r=0.756、0.893、0.735、0.667和0.653,均P<0.05);相关性分析表明染毒24 h后ROS水平与细胞凋亡率呈明显正相关(r=0.826,P<0.05)。染毒8、24 h两个时间点,染毒浓度与Bcl-2、Bax m RNA表达量存在剂量效应关系(8 h:r=-0.634和0.754,24 h:r=-0.773和0.823,P<0.05);与对照组相比,染毒24 h后染毒浓度≥1.5 mmol/L时Bcl-2 m RNA和蛋白表达明显下调,而Bax m RNA和蛋白表达明显上调(P<0.05);相关性分析表明Bax m RNA表达与ROS水平具有正相关(r=0.886和0.824,P<0.05),而Bcl-2m RNA表达与ROS水平具有负相关(r=-0.862和-0.815,P<0.05)。与对照组相比,染毒24 h后染毒浓度2.5 mmol/L时细胞色素C、活化的Caspase-3和Caspase-9蛋白表达量显著增加,而PARP-1蛋白表达量明显下降(P<0.05)。[结论]氯乙酸可致16HBE细胞发生氧化应激,在其通过氧化应激诱导细胞凋亡过程中可上调促凋亡蛋白Bax而下调抗凋亡蛋白Bcl-2表达,进一步激活线粒体依赖的凋亡通路,最终诱导细胞凋亡的发生。
[Objective] To investigate the effects of chloroacetic acid on 16HBE oxidative stress and mitochondrial apoptosis pathway in human bronchial epithelial cells. [Methods] With 16HBE cells as the research object, the experiment was divided into the control group and 0.5, 1.0, 1.5, 2.0, 2.5 mmol / L chloroacetic acid exposure group. The cell viability, The activity of superoxide dismutase (SOD) was measured. The level of reactive oxygen species (ROS) in cells was detected at 0.25,0.5,1,8,24 h after exposure. The mitochondrial membrane potential, Bcl-2 and Bax m RNA expression level. The expressions of Bcl-2, Bax, Cytochrome C, Caspase-9, Caspase-3 and PARP-1 were detected by 24 h treatment with 1.5,2.5 mmol / L chloroacetic acid. [Result] Cell viability, SOD activity and mitochondrial membrane potential decreased in a dose-dependent manner (r = -0.902, -0.732 and -0.863, P <0.05) at 24 h after exposure to chloroacetic acid. Apoptosis rate increased in a dose-dependent manner (r = 0.914, P <0.05). Compared with the control group, the cell viability decreased by 12%, 20% and 30%, and the SOD activity decreased by 9%, 21% and 30% respectively, the mitochondrial membrane potential decreased 11%, 18% and 24%, respectively, whereas the apoptotic rates were 3, 4 and 7 times that of the control group, respectively. The intracellular ROS levels increased firstly and then decreased in each dose group, reached the peak at 0.5 h after exposure, increased by 15%, 35%, 48% and 55% (P <0.05) compared with the control group, respectively Decreased after 1 h of poisoning. There was a dose-effect relationship between exposure concentration and ROS level at each time point (r = 0.756,0.893,0.735,0.667 and 0.653, all P <0.05). Correlation analysis showed that ROS level and apoptosis There was a significant positive correlation (r = 0.826, P <0.05). At 8 and 24 h of exposure, there was dose-effect relationship between the concentration of Bcl-2 and Bax m RNA (8 h: r = -0.634 and 0.754, 24 h: r = -0.773 and 0.823, P <0.05). Compared with the control group, Bcl-2 mRNA and protein expressions were significantly down-regulated and Bax m RNA and protein expressions were significantly up-regulated (P <0.05) at 24 h after exposure to ≥1.5 mmol / Correlation analysis showed that there was a positive correlation between Bax m RNA expression and ROS level (r = 0.886 and 0.824, P <0.05), while Bcl-2m RNA expression had a negative correlation with ROS level (r = -0.862 and -0.815, P <0.05 ). Compared with the control group, the expression of cytochrome C, activated Caspase-3 and Caspase-9 protein increased significantly and the expression of PARP-1 protein decreased significantly after exposure to 2.5 mmol / L for 24 h (P < 0.05). [Conclusion] Chloroacetic acid can induce oxidative stress in 16HBE cells, up-regulate pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 in oxidative stress-induced apoptosis, and further activate mitochondria-dependent apoptosis Death pathway, and ultimately induce the occurrence of apoptosis.