BPDE诱导GC-1细胞凋亡及褪黑素的保护作用  

BPDE induces apoptosis while melatonin protects in spermatogonial GC-1 cells

作　　者：姜帆[1] 张国伟[1] 敖琳[1] 曹佳[1] JIANG Fan;ZHANG Guowei;AO Lin;CAO Jia(Institute of Toxicology, College of Military Preventive Medicine, Third Military Medical University, Chongqing, 400038, Chin)

机构地区：[1]第三军医大学军事预防医学院毒理学研究所,重庆400038

出　　处：《第三军医大学学报》2017年第23期2323-2328,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金重点项目(81130051)~~

摘　　要：目的探讨7,8-二羟-9,10-环氧苯并[a]芘(benzo[a]pyrene-7,8-diol-9,10-epoxide,BPDE)对小鼠精原细胞株GC-1的细胞毒性机制及褪黑素的保护作用。方法不同浓度BPDE处理GC-1细胞,采用Annexin V/PI染色及流式细胞仪检测细胞凋亡,JC-1探针染色检测线粒体膜电位,Western blot检测细胞质Cyt C及细胞总蛋白中caspase-9/3的活化水平,DCFH-DA探针及流式细胞仪检测细胞内的活性氧(reactive oxygen species,ROS)水平。褪黑素预处理细胞后再进行BPDE染毒,采用上述方法检测褪黑素对BPDE细胞毒性的影响。结果 BPDE可提高GC-1细胞凋亡率,存在剂量-效应关系。同时,BPDE可降低细胞线粒体膜电位,促进线粒体Cyt C的释放,提高细胞中caspase-9和caspase-3蛋白的活化水平,并诱导细胞中ROS水平升高。褪黑素预处理则可降低BPDE诱导的细胞凋亡,抑制Cyt C的释放及caspase-9/3的活化,并激活Nrf-2/ARE抗氧化通路,降低细胞ROS水平。结论BPDE可诱导小鼠精原细胞GC-1线粒体途径细胞凋亡和氧化应激,而褪黑素在这一过程中起保护作用。Objective To explore the mechanism of benzo[a]pyrene-7,8-diol-9,10-epoxide （BPDE）induced cytotoxicity in mouse spermatogonial GC-1 cells and the protective role of melatonin in the process. Methods GC-1 cells were treated with various doses of BPDE. Then, cell apoptosis was detected using Annexin V/PI staining and flow cytometry analysis, and mitochondrial membrane potential was examined by JC-1 staining. After the proteins in the cytoplasm and whole cells were extracted separately, the expression of Cyt C, active caspase-9 and active caspase-3 was measured using Western blot assay. 2,7-dichloro-fluorescin diacetate （DCFH-DA） probe and flow cytometry were used to measure intracellular reactive oxygen species （ROS） level. The effect of melatonin pretreatment on the induced cytotoxicity in GC-1 cells was also studied. Results BPDE induced cell apoptosis in GC-1 cells in a dose-dependent manner. BPDE treatment also resulted in decrease in the mitochondrial membrane potential, enhancement in the release of cytoplasmic Cyt C from mitochondria, increases in the expression levels of active caspase-9 and -3, and elevation in intracellular ROS level in the GC-1 cells. Furthermore, pretreatment of melatonin significantly inhibited BPDE-induced apoptosis, release of cytoplasmic Cyt C and activation of caspase-9, and activate the Nrf-2/ARE antioxidant pathway and abolished the increase of ROS in GC-1 cells. Conclusion BPDE induces mitochondria-dependent apoptosis and oxidative stress in GC-1 cells, while melatonin protects the cells in the process.

关 键 词：7 8-二羟-9 10-环氧苯并[a]芘 褪黑素 活性氧 细胞凋亡 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学] R977.1[医药卫生—基础医学]