茴拉西坦改善阿尔茨海默病小鼠认知功能和增强突触可塑性的机制探讨

发布时间：2018-03-25 01:38

本文选题：阿尔茨海默病　切入点：东莨菪碱　出处：《河北医科大学》2017年硕士论文

【摘要】：目的:阿尔茨海默病(Alzheimer disease,AD)是最常见的老年期痴呆类型,其临床特征表现为隐袭起病,进行性的记忆和认知功能减退,多伴有人格、精神异常,是年龄相关的神经系统退行性疾病;病理学特征表现为β-淀粉样蛋白(β-amyloid,Aβ)沉积于神经元外形成大量老年斑(Senile plaques,SP)和神经元内形成以过度磷酸化的微管相关蛋白tau为主要成分的神经原纤维缠结(Neurofibrillary tangles,NFTs),同时伴有胆碱能神经元数量、突触数量的明显减少。迄今为止,AD的发病机制仍不是十分明确,当前国内外医学界主要有以下几种学说:胆碱能神经损伤学说、线粒体损伤学说、氧化应激学说、遗传与基因突变学说等,但目前无一学说能阐明其发病机制。东莨菪碱(scopolamine,SCO)是M型胆碱受体拮抗剂,可阻断大脑皮层和海马区乙酰胆碱对M受体的激动作用,从而对记忆获得产生阻抑效应,诱导痴呆,已经被广泛应用在神经营养药物的评估上。腹腔给药3mg/Kg后可造成动物记忆功能障碍。东莨菪碱用于诱导动物记忆障碍模型具有简单、经济、方便等优点,故常应用于抗AD活性化合物的初步筛选。茴拉西坦(aniracetam)作为γ-氨基丁酸的环化衍生物,为脑代谢增强药,对于改善抑郁症患者的症状方面具有长期效果,同时,茴拉西坦还有抗焦虑的作用。其主要作用机制可能是通过调节谷氨酸受体中的AMPA受体和类乙酰胆碱能系统,但国内尚未见将茴拉西坦应用东莨菪碱致痴呆模型中,本实验以东莨菪碱致痴呆作为AD的模型,探讨AMPA受体(Glu R1)、突触可塑性蛋白的表达情况,并探讨茴拉西坦是否可以通过激活谷氨酸受体磷酸化和增高突触蛋白的表达来改善AD小鼠的认知功能。方法:SPF级雄性BALB/c小鼠96只,全部动物购自北京华维通利华实验动物技术有限公司,饲养于河北省人民医院临床研究中心动物室内。由动物中心的饲养员管理,以标准的鼠粮及纯净的饮水饲养。将小鼠随机分为6组:vehicle组(生理盐水3mg/kg,vehicle group)、模型组(SCO 3mg/kg,SCO group)、低剂量组(SCO 3mg/kg+茴拉西坦40mg/kg,L-A group)、中剂量组(SCO 3mg/kg+茴拉西坦80mg/kg,M-A group)、高剂量组(SCO3mg/kg+茴拉西坦160mg/kg,H-A group)、石杉碱甲组(SCO 3mg/kg+石杉碱甲0.15mg/kg,Huper-A group),每组16只,SCO皮下注射日一次,连续给药60天,茴拉西坦和石杉碱甲在第50天灌胃给药,均为日一次,连续给药10天。造模完成后进行新物体识别实验,实验完成取小鼠海马组织,采用Western blot蛋白分析法测定ERK1/2、p-ERK1/2、Glu R1、p-Glu R1Ser845、PSD-95、SYN在海马组织蛋白表达量;采用RT-q PCR技术测定ERK1/2、Glu R1、PSD-95、SYN的m RNA表达量。采用免疫组化方法测定海马组织PSD-95、SYN的阳性细胞数。采用专用试剂盒检测乙酰胆碱酯酶活性。结果:1茴拉西坦改善东莨菪碱引起的小鼠认知功能下降与vehicle组相比,SCO组小鼠的认知指数明显降低(P0.01),这说明东莨菪碱明显降低了小鼠的认知功能,造模成功。而经茴拉西坦治疗后,与SCO组相比,M-A和H-A组小鼠认知指数均显著升高(P0.01),说明茴拉西坦可以改善小鼠的认知功能。2茴拉西坦对突触可塑性相关蛋白的影响2.1茴拉西坦激活东莨菪碱小鼠海马ERK1/2蛋白表达磷酸化6组之间的ERK1/2蛋白表达水平没有明显统计学差异(P0.05);与vehicle组相比,SCO组小鼠海马组织p-ERK1/2蛋白表达明显下降(P0.01),说明造模成功;而经茴拉西坦治疗后,与SCO组相比,M-A和H-A组海马p-ERK1/2蛋白表达水平显著升高(P0.01);阳性对照组蛋白水平p-ERK1/2表达明显升高(P0.01)。2.2茴拉西坦激活东莨菪碱小鼠海马Glu R1蛋白表达磷酸化6组之间的Glu R1蛋白表达水平没有明显统计学差异(P0.05);与vehicle组相比,SCO组小鼠海马组织p-Glu R1蛋白表达明显下降(P0.01),说明造模成功;而经茴拉西坦治疗后,与SCO组相比,H-A组海马p-Glu R1蛋白表达水平显著升高(P0.01);阳性对照组蛋白水平p-Glu R1表达明显升高(P0.01)。2.3茴拉西坦减轻东莨菪碱小鼠海马突触蛋白PSD-95、SYN表达的改变与vehicle组相比,SCO组小鼠海马组织PSD-95、SYN蛋白表达明显下降(P0.01,P0.01);而经茴拉西坦治疗后,与SCO组相比,L-A组海马PSD-95、SYN蛋白水平均显著升高(P0.01,P0.01);阳性对照组PSD-95、SYN蛋白水平表达明显升高(P0.01,P0.01)。免疫组织化学染色后可见蛋白表达水平的变化主要是在海马CA1区。3茴拉西坦上调东莨菪碱小鼠海马ERK1/2、Glu R1、SYN、PSD-95基因的表达与vehicle组相比,SCO组小鼠海马组织ERK1/2、Glu R1、SYN、PSD-95基因表达均明显下降(P0.01),而经茴拉西坦治疗后,与SCO组相比,M-A和H-A组海马ERK1/2基因水平均显著升高(P0.05,P0.01);与SCO组相比,H-A组海马Glu R1基因水平均显著升高(P0.01);与SCO组相比,L-A组海马PSD-95、SYN基因水平均即显著升高(P0.01、P0.01),阳性对照组基因水平相应基因表达均明显升高(P0.01)。4乙酰胆碱酯酶活性测定结果与vehicle组相比,石杉碱甲组乙酰胆碱酯酶活性明显下降(P0.01),而SCO组乙酰胆碱酯酶活性与vehicle组相比差异没有统计学意义(P0.05),同样,茴拉西坦治疗组对于乙酰胆碱酯酶活性的影响也没有统计学意义(P0.05)。结论:1茴拉西坦可以改善东莨菪碱引起的小鼠学习记忆能力下降。2茴拉西坦可以通过增加谷氨酸受体的磷酸化和突触相关蛋白的表达来提高东莨菪碱小鼠的学习记忆能力。
[Abstract]:Objective: Alzheimer's disease (Alzheimer disease AD) is the most common type of senile dementia, the clinical features of insidious onset, progressive memory and cognitive dysfunction, accompanied by personality, mental disorders, is a neurodegenerative disease related to age; pathological features of beta amyloid protein (beta -amyloid and A beta) deposition in neurons formed outside a large number of senile plaques (Senile plaques, SP) and neuronal neurofibrillary tangle formation by hyperphosphorylation of microtubule associated protein tau as the main component (Neurofibrillary tangles, NFTs), accompanied by the number of cholinergic neurons, significantly reduced the number of synapses so far., the pathogenesis of AD is not very clear, the medical profession at home and abroad are mainly the following: the theory of cholinergic nerve damage theory, mitochondrial damage theory, oxidative stress theory, genetics and gene mutation The theory, but there is no theory can explain its pathogenesis. Scopolamine (scopolamine, SCO) is a type M cholinergic receptor antagonist, blocked acetylcholine in the cerebral cortex and hippocampus of M receptor agonist, which have inhibitory effect on memory access, induced dementia, has been widely used in the evaluation of neurotrophic drug. After intraperitoneal Administration of 3mg/Kg can cause memory impairment induced by scopolamine in animal. The animal model of memory disorder is simple, economic, convenient, preliminary screening is often used in anti AD active compounds. Lacita fennel (aniracetam) as ring derivatives of gamma aminobutyric acid, brain metabolism drugs, has long the effect in improving the symptoms of depression at the same time, Ra Laci Staw and anti anxiety. The main mechanism may be through the regulation of glutamate receptor in AMPA receptor And the cholinergic system, but there is no dementia model induced by scopolamine in the application of Ra Laci Staw, in this experiment, scopolamine induced dementia as the AD model, to investigate the AMPA receptor (Glu R1), the expression of synaptic plasticity protein, and to explore the cognitive function of Ra Laci Staw whether by expression of activation of glutamate receptor phosphorylation and increased synaptic to improve the AD protein in mice. Methods: SPF male BALB/c 96 mice all animal was purchased from Beijing Hua Lihua Experimental Animal Technology Co. Ltd., raised in Hebei People's Hospital clinical research laboratory animal research center. By the breeder management of Animal Center, feeding with the standard rat food and clean drinking water. The mice were randomly divided into into 6 groups: group vehicle (normal saline 3mg/kg, vehicle group), model group (SCO 3mg/kg, SCO group), low dose group (SCO 3mg/kg+ 40mg/kg L-A group La zetham fennel,), in Dose group (SCO 3mg/kg+ 80mg/kg M-A group aniracetam, high dose group (SCO3mg/kg+), aniracetam 160mg/kg, H-A group), huperzine a group (SCO 3mg/kg+ huperzine 0.15mg/kg, Huper-A group), 16 rats in each group, SCO subcutaneous injection once a day, continuous administration for 60 days, fennel Tracy Tanzania and huperzine A in fiftieth days were administered orally, once a day, continuous administration for 10 days. After modeling the new object recognition experiment, experiment the mice hippocampus, determination of ERK1/2, analysis by using the method of Western blot protein p-ERK1/2, Glu R1, p-Glu R1Ser845, PSD-95, expression SYN in hippocampus protein; Determination of ERK1/2, using RT-q PCR technology Glu R1, PSD-95, RNA SYN. The expression of M was measured by immunohistochemical method of PSD-95 in the hippocampus, the number of SYN positive cells. Acetylcholinesterase activity was measured by special kit. Results: 1 aniracetam scopolamine induced small In cognitive decline compared with the vehicle group, cognitive index in group SCO were significantly lower (P0.01), indicating that scopolamine significantly reduces the cognitive function in mice, animal model. By Lacita fennel after treatment, compared with group SCO, M-A and H-A group were significantly elevated both cognitive index (P0.01), description effect of aniracetam can improve the cognitive function of.2 mice Lacita fennel proteins related to synaptic plasticity 2.1 aniracetam activation of scopolamine in mice ERK1/2 protein expression in hippocampus phosphorylation between the 6 groups in the expression level of ERK1/2 protein had no obvious statistical difference (P0.05); compared with vehicle group, the expression of SCO p-ERK1/2 protein in the hippocampus of mice significantly decreased (P0.01), indicating that the model was successful; by aniracetam after treatment, compared with the SCO group, the expression level of M-A and p-ERK1/2 protein in hippocampus of H-A group increased significantly (P0.01); the protein level of p-ERK1/ in the positive control group 2 expression was significantly increased (P0.01).2.2 aniracetam activation of scopolamine in mice hippocampal Glu protein expression of R1 phosphorylation of Glu between the 6 groups R1 protein expression level showed no statistically significant differences (P0.05); compared with vehicle group, SCO p-Glu expression of R1 protein in hippocampus of mice decreased significantly (P0.01), indicating that the model was successful; and by aniracetam after treatment, compared with the SCO group, the expression level of H-A in group p-Glu significantly increased R1 protein (P0.01) positive control group; p-Glu protein level significantly increased the expression of R1 (P0.01).2.3 aniracetam reduce mouse hippocampal synaptic protein PSD-95 induced by scopolamine, the change of SYN expression compared with vehicle group, hippocampus SCO mice in group PSD-95, SYN protein expression was significantly decreased (P0.01, P0.01); and by aniracetam after treatment, compared with the SCO group, L-A group, PSD-95 in hippocampal SYN protein levels were significantly increased (P0.01, P0.01); the positive control group PSD-95, SYN protein The level of expression was significantly increased (P0.01, P0.01). Immunohistochemical staining showed protein expression level changes mainly in the hippocampal CA1 region.3 upregulation of scopolamine in mice hippocampus ERK1/2 Hui Lacita, Glu R1, SYN, PSD-95 gene expression compared with vehicle group, SCO group of mice sea horse tissue ERK1/2, Glu R1, SYN, PSD-95 gene expression decreased significantly (P0.01), and by aniracetam after treatment, compared with the SCO group, M-A group and H-A ERK1/2 gene in the hippocampus were significantly increased (P0.05, P0.01); compared with SCO group, the level of H-A group in hippocampus Glu of R1 gene was significantly elevated (P0.01); compared with the SCO group. In group L-A, PSD-95, SYN gene levels were increased significantly (P0.01, P0.01), the corresponding gene expression level of gene positive control group were significantly higher (P0.01).4 determination of acetylcholinesterase activity compared with vehicle group, huperzine a group of acetylcholinesterase activity decreased obviously (P0.0 1), and SCO group acetylcholinesterase activity compared with vehicle group, the difference was not statistically significant (P0.05). Similarly, Ra Laci Staw treatment group effect on acetylcholinesterase activity was also not statistically significant (P0.05). Conclusion: 1 aniracetam induced by scopolamine can improve the learning and memory ability of mice decreased.2 aniracetam can improve learning and memory the ability of scopolamine in mice by increasing the expression of synapse associated protein phosphorylation and glutamate receptors.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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