Meng T, Guo H J, Yao Y, Mi Z H, Tian Y, Yu J Z
Institute of Brain Science/The Datong Key Laboratory of Molecular and Cellular Immunology, Shanxi Datong University, Datong 037009, China.
Department of Neurology, First Affiliated Hospital of Shanxi Datong University, Datong 037006, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2024 Sep 20;42(9):656-667. doi: 10.3760/cma.j.cn121094-20231010-00080.
To investigate the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells (BEAS-2B cells), and to study the intervention effect and mechanism of N-acetylcysteine (NAC) on ultrafine carbon black-induced oxidative damage in BEAS-2B cells. In March 2023, BEAS-2B cells were used as research object, an in vitro airway model exposed to ultrafine carbon black was constructed. A control group and three carbon black exposure groups (50, 100, 200 μg/ml) were set up, and the cells were treated with corresponding concentrations of ultrafine carbon black for 24 hours. In addition, the experiment was divided into control group, NAC+ control group, 100 μg/ml carbon black exposure group and NAC+ exposure group. The corresponding groups were treated with 2 mmol/L NAC for 1 h and 100 μg/ml ultrafine carbon black for 24 h, respectively. Cell viability was measured by CCK-8 assay. Intracellular reactive oxygen species (ROS) level was detected by chemical fluorescence method. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), as well as the content of malondialdehyde (MDA) were detected by colorimetry. The mRNA and protein expressions of autophagy-related genes[Atg5, Atg7, Beclin1, microtubule-associated protein light chain 3B (LC3B), p62 and lysosome-associated membrane protein 2 (LAMP2) ] and apoptosis-related genes [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase3, Caspase9 and poly (ADP-ribose) polymerase 1 (PARP1) ] were determined by fluorescence quantitative PCR and Western blot. Cell apoptosis was determined by flow cytometry. Compared with the control group, the relative survival rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly decreased, the levels of ROS and MDA were significantly increased, and the activities of SOD, GSH-Px and CAT were significantly decreased (<0.05). The relative survival rate, ROS and MDA levels, SOD, GSH-Px and CAT activities were significantly correlated with the exposure dose of ultrafine carbon black ((s)=-0.755, 0.826, 0.934, -0.810, -0.880, -0.840, <0.05). Compared with the control group, the relative expression levels of Atg5, Atg7, Beclin1, LC3B, p62, LAMP2, Bax, Caspase3, Caspase9, PARP1 mRNA and Atg5, Atg7, Beclin1, LC3BⅡ, p62, LAMP2, Bax, cleaved Caspase3 (C-Caspase3), cleaved Caspase9 (C-Caspase9), cleaved PARP1 (C-PARP1) protein and the ratio of LC3BⅡ/LC3BⅠ in 50, 100 and 200 μg/ml carbon black exposure groups were significantly increased, while the relative expression levels of Bcl-2 mRNA and protein were significantly decreased (<0.05). The changes of the above indexes were significantly correlated with the exposure dose of carbon black ((s)=0.892, 0.879, 0.944, 0.892, 0.828, 0.880, 0.814, 0.794, 0.931, 0.918, 0.813, 0.866, 0.774, 0.695, 0.918, 0.761, 0.794, 0.944, 0.833, 0.866, 0.905, -0.886, -0.748, <0.05). Compared with 100 μg/ml carbon black exposure group, the relative survival rate, the activities of SOD, GSH-Px and CAT in NAC+exposure group were significantly increased, while the levels of ROS and MDA were significantly decreased, and the relative expression levels of LC3B, p62 and Caspase3 mRNA and protein as well as the ratio of LC3BⅡ/LC3BⅠ were significantly decreased, and the differences were statistically significant (<0.05). Compared with the control group, the apoptosis rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly increased (<0.05), and there was a significant positive correlation between ultrafine carbon black exposure dose and cell apoptosis rate ((s)=0.944, <0.05). While compared with 100 μg/ml carbon black exposure group, the apoptosis rate of NAC+exposure group was significantly decreased, and the difference was statistically significant (<0.05) . Cell autophagy and apoptosis may be important pathophysiological mechanisms of ultrafine carbon black-induced oxidative damage in BEAS-2B cells. NAC can alleviate the occurrence of BEAS-2B cell damage caused by ultrafine carbon black by regulating oxidative stress and the cascading autophagy and apoptosis pathways.
探讨超细炭黑诱导人支气管上皮细胞（BEAS-2B细胞）自噬和凋亡的分子机制，研究N-乙酰半胱氨酸（NAC）对超细炭黑诱导的BEAS-2B细胞氧化损伤的干预作用及机制。2023年3月，以BEAS-2B细胞为研究对象，构建暴露于超细炭黑的体外气道模型。设对照组和3个炭黑暴露组（50、100、200μg/ml），用相应浓度的超细炭黑处理细胞24小时。此外，实验分为对照组、NAC+对照组、100μg/ml炭黑暴露组和NAC+暴露组。相应组分别用2mmol/L NAC处理1小时和100μg/ml超细炭黑处理24小时。采用CCK-8法检测细胞活力。用化学荧光法检测细胞内活性氧（ROS）水平。用比色法检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）的活性以及丙二醛（MDA）含量。用荧光定量PCR和蛋白质印迹法检测自噬相关基因[自噬相关蛋白5（Atg5）、自噬相关蛋白7（Atg7）、Beclin1、微管相关蛋白轻链3B（LC3B）、p62和溶酶体相关膜蛋白2（LAMP2）]和凋亡相关基因[B细胞淋巴瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）、半胱天冬酶3（Caspase3）、半胱天冬酶9（Caspase9）和聚（ADP-核糖）聚合酶1（PARP1）]的mRNA和蛋白表达。用流式细胞术检测细胞凋亡。与对照组比较，50、100、200μg/ml炭黑暴露组BEAS-2B细胞相对存活率显著降低，ROS和MDA水平显著升高，SOD、GSH-Px和CAT活性显著降低（P<0.05）。相对存活率、ROS和MDA水平、SOD、GSH-Px和CAT活性与超细炭黑暴露剂量显著相关（r=-0.755、0.826、0.934、-0.810、-0.880、-0.840，P<0.05）。与对照组比较，50、100、200μg/ml炭黑暴露组Atg5、Atg7、Beclin1、LC3B、p62、LAMP2、Bax、Caspase3、Caspase9、PARP1 mRNA及Atg5、Atg7、Beclin1、LC3BⅡ、p62、LAMP；Bax、裂解的半胱天冬酶3（C-Caspase3）、裂解的半胱天冬酶9（C-Caspase9）、裂解的PARP1（C-PARP1）蛋白相对表达水平及LC3BⅡ/LC3BⅠ比值显著升高，而Bcl-2 mRNA和蛋白相对表达水平显著降低（P<0.05）。上述指标变化与炭黑暴露剂量显著相关（r=0.892、0.879、0.944、0.892、0.828、0.880、0.814、0.794、0.931、0.918、0.813、0.866、0.774、0.695、0.918、{0.761、0.794、0.944、0.833、0.866、0.905、-0.886、-0.748，P<0.05}）。与100μg/ml炭黑暴露组比较，NAC+暴露组相对存活率、SOD、GSH-Px和CAT活性显著升高，而ROS和MDA水平显著降低，LC3B、p62和Caspase3 mRNA和蛋白相对表达水平及LC3BⅡ/LC3BⅠ比值显著降低，差异有统计学意义（P<0.05）。与对照组比较，50、100、200μg/ml炭黑暴露组BEAS-2B细胞凋亡率显著升高（P<0.05），超细炭黑暴露剂量与细胞凋亡率呈显著正相关（r=0.944，P<0.05）。而与100μg/ml炭黑暴露组比较，NAC+暴露组凋亡率显著降低，差异有统计学意义（P<0.05）。细胞自噬和凋亡可能是超细炭黑诱导BEAS-2B细胞氧化损伤的重要病理生理机制。NAC可通过调节氧化应激及自噬和凋亡级联途径减轻超细炭黑所致BEAS-2B细胞损伤的发生。
