二苯乙烯苷对α-突触核蛋白过表达和聚集的影响及其作用机制

发布时间：2018-05-14 03:22

本文选题：二苯乙烯苷 + α-突触核蛋白　；参考：《首都医科大学》2017年硕士论文

【摘要】：目的α-突触核蛋白(a-synuclein,α-syn)在多种神经退行性疾病的发病机制中起着重要作用,这些疾病包括帕金森病、路易小体痴呆、阿尔茨海默病、多系统萎缩等,又统称为突触核蛋白病。聚集的α-Syn是路易小体的重要组成部分,而且突变的α-syn或多倍体α-syn可导致家族性帕金森病。但目前这些疾病都缺乏有效的治疗药物。二苯乙烯苷(2,3,5,4’-tetrahydroxy-stilbene glucoside,TSG)是中药何首乌的主要有效成分和标志成分。我们的前期研究发现TSG能够抑制APP转基因阿尔茨海默病小鼠模型海马和大脑皮层α-syn过表达和聚集,抑制老年小鼠纹状体和海马α-syn过表达和聚集。本研究的目的是在A53T突变型α-syn转基因小鼠模型和α-syn基因转染神经细胞模型上,进一步研究TSG对α-syn过表达和聚集的影响及其在翻译后修饰和蛋白降解方面的作用机制。方法1.应用6月龄A53T突变型α-syn转基因小鼠,灌胃给予TSG 6个月或9个月。应用爬杆试验和筑巢试验检测小鼠的运动能力,Y迷宫试验检测小鼠的记忆能力;应用免疫组化方法观察小鼠脑内酪氨酸羟化酶(TH)标记的DA能神经元、Iba-1标记的小胶质细胞;用Western blot法检测小鼠脑中α-syn单体和寡聚体、磷酸化a-syn、Parkin、Beclin1、Atg、LC3的表达。2.采用α-syn基因转染的SH-SY5Y细胞,与TSG孵育24 h后,换含小剂量鱼藤酮的培养液继续孵育24 h。应用CCK8检测细胞活性;免疫荧光双标法检测α-syn的表达;Western blot法检测α-syn、磷酸化α-syn、磷酸化PP2A的表达。结果1.12月龄和15月龄a53tα-syn转基因小鼠在爬杆试验中的潜伏期延长,筑巢行为评分减低,y迷宫自发性交替反应减低;tsg灌胃给药6个月或9个月能够明显缩短12月龄和15月龄模型小鼠在爬杆试验中的潜伏期,增高筑巢行为评分和y迷宫自发性交替反应,表明tsg能够改善α-syn转基因小鼠的运动功能和记忆能力。2.15月龄a53tα-syn转基因模型组小鼠黑质致密部th标记的da能神经元数量减少,在黑质和纹状体部位iba-1标记的小胶质细胞数量增高。tsg灌胃给药9个月能够增加模型小鼠黑质致密部da能神经元数量,减少黑质和纹状体部位小胶质细胞数量,表明tsg具有神经保护作用。3.15月龄a53tα-syn转基因小鼠大脑皮层α-syn单体和寡聚体的表达增高;tsg能够减低模型小鼠大脑皮层α-syn单体和寡聚体的表达水平,表明tsg对α-syn转基因小鼠脑内α-syn的过表达和聚集有抑制作用。4.在作用机制方面,我们发现15月龄a53tα-syn转基因模型组小鼠纹状体、大脑皮层中的α-syn在ser129位点的磷酸化水平增高;tsg能够降低模型小鼠纹状体和大脑皮层中α-syn的磷酸化水平(ser129位点),提示tsg能够改善α-syn的翻译后修饰,这可能是tsg抑制α-syn聚集的作用机制之一。5.tsg能够增高15月龄a53tα-syn转基因小鼠大脑皮层及小脑中parkin蛋白、atg5和lc3表达;提示tsg可能通过上调泛素-蛋白酶体系统(usp)和自噬途径从而增强α-syn的降解,这可能是tsg抑制α-syn过表达和聚集的另一个作用机制。6、在体外实验中,tsg能够抑制α-syn基因转染sh-sy5y细胞内α-syn过表达,降低鱼藤酮复合α-syn基因转染细胞模型中α-syn在ser129位点的磷酸化水平和pp2a的磷酸化水平,提示tsg可能通过下调pp2a的磷酸化引起pp2a活性增高,这可能是TSG抑制α-syn磷酸化的作用机制之一。结论TSG灌胃给药能够改善15月龄A53Tα-syn转基因小鼠的运动能力和记忆功能,增高黑质多巴胺能神经元数量,抑制黑质和纹状体小胶质细胞激活,抑制脑内α-syn过表达和聚集;其作用机制可能涉及通过下调PP2A磷酸化而抑制α-syn的磷酸化,通过上调泛素-蛋白酶体系统和自噬途径而增强α-syn的降解。结果提示TSG可能有利于防治突触核蛋白相关的神经退行性疾病。
[Abstract]:Objective alpha synuclein (a-synuclein, alpha -syn) plays an important role in the pathogenesis of a variety of neurodegenerative diseases. These diseases include Parkinson's disease, Louis's dementia, Alzheimer's disease, multi system atrophy and so on. They are also known as synuclein. The aggregated alpha -Syn is an important part of the Louis corpuscle, and the mutation is alpha. -syn or polyploid alpha -syn can lead to familial Parkinson's disease. But these diseases are currently lacking in effective therapeutic drugs. Two 2,3,5,4 '-tetrahydroxy-stilbene glucoside (TSG) is the main active component and marker of Polygonum multiflorum. Our preliminary study showed that TSG can inhibit APP transgenic Alzheimer's disease. The overexpression and aggregation of alpha -syn in the hippocampus and cerebral cortex of the rat model inhibit the overexpression and accumulation of alpha -syn in the striatum and hippocampus of the aged mice. The purpose of this study was to further study the effect of TSG on the over expression and aggregation of alpha -syn in the A53T mutated alpha -syn transgenic mouse model and the transfection of the alpha -syn gene into the neural cell model and the posttranslational repair. The mechanism of role of ornamentation and protein degradation. Method 1. using 6 month old A53T mutated alpha -syn transgenic mice, gavage was given to TSG for 6 months or 9 months. The exercise ability of mice was detected by climbing pole test and nesting test, the memory ability of mice was detected by Y maze test, and the immunohistochemical method was used to observe the tyrosine hydroxylase (TH) mark in the brain of mice. The DA neurons and Iba-1 labeled microglia were recorded, and the Western blot method was used to detect the alpha -syn monomer and oligomer in the brain of mice, phosphorylated a-syn, Parkin, Beclin1, Atg, LC3, and.2. using alpha -syn gene transfected cells. After incubating 24, the cells were incubated with small dose of rotenone and continued to incubate 24 applied detection cells. The expression of alpha -syn was detected by immunofluorescence double labeling; Western blot method was used to detect the expression of alpha -syn, phosphorylated alpha -syn, and phosphorylated PP2A. Results the incubation period of 1.12 month old and 15 month old a53t alpha -syn transgenic mice was prolonged in the climbing pole test, the score of nesting behavior was reduced, the spontaneous alternating response to the Y maze decreased, and TSG was administered for 6 months or 9 months by TSG. It can significantly shorten the incubation period of 12 month old and 15 month old model mice in the climbing pole test, increase the nest behavior score and the spontaneous alternating reaction of Y maze, which indicates that TSG can improve the motor function and memory ability of the -syn transgenic mice with.2.15 month old a53t alpha -syn transgenic model group, and the number of Da energy neurons marked in the dense part of the substantia nigra of the -syn transgenic mice Decrease, the increased number of microglia labeled by Iba-1 in substantia nigra and striatum.Tsg can increase the number of DA neurons in the dense part of the substantia nigra and decrease the number of microglia in the substantia nigra and striatum of the model mice for 9 months, indicating that TSG has neuroprotective effect on the cerebral cortex alpha -syn of.3.15 month old a53t alpha -syn transgenic mice. The expression of monomers and oligomers increased; TSG could reduce the expression level of alpha -syn monomers and oligomers in the cerebral cortex of the model mice, indicating that TSG has inhibitory effect on the over expression and aggregation of alpha -syn in the brain of alpha -syn transgenic mice. We found that the 15 month old a53t alpha -syn transgenic model group has the striatum, the cerebral cortex in the mice. The level of phosphorylation of alpha -syn at ser129 loci increases, and TSG can reduce the phosphorylation level of alpha -syn in the striatum and cerebral cortex of model mice (ser129 site), suggesting that TSG can improve posttranslational modification of alpha -syn, which may be one of the mechanisms of TSG inhibition of alpha -syn aggregation, and.5.tsg can increase the brain of 15 month old a53t alpha -syn transgenic mice. The expression of parkin protein, ATG5 and LC3 in the cortex and cerebellum, suggesting that TSG may enhance the degradation of alpha -syn by up regulation of the ubiquitin proteasome system (USP) and autophagy pathway, which may be another mechanism of inhibition of the over expression and aggregation of alpha -syn by TSG. In vitro, TSG can inhibit alpha -syn gene transfection into SH-SY5Y cells. Expression, reduce the phosphorylation level of alpha -syn at ser129 site and the phosphorylation level of PP2A in the cell model of rotenone compound alpha -syn gene transfection, suggesting that TSG may increase the activity of PP2A by down-regulation of PP2A phosphorylation, which may be one of the mechanisms of TSG inhibition of alpha -syn phosphorylation. Conclusion TSG can improve 15 month old A53T alpha -s. The exercise ability and memory function of YN transgenic mice, increase the number of dopaminergic neurons in substantia nigra, inhibit the activation of substantia nigra and striatum microglia, inhibit the over expression and aggregation of alpha -syn in the brain; its mechanism may involve the inhibition of phosphorylation of alpha -syn by down-regulation of PP2A phosphorylation, by up regulation of the ubiquitin proteasome system and autophagy. The results suggest that TSG may be beneficial to the prevention of synuclein related neurodegenerative diseases. The results suggest that -syn may play an important role in the prevention and treatment of neurodegenerative diseases associated with synuclein.

【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741

【参考文献】