原花青素对鱼藤酮诱导PC12细胞损伤的保护作用及机制研究
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摘要
帕金森症(Parkinsons disease,PD)是一种老年人群常见的中枢神经系统变性病,因发病率明显上升,其病因与防治策略亟待阐明.文中以多巴胺能神经元模型细胞PC12为材料,以致PD毒性物质鱼藤酮(Rotenone,Rot)处理细胞,检测原花青素(Grape seeds procyanidins,GSP)对Rot引起细胞毒性的防护作用,并探讨其机理.MTT法显示,GSP可减弱Rot对细胞生长的抑制作用,其效果具有浓度和时间依赖性;倒置显微镜观察吉姆萨染色和荧光染料Hoechst 33342染色后显示,Rot处理细胞由平铺多角形变为皱缩,突起回缩,细胞核染色质凝集,部分细胞核碎裂,呈现一定凋亡特征;GSP预处理对细胞和细胞核形态的改变有明显保护作用.DCFH-DA、Rhodamine 123染色后经流式细胞术分析及荧光显微镜观察显示,GSP抑制了Rot所致细胞内活性氧水平升高,同时明显改善Rot引起的线粒体膜电势降低;Annexin V/PI双染分析发现GSP联合处理明显抑制了Rot所致细胞凋亡.结果表明,GSP可能通过降低细胞ROS生成、恢复线粒体膜电势水平而保护细胞,抑制Rot引起的细胞凋亡,可能在防治帕金森病方面发挥作用.
Parkinson's disease(PD)is a common neurodegenerative disorder in elderly population.On account of increasing the incidence rate,its etiology and prevention strategy must be clarified.In this study,dopaminergic neuronal cell model PC12 were treated with rotenone(Rot),and the inhibitory effect of grape seeds procyanidins(GSP)on Rot cytotoxicity was investigated.MTT assay showed that GSP can attenuate the inhibitory effect of Rot on cell growth in dose-and time-dependent pattern.Under inverted microscope,Giemsa and Hoechst 33342 stained cells showed that Rot can damage cells significantly on cell morphology.Cell is changed from flat polygons to shrinkage,neurites is retracted,nuclear chromatin is agglutinated,and some nuclei are fragmented,showing a certain apoptosis characteristics. GSP pre-treatment has obvious protective activity on the changes of cell and nuclear morphology.After staining with DCFH-DA and Rhodamine 123,flow cytometry analysis and fluorescence microscopy showed that after GSP treatment,the content of reactive oxygen species(ROS)in cells treated with Rot is decreased significantly and the mitochondria membrane potential(MMPs)are increased.Annexin V/PI double staining test exhibited that GSP is reduced apoptosis by Rot.These results indicated that GSP can protect cells,inhibit the apoptosis induced by Rot in PC12 cell through down-regulating the production of ROS and restoring mitochondrial membrane.GSP may play a role in the prevention and treatment of Parkinson's disease.
Parkinson's disease(PD)is a common neurodegenerative disorder in elderly population.On account of increasing the incidence rate,its etiology and prevention strategy must be clarified.In this study,dopaminergic neuronal cell model PC12 were treated with rotenone(Rot),and the inhibitory effect of grape seeds procyanidins(GSP)on Rot cytotoxicity was investigated.MTT assay showed that GSP can attenuate the inhibitory effect of Rot on cell growth in dose-and time-dependent pattern.Under inverted microscope,Giemsa and Hoechst 33342 stained cells showed that Rot can damage cells significantly on cell morphology.Cell is changed from flat polygons to shrinkage,neurites is retracted,nuclear chromatin is agglutinated,and some nuclei are fragmented,showing a certain apoptosis characteristics. GSP pre-treatment has obvious protective activity on the changes of cell and nuclear morphology.After staining with DCFH-DA and Rhodamine 123,flow cytometry analysis and fluorescence microscopy showed that after GSP treatment,the content of reactive oxygen species(ROS)in cells treated with Rot is decreased significantly and the mitochondria membrane potential(MMPs)are increased.Annexin V/PI double staining test exhibited that GSP is reduced apoptosis by Rot.These results indicated that GSP can protect cells,inhibit the apoptosis induced by Rot in PC12 cell through down-regulating the production of ROS and restoring mitochondrial membrane.GSP may play a role in the prevention and treatment of Parkinson's disease.
引文
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[2]孙丽华,江月仙,王巧能.天然抗氧化剂原花青素的保健功能及其应用[J].食品研究与开发,2004,25(2):109.
[3]刘国安,冯筱璐,牛娟,等.原花青素抗氧化活性及对细胞氧化损伤的保护作用[J].西北师范大学学报(自然科学版),2011,47(6):58.
[4]LEVITES Y,AMIT T,MANDEL S.Neuroprotection and neurorescue againstαβtoxicity and PKC-dependent release of non-amyloidogenic soluble precursor protein by green tea polyphenol(-)-epigallocatechin-3-gallate[J].The FASEB Journal,2003,17(8):952.
[5]TANNER C,KAMEL F,ROSS G.Rotenone,paraquat,and Parkinson's disease[J].Environmental Health Perspectives,2011,119(6):866.
[6]GIBELLINI L,PINTI M,NASI M,et a1.Interfering with ROS metabolism in cancer cells:the potential role of quercetin[J].Cancers,2010,2(2):1288.
[7]HAN X,ZHU S,WANG B,et a1.Antioxidant action of 7,8-dihydroxyflavone protects PC12cells against 6-hydroxydopamine-induced cytotoxicity[J].Neurochemistry International,2014,64(1):18.
[8]MONDAL A,BENNETT L L.Resveratrol enhances the efficacy of sorafenib mediated apoptosis in human breast cancer MCF7cells through ROS,cell cycle inhibition,caspase 3and PARP cleavage[J].Biomedicine&Pharmacotherapy,2016,84:1906.
[9]SCHAFER Z,GRASSIAN A,SONG L.Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment[J].Nature,2009,461:109.
[10]LOPEZ J,TAIT S.Mitochondrial apoptosis:killing cancer using the enemy within[J].British Journal of Cancer,2015,112(6):957.
[11]WANG N,FENG Y,ZHU M,et al.Berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells:the cellular mechanism[J].Journal of Cellular Biochemistry,2010,111(6):1426.
[12]ROBERTS D P,LEWIS S J G.Considerations for general anaesthesia in Parkinson's disease[J].Journal of Clinical Neuroscience,2018,48:34.
[13]CHEN H,DING D,WANG J,et al.Parkinson's disease research in a prospective cohort in China[J].Parkinsonism and Related Disorders,2015,21(10):1200.
[14]SCHAPIRA A H,JENNER P.Etiology and pathogenesis of Parkinson's disease[J].Mov Disord,2011,26(6):1049.
[15]王炳,方晓雨,高峰,等.五味子酚对槟榔碱致帕金森病模型小鼠的保护作用[J].齐齐哈尔医学院学报,2017,38(16):1861.
[16]孟凛冽,李峰,伞勇智,等.帕金森病氧化应激机制及抗氧化药物治疗进展[J].现代生物医学进展,2015,15(2):380.
[17]邓勇,逯军.鱼藤酮致线粒体氧化损伤的作用和机制[J].中南大学学报(医学版),2014,39(10):1093.
[18]石碧,狄莹.植物多酚[M].北京:科学出版社,2000:124.
[19]GAO X,CASSIDY X,SCHWARZSCHILD A,et a1.Habitual intake of dietary flavonoids and risk of Parkinson disease[J].Neurology,2012,78(15):1138.
[20]蔡洪斌,王峰,张义军.原花青素对阿尔茨海默病模型大鼠脑组织的预防保护作用[J].中国老年学杂志,2011,31(22):4408.