姜黄素通过激活自噬保护帕金森病多巴胺能神经元的研究  

Protective effect of curcumin on dopamine neurons of Parkinson＇s disease by activating autophagy

作　　者：吴忧 徐彬 梁顺利 张荣博 徐林胜 Wu You;Xu Bin;Liang Shunli;Zhang Rongbo;Xu Linsheng(Department of Neurology,Second Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310005,China)

机构地区：[1]浙江中医药大学附属第二医院神经内科,杭州310005

出　　处：《中华神经医学杂志》2018年第10期973-980,共8页Chinese Journal of Neuromedicine

摘　　要：目的探讨姜黄素是否通过激活自噬保护帕金森病（PD）多巴胺能（DA）神经元及其激活自噬的具体机制。方法采用1-甲基-4-苯基-四氢吡啶离子（MPP＋）处理人神经母细胞瘤SH-SY5Y细胞建立PD细胞模型；同时采用1-甲基-4-苯基-1, 2, 3, 6-四氢吡啶（MPTP）腹腔注射C57BL/6雄性小鼠建立PD动物模型；细胞及小鼠分别分为正常对照组、模型组、姜黄素组、姜黄素＋3-MA组、3-MA组。姜黄素组、姜黄素＋3-甲基腺嘌呤（3-MA）组和3-MA组细胞在MPP＋处理的同时分别将姜黄素（40 μmol/L）、姜黄素（40 μmol/L）＋3-MA（4 mmol/L）、3-MA（4 mmol/L）添加于培养基中孵育。姜黄素组、姜黄素＋3-MA组、3-MA组小鼠在MPTP腹腔注射的同时分别予姜黄素（80 mg/kg）、姜黄素（80 mg/kg）＋3-MA（2 mg/kg）、3-MA（2 mg/kg）腹腔注射，1次/d。正常对照组同时腹腔注射无菌生理盐水（30 mL/kg）。各组细胞在药物处理48 h后分别进行酪氨酸羟化酶（TH）免疫荧光染色。采用Western blotting实验检测α-突触核蛋白（α-Syn）、自噬相关蛋白溶酶体相关膜蛋白2A（LAMP2A）和微管相关蛋白1轻链3（LC3）的蛋白表达，并分离核浆蛋白测定核转录因子EB（TFEB）蛋白在胞浆和胞核中的表达；采用RT-PCR检测α-Syn mRNA表达。各组小鼠在最后1次药物注射后14 d时处死取中脑黑质脑组织行TH免疫组化染色。采用Western blotting实验检测α-Syn和LC3的蛋白表达。结果（1）与模型组比较，姜黄素组细胞和小鼠中脑黑质TH阳性细胞数目增多（即DA神经元存活数目增多），细胞内α-Syn蛋白及mRNA表达减少，小鼠中脑α-Syn蛋白表达减少，差异均有统计学意义（P〈0.05）。同时，姜黄素组细胞内自噬蛋白LAMP2A和LC3-Ⅱ表达增加，小鼠中脑LC3-Ⅱ蛋白表达增加，差异均有统计学意义（P〈0.05）。（2）与姜黄素组比较，姜黄素＋3-MA组细胞及小鼠中脑黑�ObjectiveTo investigate whether curcumin protects dopamine neurons in Parkinson＇s disease （PD） by activating autophagy and discuss its specific mechanism.MethodsIn vitro, SH-SY5Y human neuroblastoma cells were treated with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine （MPP＋） to induce PD cell models. In vivo, C57BL/6 male mice were performed intraperitoneal injection of 30 mg/kg 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine （MPTP） once daily for 7 d continuously to establish PD models. SH-SY5Y human neuroblastoma cells and C57BL/6 male mice were, respectively, divided into normal control group, model group, curcumin group, curcumin and 3-methyladenine （3-MA） group, and 3-MA group. Curcumin was used as intervention treatment, and 3-MA, an autophagy inhibitor, was used individually or concurrently with curcumin as intervention controls： curcumin （40 μmol/L）, curcumin （40 μmol/L）＋3-MA （4 mmol/L）, and 3-MA （4 mmol/L） were added into the cells of curcumin group, curcumin and 3-MA group, and 3-MA group at the time of MPP＋ addition; curcumin （80 mg/kg）, curcumin （80 mg/kg）＋3-MA （2 mg/kg）, and 3-MA （2 mg/kg） were added into the mice of curcumin group, curcumin and 3-MA group, and 3-MA group at the time of MPTP injection once daily. In the normal control group, sterile saline was injected into the abdomen simultaneously （30 mL/kg）. In cell groups, after 48 h of drug treatment, the number of surviving dopamine neurons was detected by tyrosine hydroxylase （TH） immunofluorescence; Western blotting was used to detect protein expressions of α-synuclein （α-Syn）, and autophagy related proteins lysosome-associated membrane protein 2A （LAMP2A） and microtubule-associated protein 1 light chain 3 （LC3）; transcription factor EB （TFEB） levels in the cytoplasm and nucleus were detected separately by Western blotting; reverse transcription （RT）-PCR was used to detect the α-Syn mRNA expression. In mice groups, 14 d after the last injection, mi

关 键 词：帕金森病 姜黄素 自噬 Α-突触核蛋白 核转录因子EB 

分 类 号：R742.5[医药卫生—神经病学与精神病学]