硫辛酸对果蝇帕金森病模型神经元的作用及其机制  

Effect of lipoic acid on neurons in Drosophila model of Parkinson's disease and its mechanism

作　　者：熊江福 张祥明 石彩群 张春林 XIONG Jiangfu;ZHANG Xiangming;SHI Caiqun;ZHANG Chunlin(Department of Biology,School of Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院生物学系,贵州贵阳550025

出　　处：《贵州医科大学学报》2023年第9期993-1002,共10页Journal of Guizhou Medical University

基　　金：国家自然科学基金(82160234);贵州省科技厅科技计划项目(黔科合基础-ZK〔2021〕一般113);贵州省教育厅特色领域项目(黔教合KY字〔2021〕066);2019年国家级、省级大学生创新训练项目(201910660008);2020年省级大学生创新训练项目(S202010660065)。

摘　　要：目的探讨α-硫辛酸(α-LA)对SNCA.A30P突变转基因帕金森病(PD)果蝇模型神经元的作用及其作用机制。方法以Elav-GAL4>UAS-SNCA.A30P果蝇品系作为模型(PD)组,Elav-GAL4果蝇品系作为对照(control)组,Elav-GAL4>UAS-SNCA.A30P果蝇品系培养于α-LA加药培养基中作为干预实验(LA)组;敲降Atg5(Atg5 RNAi)或Atg8a(Atg8a RNAi)后,Atg5 RNAi和Atg8a RNAi的PD果蝇培养于α-LA加药培养基中作为Atg5降低+LA组和Atg8a降低+LA组;观察α-LA对果蝇上爬能力和寿命的影响,检测各组果蝇脑部活性氧(ROS)、谷胱甘肽(GSH)、丙二醛(MDA)、超氧化物歧化酶(SOD)及三磷酸腺苷(ATP)含量,免疫荧光分析果蝇大脑多巴胺(DA)神经元的分布,Western blot检测果蝇脑部组织中酪氨酸羟化酶(TH)、α-突触核蛋白(α-syn)、线粒体氧化呼吸链复合物Ⅰ亚单位NADH脱氢酶(泛醌)铁硫蛋白3(NDUFS3)、自噬相关蛋白5(Atg5)及自噬相关蛋白8a(Atg8a)的表达。结果α-LA能够提高PD果蝇存活率(P<0.05)及上爬能力(P<0.001);α-LA可降低ROS(P<0.05)、MDA含量(P<0.05)以及α-syn表达水平(P<0.01),提高GSH(P<0.05)、SOD(P<0.01)、ATP含量(P<0.01)、TH(P<0.05)、NDUFS3(P<0.05)、Atg5(P<0.05)以及Atg8a(P<0.05)的表达;与PD组比较,Atg5降低+LA组和Atg8a降低+LA组果蝇脑内α-syn含量无明显变化(P>0.05)。结论α-LA对PD果蝇模型具有神经元保护作用,其机制可能是通过增强细胞自噬、降低α-syn的表达水平,从而降低α-syn的神经毒性。Objective To investigate the protective effect and mechanism ofα-lipoic acid on neurons in Drosophila model of SNCA.A30P mutated transgenic with Parkinson's disease.Methods Elave-GAL4>UAS-SNCA.A30P strain Drosophila was used as PD model group.The Elave-GAL4 strain Drosophila was used as the control group.Elave-GAL4>UAS-SNCA.A30P strain Drosophila was maintained in normal medium mixed withα-LA as treatment group.After Atg5(Atg5 RNAi)or Atg8a(Atg8a RNAi)was knocked down,PD model group of Atg5 RNAi and Atg8a RNAi was cultivated in normal medium mixed withα-LA for further grouping into Atg5 lowered+LA group and Atg8a lowered+LA group.The effects ofα-LA on the climbing ability and lifespan of Drosophila were observed.Then,measuring the content of reactive oxygen species(ROS),glutathione(GSH),malondialdehyde(MDA),superoxide dismutase(SOD),and adenosine triphosphate(ATP)in Drosophila's brain.dopamine(DA)neuron distribution were detected by immunofluorescence.Furthermore,the expression of tyrosine hydroxylase(TH),α-synuclein(α-syn),ADH dehydrogenase(ubiquinone)ferrithionein 3(NDUFS3),and autphagy related 5(Atg5),autophagy related 8a(Atg8a)in Drosophila's brain were detected by Western blot.Resultsα-LA could successfully improve survival rate(P<0.05)and climbing ability in PD Drosophila(P<0.001).Theα-LA could significantly decrease the content of ROS(P<0.05),MDA(P<0.05),andα-syn expression(P<0.01),but increase the content of GSH(P<0.05),SOD(P<0.01),ATP(P<0.01).Theα-LA could promote the expression of TH(P<0.05),NDUFS3(P<0.05),Atg5(P<0.05),and Atg8a(P<0.05).Compared with PD group,α-syn content in Drosophila brain of Atg5 lowered+LA group and Atg8a lowered+LA group showed no significant change(P>0.05).Conclusionα-LA has neuroprotective effect on PD Drosophila model,and its mechanism may be to enhance autophagy and reduce the expression level ofα-syn to reduce the neurotoxicity ofα-syn.

关 键 词：硫辛酸 帕金森病 果蝇模型 Α-突触核蛋白 自噬 多巴胺神经元 

分 类 号：R392-33[医药卫生—免疫学] R741.2[医药卫生—基础医学] R-331