熊江福,张祥明,石彩群,张春林
XIONG Jiangfu,ZHANG Xiangming,SHI Caiqun,ZHANG Chunlin

• 1:贵州医科大学基础医学院生物学系

摘要(Abstract)：

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 of α-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 with α-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 with α-LA for further grouping into Atg5 lowered+LA group and Atg8a lowered+LA group. The effects of α-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. Results α-LA could successfully improve survival rate(P<0.05) and climbing ability in PD Drosophila(P<0.001). The α-LA could significantly decrease the content of ROS(P<0.05), MDA(P<0.05), and α-syn expression(P<0.01), but increase the content of GSH(P<0.05), SOD(P<0.01), ATP(P<0.01). The α-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). Conclusion α-LA has neuroprotective effect on PD Drosophila model, and its mechanism may be to enhance autophagy and reduce the expression level of α-syn to reduce the neurotoxicity of α-syn.

关键词(KeyWords)： 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
α-lipoic acid;Parkinson's disease;Drosophila model;α-synuclein;autophagy;dopaminergic neuron

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 熊江福,张祥明,石彩群,张春林
XIONG Jiangfu,ZHANG Xiangming,SHI Caiqun,ZHANG Chunlin

DOI: 10.19367/j.cnki.2096-8388.2023.09.001

参考文献(References)：

• [1] REICH S G,SAVITT J M.Parkinson's disease[J].Medical Clinics of North America,2019,103(2):337-350.
• [2] TOLOSA E,GARRIDO A,SCHOLZ S W,et al.Challenges in the diagnosis of Parkinson's disease[J].The Lancet Neurology,2021,20(5):385-397.
• [3] POEWE W,SEPPI K,MARINI K,et al.New hopes for disease modification in Parkinson's disease[J].Neuropharmacology,2020,171:108085.
• [4] EMAMZADEH F N,SURGUCHOV A.Parkinson's disease:biomarkers,treatment,and risk factors[J].Frontiers in Neuroscience,2018,12:612.
• [5] HELY M A,MORRIS J G L,REID W G J,et al.Sydney multicenter study of Parkinson's disease:Non-L-dopa-responsive problems dominate at 15 years[J].Movement Disorders,2005,20(2):190-199.
• [6] VIJIARATNAM N,SIMUNI T,BANDMANN O,et al.Progress towards therapies for disease modification in Parkinson's disease[J].The Lancet Neurology,2021,20(7):559-572.
• [7] ARAUJO D P,LOBATO R F G,CAVALCANTI J R L P,et al.The contributions of antioxidant activity of lipoic acid in reducing neurogenerative progression of Parkinson's disease:a review[J].The International Journal of Neuroscience,2011,121(2):51-57.
• [8] SEIFAR F,KHALILI M,KHALEDYAN H,et al.α-Lipoic acid,functional fatty acid,as a novel therapeutic alternative for central nervous system diseases:a review[J].Nutritional Neuroscience,2019,22(5):306-316.
• [9] ZAITONE S A,ABO-ELMATTY D M,SHAALAN A A.Acetyl-l-carnitine and α-lipoic acid affect rotenone-induced damage in nigral dopaminergic neurons of rat brain,implication for Parkinson's disease therapy[J].Pharmacology Biochemistry and Behavior,2012,100(3):347-360.
• [10]李成朋，韩飞，杨梅，等.硫辛酸对6-羟多巴胺诱导的帕金森模型大鼠的保护作用[J].贵州医科大学学报，2017,42(3):249-252.
• [11]邰胜燕，翟素珍，张祥明，等.α硫辛酸抑制6-羟基多巴胺诱导的PC12细胞中α突触核蛋白(α-synuclein)表达及机制[J].细胞与分子免疫学杂志，2018,34(9):812-817.
• [12]SULZER D,EDWARDS R H.The physiological role of α-synuclein and its relationship to Parkinson's disease[J].Journal of Neurochemistry,2019,150(5):475-486.
• [13]ROCHA E M,DEMIRANDA B,SANDERS L H.α-synuclein:pathology,mitochondrial dysfunction and neuroinflammation in Parkinson's disease[J].Neurobiology of Disease,2018,109:249-257.
• [14]TANG Q L,GAO P,ARZBERGER T,et al.α-synuclein defects autophagy by impairing SNAP29-mediated autophagosome-lysosome fusion[J].Cell Death & Disease,2021,12(10):854.
• [15]ZHAO H,ZHAO X C,LIU L J,et al.Neurochemical effects of the R form of α-lipoic acid and its neuroprotective mechanism in cellular models of Parkinson's disease[J].The International Journal of Biochemistry & Cell Biology,2017,87:86-94.
• [16]JIA J,GONG X Y,ZHAO Y,et al.Autophagy enhancing contributes to the organ protective effect of α-lipoic acid in septic rats[J].Frontiers in Immunology,2019,10:1491.
• [17]NICHOLS C D.Drosophila melanogaster neurobiology,neuropharmacology,and how the fly can inform central nervous system drug discovery[J].Pharmacology & Therapeutics,2006,112(3):677-700.
• [18]WHITWORTH A J.Drosophila models of Parkinson's disease[J].Advances in Genetics,2011,73:1-50.
• [19]BIER E.Drosophila,the golden bug,emerges as a tool for human genetics[J].Nature Reviews Genetics,2005,6(1):9-23.
• [20]MONASTIRIOTI M.Biogenic amine systems in the fruit fly Drosophila melanogaster[J].Microscopy Research and Technique,1999,45(2):106-121.
• [21]POZODEVOTO V M,FALZONE T L.Mitochondrial dynamics in Parkinson’s disease:a role for α-synuclein[J].Disease Models & Mechanisms,2017,10(9):1075-1087.
• [22]WANG L,ZHAO Z J,ZHAO L,et al.Lactobacillus plantarum DP189 reduces α-SYN aggravation in MPTP-induced Parkinson's disease mice via regulating oxidative damage,inflammation,and gut microbiota disorder[J].Journal of Agricultural and Food Chemistry,2022,70(4):1163-1173.
• [23]SONG G,LIU Z G,WANG L F,et al.Protective effects of lipoic acid against acrylamide-induced neurotoxicity:involvement of mitochondrial energy metabolism and autophagy[J].Food & Function,2017,8(12):4657-4667.
• [24]POSTUMA R B,AARSLAND D,BARONE P,et al.Identifying prodromal Parkinson's disease:pre-Motor disorders in Parkinson's disease[J].Movement Disorders,2012,27(5):617-626.
• [25]FLEMING A,BOURDENX M,FUJIMAKI M,et al.The different autophagy degradation pathways and neurodegeneration[J].Neuron,2022,110(6):935-966.
• [26]HOU X,WATZLAWIK J O,FIESEL F C,et al.Autophagy in Parkinson's disease[J].Journal of Molecular Biology,2020,432(8):2651-2672.

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