摘要
目的探讨PARP-1在鱼藤酮(rotenone)诱导大鼠PC12细胞线粒体DNA(mtDNA)损伤中的作用。方法不同浓度(0、0.1、0.5、1.0、1.5μmol/L)鱼藤酮处理PC12细胞,TaqMan探针法PCR检测mtDNA缺失,qPCR法检测mtDNA拷贝数,Western blot检测PARP-1蛋白在PC12细胞内和线粒体内的表达水平。PARP-1选择性抑制剂PJ34预处理鱼藤酮染毒的PC12细胞,采用CCK-8法检测细胞增殖活力,TaqMan探针法PCR检测mtDNA缺失,qPCR法检测mtDNA拷贝数,四甲基罗丹明乙酯(TMRM)染色检测线粒体膜电位,DCFH-DA探针检测细胞内活性氧(reactive oxygen species,ROS)水平。结果 TaqMan探针法PCR和qPCR法检测结果显示,与对照组(0μmol/L鱼藤酮)比较,鱼藤酮处理可引起PC12细胞线粒体DNA缺失增加和线粒体DNA拷贝数降低(P<0.05)。同时,细胞和线粒体内的PARP-1蛋白表达被激活,Western bolt检测结果显示,PARP-1蛋白表达水平随鱼藤酮浓度增加而增加。与鱼藤酮单独处理组比较,PJ34预处理可提高鱼藤酮染毒引起的PC12细胞的活力下降(P<0.05),并降低线粒体DNA缺失和提高线粒体DNA拷贝数水平(P<0.05)。TMRM染色结果显示,PJ34预处理后线粒体膜电位水平提高(P<0.05);DCFH-DA探针检测结果显示,PJ34预处理后细胞内ROS水平下降(P<0.05)。结论鱼藤酮可诱导PC12细胞线粒体DNA的损伤,激活PC12细胞和线粒体内PARP-1蛋白的表达,PARP-1选择性抑制剂PJ34对鱼藤酮诱导的多巴胺神经元PC12细胞线粒体DNA的损伤具有保护作用。
Objective To explore the effect of poly(ADP-ribose)polymerase-1(PARP-1)on mitochondrial DNA(mtDNA)damage induced by rotenone in rat adrenal medulla pheochromocytoma PC12 cells.Methods PC12 cells were treated with various dose of rotenone.The mtDNA common deletion(CDs)was determined by TaqMan assay,and mtDNA copy number was detected by quantitative polymerase chain reaction(qPCR).Western blotting was performed to detect the protein levels of PARP-1 in whole cell and mitochondria.After the rotenone exposed PC12 cells were treated with PJ34(PARP-1 selective inhibitor),cell viability was detected by CCK-8 assay,mitochondrial membrane potential was examined by TMRM staining,intracellular reactive oxygen species(ROS)level was tested by 2,7-dichlorofluorescin diacetate(DCFH-DA)probe.Results TaqMan and qPCR assays showed that rotenone treatment induced increased mtDNA deletion and decreased mtDNA copy number in PC12 cells when compared to control cells(P<0.05).Meanwhile,PARP-1 was activated by rotenone treatment in the whole cells and mitochondria,and Western boltting showed that the protein level of PARP-1 was upregulated in a dose-dependent manner.Furthermore,compared with the rotenone group,PJ34 pretreatment enhanced the cell viability significantly in PC12 cells(P<0.05),abolished mtDNA CDs and increased mtDNA copy number(P<0.05).Additionally,PJ34 pretreatment caused the improvement of mitochondrial membrane potential and reduced ROS production when compared with the rotenone cells(P<0.05).Conclusion Rotenone induces mitochondrial DNA damage,which further activates the expression of PARP-1 protein,especially mitochondria in PC12 cells.And PJ34,as the PARP-1 selective inhibitor has a protective effect on rotenone-induced mitochondrial DNA damage in PC12 cells.
作者
肖靖淞
侯贵钟
赛燕
张爱华
曹佳
XIAO Jingsong;HOU Guizhong;SAI Yan;ZHANG Aihua;CAO Jia(Department of Toxicology,College of Public Health,Guizhou Medical University,Guiyang,Guizhou Province,550025;Institute of Toxicology,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2019年第8期778-785,共8页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81473006)~~
