孟涛, 苗盼盼, 杨墨, 贾强, 戴宇飞. 氯乙酸致人支气管上皮细胞16HBE的凋亡机制研究[J]. 环境与职业医学, 2016, 33(9): 858-864. DOI: 10.13213/j.cnki.jeom.2016.16346
引用本文: 孟涛, 苗盼盼, 杨墨, 贾强, 戴宇飞. 氯乙酸致人支气管上皮细胞16HBE的凋亡机制研究[J]. 环境与职业医学, 2016, 33(9): 858-864. DOI: 10.13213/j.cnki.jeom.2016.16346
MENG Tao, MIAO Pan-pan, YANG Mo, JIA Qiang, DAI Yufei. Apoptosis Mechanism of Human Bronchial Epithelial 16HBE Cells Induced by Chloroacetic Acid[J]. Journal of Environmental and Occupational Medicine, 2016, 33(9): 858-864. DOI: 10.13213/j.cnki.jeom.2016.16346
Citation: MENG Tao, MIAO Pan-pan, YANG Mo, JIA Qiang, DAI Yufei. Apoptosis Mechanism of Human Bronchial Epithelial 16HBE Cells Induced by Chloroacetic Acid[J]. Journal of Environmental and Occupational Medicine, 2016, 33(9): 858-864. DOI: 10.13213/j.cnki.jeom.2016.16346

氯乙酸致人支气管上皮细胞16HBE的凋亡机制研究

Apoptosis Mechanism of Human Bronchial Epithelial 16HBE Cells Induced by Chloroacetic Acid

  • 摘要: 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 mRNA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 mRNA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 mRNA和蛋白表达明显下调,而Bax mRNA和蛋白表达明显上调(P<0.05);相关性分析表明Bax mRNA表达与ROS水平具有正相关(r=0.886和0.824,P<0.05),而Bcl-2mRNA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。

     

    Abstract: Objective

    To examine the chloroacetic acid induced oxidative stress and the related effect on mitochondrial pathway related apoptosis in human normal bronchial epithelial 16HBE cells.

    Method

    Cell viability, apoptosis, and superoxide dismutase (SOD) activity were determined in 16HBE cells exposed to 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mmol/L chloroacetic acid for 24 h in vitro. Reactive oxygen species (ROS) were assayed after 16HBE cells were treated with the above-mentioned concentrations for 0.25, 0.5, 1, 8, 24 h, respectively. Mitochondrial membrane potential and expression levels of Bcl-2 and Bax mRNA were measured after 8 and 24 h of treatment. Expression levels of apoptosis-related proteins including Bcl-2, Bax, cytochrome C, Caspase-9, Caspase-3, and PARP-1 were measured after 16HBE cells treated with 1.5 and 2.5 mmol/L chloroacetic acid for 24 h.

    Result

    The cell viability rate, SOD activity, and mitochondrial membrane potential decreased in a dose-dependent manner with increasing concentrations of chloroacetic acid (r=-0.902, -0.732, and -0.863, respectively, P<0.05); but the cell apoptosis rate increased in a dose-dependent manner (r=0.914, P<0.05). In comparison with the control group, remarkable reductions of 12%, 20%, and 30% for cell viability, 9%, 21%, and 30% for SOD activity, and 11%, 18%, and 24% for mitochondrial membrane potential were found in the 1.5, 2.0, and 2.5 mmol/L chloroacetic acid treated groups (P<0.05) respectively; moreover, the cell apoptosis rates were 3, 4, and 7 times of the control group respectively. The intracellular ROS levels increased first and then decreased; peak values shown after 0.5 h of exposure in the 1.0, 1.5, 2.0, 2.5 mmol/L treatment groups, and significantly increased by 15%, 35%, 48%, and 55% respectively in comparison with the controls (P<0.05); the ROS levels in various concentration groups gradually dropped after 1h. There were significant dose-response relationships between ROS levels and concentrations of chloroacetic acid at each designed time point of exposure (r=0.756, 0.893, 0.735, 0.667, and 0.653, P<0.05). There was a distinct positive correlation between cell apoptosis rate and ROS level (r=0.826, P<0.05). After 8 and 24 h of exposure, the Bcl-2 and Bax mRNA expression levels both showed dose-response relationships with exposure concentrations (r=-0.634 and 0.754 at 8 h, r=-0.773 and 0.823 at 24 h, P<0.05). After 24 h of exposure, there was a significant decrease of the Bcl-2 mRNA and protein expression levels but a remarkable increase of the Bax mRNA and protein expression levels in 1.5 and 2.5 mmol/L treatment groups in contrast to the controls (P<0.05). There were significant positive correlations between the ROS level and the mRNA expression level of Bax (r=0.886 and 0.824, P<0.05), and negative correlations between the ROS level and the mRNA expression level of Bcl-2 (r=-0.862 and -0.815, P<0.05). After 24 h of exposure, compared with the control group, the protein expression levels of cytochrome C, activated Caspase-3, and activated Caspase-9 were distinctly increased and the PARP-1 protein level was significantly decreased in the 2.5 mmol/L chloroacetic acid treated group (P<0.05).

    Conclusion

    Chloroacetic acid could trigger oxidative stress in 16HBE cells and result in cell apoptosis via activating mitochondrial pathway with elevated expression level of Bax and decreased expression level of Bcl-2.

     

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