超细炭黑诱导人支气管上皮细胞自噬和凋亡的分子机制及N-乙酰半胱氨酸的干预效果研究  

Study on the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells and the intervention effect of N-acetylcysteine

作　　者：孟涛 郭华杰 姚艳 米中华 田洋 尉杰忠[2] Meng Tao;Guo Huajie;Yao Yan;Mi Zhonghua;Tian Yang;Yu Jiezhong(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)

机构地区：[1]山西大同大学脑科学研究所/分子细胞免疫学大同市重点实验室,大同037009 [2]山西大同大学附属第一医院神经内科,大同037006

出　　处：《中华劳动卫生职业病杂志》2024年第9期656-667,共12页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金青年项目(81502845);山西省"四个一批"科技兴医创新计划项目(2021XM17);山西省基础研究计划项目(202103021224311);大学生创新创业训练计划项目(20220808、XDC2021135)。

摘　　要：目的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,并研究N-乙酰半胱氨酸(NAC)对超细炭黑致BEAS-2B细胞氧化损伤的干预效果和机制。方法于2023年3月,以BEAS-2B细胞为研究对象,构建超细炭黑暴露气道体外模型。实验先设对照组和3个炭黑暴露剂量组(50、100、200μg/ml),用相应浓度超细炭黑处理细胞24 h;再设对照组2、NAC+对照组、100μg/ml炭黑暴露组、NAC+暴露组,相应组别分别用2 mmol/L NAC处理细胞1 h和100μg/ml超细炭黑处理24 h。以CCK-8法检测细胞活力;化学荧光法检测细胞内活性氧(ROS)水平;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)活力及丙二醛(MDA)含量;荧光定量PCR法和免疫印迹法测定自噬相关基因[Atg5、Atg7、Beclin1、微管相关蛋白轻链3B(LC3B)、p62、溶酶体关联膜蛋白2(LAMP2)]、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)、多聚ADP核糖转移酶1(PARP1)]mRNA和蛋白相对表达;流式细胞术测定细胞凋亡情况。结果与对照组比较,50、100、200μg/ml炭黑暴露剂量组BEAS-2B细胞相对存活率明显降低,ROS和MDA水平明显增加,而SOD、GSH-Px和CAT活力明显降低(P<0.05),超细炭黑暴露剂量与细胞相对存活率、ROS和MDA水平以及SOD、GSH-Px和CAT活力呈明显相关性(r s=-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炭黑暴露剂量组Atg5、Atg7、Beclin1、LC3B、p62、LAMP2、Bax、Caspase3、Caspase9、PARP1 mRNA和Atg5、Atg7、Beclin1、LC3BⅡ、p62、LAMP2、Bax、裂解半胱天冬酶3(C-Caspase3)、裂解半胱天冬酶9(C-Caspase9)、裂解多聚ADP核糖转移酶1(C-PARP1)蛋白相对表达水平以及LC3BⅡ/LC3BⅠ比值明显增加,而Bcl-2 mRNA和蛋白相对表达水平明显降低(P<0.05),超细炭黑暴露剂量与上述指标的变化呈明显相�Objective 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.Methods 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(50,100,200μg/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,100μg/ml carbon black exposure group and NAC+exposure group.The corresponding groups were treated with 2 mmol/L NAC for 1 h and 100μg/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.Results Compared with the control group,the relative survival rates of BEAS-2B cells in 50,100,200μg/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(P<0.05).The relative survival rate,ROS and MDA levels,SOD,GSH-Px and CAT activities were significantly correlated with the expos

关 键 词：细胞凋亡 炭黑 超细颗粒物 支气管上皮细胞 氧化应激 自噬 

分 类 号：R392[医药卫生—免疫学]