PARP-1在鱼藤酮诱导PC12细胞线粒体DNA损伤中的作用
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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.
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.
引文
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[18] CZAPSKI G A,CIE.Inhibition of poly(ADP-ribose) polymerase-1 alters expression of mitochondria-related genes in PC12 cells:relevance to mitochondrial homeostasis in neurodegenerative disorders[J].Biochim Biophys Acta Mol Cell Res,2018,1865(2):281-288.DOI:10.1016/j.bbamcr.2017.11.003.
[19] SZCZESNY B,BRUNYANSZKI A,OLAH G,et al.Opposing roles of mitochondrial and nuclear PARP1 in the regulation of mitochondrial and nuclear DNA integrity:implications for the regulation of mitochondrial function[J].Nucleic Acids Res,2014,42(21):13161-13173.DOI:10.1093/nar/gku1089.
[20] 王莹颖,刘文景,宁瑶,等.PARP抑制剂的作用机制和研究进展[J].中国新药杂志,2018,27(3):306-313.WANG Y Y,LIU W J,NING Y,et al.Progress in the research of PARP inhibitors and their mechanisms of action[J].Chin J New Drug,2018,27(3):306-313.
[21] SONI A,LI F H,WANG Y,et al.Inhibition of PARP1 by BMN673 effectively sensitizes cells to radiotherapy by upsetting the balance of repair pathways processing DNA double-strand breaks[J].Mol Cancer Ther,2018,17(10):2206-2216.DOI:10.1158/1535-7163.MCT-17-0836.
[22] EL AMKI M,LEROUET D,GARRAUD M,et al.Improved reperfusion andvasculoprotection by the poly(ADP-Ribose)Polymerase inhibitor PJ34 after stroke and thrombolysis in mice[J].Mol Neurobiol,2018,55(12):9156-9168.DOI:10.1007/s12035-018-1063-3.
[2] PENG K G,TAO Y,ZHANG J,et al.Resveratrol regulates mitochondrial biogenesis and Fission/Fusion to attenuate rotenone-induced neurotoxicity[J].Oxid Med Cell Longev,2016,2016:6705621.DOI:10.1155/2016/6705621.
[3] SILVA R,AGUIAR T Q,OLIVEIRA R,et al.Light exposure during growth increases riboflavin production,reactive oxygen species accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains[J].FEMS Yeast Res,2019,19(1).DOI:10.1093/femsyr/foy114.
[4] ZAPICO S C,UBELAKER D H.MtDNA mutations and their role in aging,diseases and forensic sciences[J].Aging Dis,2013,4(6):364-380.DOI:10.14336/AD.2013.0400364.
[5] BENDER A,KRISHNAN K J,MORRIS C M,et al.High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease[J].Nat Genet,2006,38(5):515-517.DOI:10.1038/ng1769.
[6] LANGELIER M F,SERVENT K M,ROGERS E E,et al.A third zinc-binding domain of human poly(ADP-ribose) polymerase-1 coordinates DNA-dependent enzyme activation[J].J Biol Chem,2008,283(7):4105-4114.DOI:10.1074/jbc.m708558200.
[7] ROSSI M N,CARBONE M,MOSTOCOTTO C,et al.Mitochondrial localization of PARP-1 requires interaction with mitofilin and is involved in the maintenance of mitochondrial DNA integrity[J].J Biol Chem,2009,284(46):31616-31624.DOI:10.1074/jbc.M109.025882.
[8] MARTIRE S,MOSCA L,D′ERME M.PARP-1 involvement in neurodegeneration:A focus on Alzheimer’s and Parkinson’s diseases[J].Mech Ageing Dev,2015,146-148:53-64.DOI:10.1016/j.mad.2015.04.001.
[9] 刘疏影,陈彪.帕金森病流行现状[J].中国现代神经疾病杂志,2016,16(2):98-101.DOI:10.3969/j.issn.1672-6731.2016.02.007.LIU S Y,CHEN B.Epidemiology of Parkinson’s disease[J].Chin J Contemp Neurol Neurosurg,2016,16(2):98-101.DOI:10.3969/j.issn.1672-6731.2016.02.007.
[10] WINKLHOFER K F,HAASS C.Mitochondrial dysfunction in Parkinson’s disease[J].Biochim Biophys Acta,2010,1802(1):29-44.DOI:10.1016/j.bbadis.2009.08.013.
[11] GREENAMYRE J T,BETARBET R,SHERER T B.The rotenone model of Parkinson’s disease:genes,environment and mitochondria[J].Parkinsonism Relat Disord,2003,9(Suppl 2):S59-S64.
[12] GAGNE J P,ROULEAU M,POIRIER G G.PARP-1 activation:bringing the pieces together[J].Science,2012,336(6082):678-679.DOI:10.1126/science.1221870.
[13] ZHEN Y L,YU Y H.Proteomic analysis of the downstream signaling network of PARP1[J].Biochemistry,2018,57(4):429-440.DOI:10.1021/acs.biochem.7b01022.
[14] HEGEDüS C,VIRáG L.Inputs and outputs of poly(ADP-ribosyl)ation:Relevance to oxidative stress[J].Redox Biol,2014,2:978-982.DOI:10.1016/j.redox.2014.08.003.
[15] 赵伟,王永伟,韦冠山,等.PARP-1介导的自噬流受阻在大鼠心肌细胞缺血再灌注损伤中的作用[J].南方医科大学学报,2018,38(8):975-979.DOI:10.3969/j.issn.1673-4254.2018.08.12.ZHAO W,WANG Y W,WEI G S,et al.Role of poly(ADP-ribose) polymerases-1-mediated blockade of autophagy in ischemia/reperfusion injury of rat cardiomyocytes[J].J South Med Univ,2018,38(8):975-979.DOI:10.3969/j.issn.1673-4254.2018.08.12.
[16] D?LLE C,FL?NES I,NIDO G S,et al.Defective mitochondrial DNA homeostasis in the substantia nigra in parkinson disease[J].Nat Commun,2016,7:13548.DOI:10.1038/ncomms13548.
[17] BRISTON T,HICKS A R.Mitochondrial dysfunction and neurodegenerative proteinopathies:mechanisms and prospects for therapeutic intervention[J].Biochem Soc Trans,2018,46(4):829-842.DOI:10.1042/BST20180025.
[18] CZAPSKI G A,CIE.Inhibition of poly(ADP-ribose) polymerase-1 alters expression of mitochondria-related genes in PC12 cells:relevance to mitochondrial homeostasis in neurodegenerative disorders[J].Biochim Biophys Acta Mol Cell Res,2018,1865(2):281-288.DOI:10.1016/j.bbamcr.2017.11.003.
[19] SZCZESNY B,BRUNYANSZKI A,OLAH G,et al.Opposing roles of mitochondrial and nuclear PARP1 in the regulation of mitochondrial and nuclear DNA integrity:implications for the regulation of mitochondrial function[J].Nucleic Acids Res,2014,42(21):13161-13173.DOI:10.1093/nar/gku1089.
[20] 王莹颖,刘文景,宁瑶,等.PARP抑制剂的作用机制和研究进展[J].中国新药杂志,2018,27(3):306-313.WANG Y Y,LIU W J,NING Y,et al.Progress in the research of PARP inhibitors and their mechanisms of action[J].Chin J New Drug,2018,27(3):306-313.
[21] SONI A,LI F H,WANG Y,et al.Inhibition of PARP1 by BMN673 effectively sensitizes cells to radiotherapy by upsetting the balance of repair pathways processing DNA double-strand breaks[J].Mol Cancer Ther,2018,17(10):2206-2216.DOI:10.1158/1535-7163.MCT-17-0836.
[22] EL AMKI M,LEROUET D,GARRAUD M,et al.Improved reperfusion andvasculoprotection by the poly(ADP-Ribose)Polymerase inhibitor PJ34 after stroke and thrombolysis in mice[J].Mol Neurobiol,2018,55(12):9156-9168.DOI:10.1007/s12035-018-1063-3.