炭黑对人支气管上皮细胞细胞器功能和凋亡的影响及机制
Effects and mechanism of exposure to carbon black on organelle function and apoptosis of human bronchial epithelial cells
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摘要:
背景 国际癌症研究机构（IARC）1996年把炭黑归类为人类可能致癌物，但炭黑暴露导致不良健康效应的机制仍不十分清楚，氧化应激通常被认为是机制之一，氧化应激下游可能进一步诱导了其他生物学事件的出现，如细胞器功能紊乱、凋亡等。
目的 本研究通过研究炭黑暴露对人支气管上皮细胞氧化应激、线粒体和溶酶体功能及凋亡的影响，探讨炭黑暴露导致不良健康效应的病理生理机制。
方法 体外培养人支气管上皮细胞Beas-2B，取对数生长期的细胞用于实验。细胞增殖实验设对照组和4个炭黑剂量组（25、50、100、200 μg/mL），其余实验均设对照组、3个炭黑剂量组（25、50、100 μg/mL）及N-乙酰半胱氨酸（NAC，2 mmol/L）+50 μg/mL炭黑组。用不同浓度的炭黑染毒液暴露24 h后，用CCK-8检测细胞的存活率，用特异性荧光探针及高内涵筛选系统检测并分析细胞内活性氧（ROS）、线粒体膜电位（MMP）及溶酶体pH值水平，用流式细胞术检测细胞凋亡率。
结果 50、100、200 μg/mL炭黑剂量组与对照组相比，细胞存活率下降（均P < 0.05）。50、100 μg/mL炭黑剂量组与对照组相比，细胞内ROS水平升高，MMP水平下降，溶酶体pH值升高，细胞凋亡率升高（均P < 0.05）。此外，NAC+50 μg/mL炭黑组与单纯50 μg/mL炭黑剂量组相比，细胞内ROS水平下降，MMP水平升高，溶酶体pH值和细胞凋亡率下降（均P < 0.05）。
结论 炭黑暴露人支气管上皮细胞引起氧化应激反应，而氧化应激又引起了线粒体MMP水平下降及溶酶体pH值的上升，上述细胞器功能的紊乱可能进一步诱导了细胞凋亡的发生。
Abstract:Background Carbon black was classified as a possible carcinogen to human by the International Agency for Research on Cancer (IARC) in 1996, but the mechanism by which it causes adverse health effects is still not fully understood. Oxidative stress is usually considered as one of the mechanisms involved, which might further trigger some biological events like organelle dysfunction or apoptosis.
Objective This study is designed to investigate the effects of carbon black on oxidative stress, mitochondria, lysosomes, and apoptosis in human bronchial epithelial cells, aiming to understand the pathophysiological mechanism by which carbon black induces adverse health outcomes.
Methods In vitro cultured human bronchial epithelial cells Beas-2B in logarithmic growth phase were prepared for the following experiments. One control group and four carbon black groups (25, 50, 100, and 200 μg/mL) were set for cell viability assay, and one control group, three carbon black groups (25, 50, and 100 μg/mL), and one N-acety-L-cysteine (NAC, 2 mmol/L) + 50 μg/mL carbon black group for the rest assays. After exposure to designed concentrations of carbon black for 24 h, the cell viability was determined by CCK-8; the intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and lysosomal pH were detected by high content screening system with specific molecular probes; the apoptosis rate was examined by flow cytometry.
Results The cell viabilities of the 50, 100, and 200 μg/mL carbon black groups were decreased significantly compared with the control group (Ps < 0.05). The intracellular ROS, lysosomal pH, and apoptosis rates were increased while the MMP levels were decreased in the 50 and 100 μg/mL carbon black groups compared with the control group (Ps < 0.05). The intracellular ROS, lysosomal pH, and apoptosis rate were decreased while the MMP level was increased in the NAC + 50 μg/mL carbon black group compared with the 50 μg/mL carbon black group (Ps < 0.05).
Conclusion Carbon black exposure could induce oxidative stress in human bronchial epithelial cells, and oxidative stress could subsequently cause a decrease in MMP and an increase in lysosomal pH; such organelle dysfunction might further trigger cell apoptosis.
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