摘要
目的探讨褪黑素(melatonin,MT)在1-甲基-4-苯基-吡啶离子(1-methyl-4-phenylpyridinium ion,MPP^(+))诱导的帕金森病体外模型中的作用及分子机制。方法将MN9D细胞分为对照组、MPP^(+)组、MT组、治疗组。采用细胞计数试剂盒8检测MT和MPP^(+)对细胞活力的影响;采用线粒体膜电位检测试剂盒(JC-1)评价线粒体功能;Hoechst/PI双染法检测细胞凋亡;通过免疫荧光蛋白质印迹法(Western blotting)检测凋亡相关蛋白[Cleaved-Caspase 3和细胞色素C(cytochrome C,CytC)]以及泛醌―细胞色素C还原酶核心蛋白1(ubiquinol-cytochrome C reductase core protein 1,UQCRC1)蛋白的表达;采用干扰RNA技术沉默MN9D细胞中UQCRC1的表达,然后采用Western blotting检测凋亡相关蛋白表达。结果MT可以减轻MPP^(+)诱导的细胞活力下降(P<0.05);恢复MPP^(+)造成的线粒体膜电位下降(P<0.05);减少MPP^(+)诱导的凋亡细胞数量(P<0.05);抑制凋亡蛋白(CytC和Cleaved-Caspase3)的表达(P<0.05);上调UQCRC1的表达(P<0.05)。沉默UQCRC1后,MT组和治疗组的UQCRC1表达均下降(P<0.05);MT对MPP^(+)诱导细胞凋亡的保护作用下降(P<0.05);凋亡蛋白(CytC和Cleaved-Caspase3)的表达增加(P<0.05)。结论MT对MPP^(+)诱导的多巴胺能神经元损伤具有保护作用,其机制可能是通过上调UQCRC1抑制神经元凋亡。
Objective To investigate the effects of melatonin in an in vitro model of Parkinson's disease induced by 1-methyl-4-phenylpyridinium ion(MPP^(+)) and the molecular mechanisms.Methods MN9D cells were divided into control group,MPP^(+) group,melatonin group,and treatment group.The effects of melatonin and MPP^(+) on cell viability were determined using cell counting kit-8.Mitochondrial function was evaluated using the mitochondrial membrane potential assay kit JC-1.Cell apoptosis was determined with Hoechst/propidium iodide double staining.Immunofluorescence assay and Western blotting were used to measure the expression of apoptotic proteins [cleaved caspase-3 and cytochrome C(CytC)] and ubiquinol-cytochrome c reductase core protein 1(UQCRC1).RNA interference technology was used to silence UQCRC1 expression in MN9D cells to measure the expression of the apoptotic proteins by Western blotting.Results Melatonin significantly inhibited MPP^(+)-induced decreases in cell viability and mitochondrial membrane potential,significantly reduced the number of MPP^(+)-induced apoptotic cells,significantly down-regulated the expression of CytC and cleaved-caspase 3,and significantly up-regulated the expression of UQCRC1(all P<0.05).After silencing UQCRC1,the expression of UQCRC1 in the melatonin group and the treatment group was significantly decreased;the protective effect of melatonin against MPP^(+)-induced apoptosis was significantly decreased;and the expression of CytC and cleaved-caspase 3was significantly increased(all P<0.05).Conclusions Melatonin has a protective effect against MPP^(+)-induced damage to dopaminergic neurons,possibly by upregulating UQCRC1 to inhibit neuronal apoptosis.
作者
李傲涵
曾炼
刘颖
张振
胡鹏超
丁旭东
罗辉宇
LI Aohan;ZENG Lian;LIU Ying;ZHANG Zhen;HU Pengchao;DING Xudong;LUO Huiyu(Department of Anesthesiology,Xiangyang No.1 People's Hospital,Hubei University of Medicine,Xiangyang,Hubei 441000,China;Central Liaboratory,Xiangyang No.1 People's Hospital,Hubei University of Medicine,Xiangyang,Hubei 441000,China;Department of Rehabilitation Medicine,Xiangyang No.1 People's Hospital,Hubei University of Medicine,Xiangyang,Hubei 441000,China;Hubei Clinical Research Center of Parkinson's Disease,Xiangyang,Hubei 441000,China)
出处
《国际神经病学神经外科学杂志》
2023年第3期26-31,共6页
Journal of International Neurology and Neurosurgery
基金
湖北省科技计划项目(2021CFB582)
襄阳市科技局项目(2021ZD13)
襄阳市第一人民医院院级科研平台(XYY2022P02)。
作者简介
李傲涵,男,硕士,住院医师。Email:446210721@qq.com;通信作者:罗辉宇,男,博士,主任医师。Email:603983267@qq.com。
