【作者】 魏大巧；

【导师】 白洁；

【作者基本信息】 昆明理工大学 ， 环境科学， 2015， 博士

【摘要】 阿尔茨海默症,又称老年痴呆症,高发于50岁以上的人群,是由环境及遗传等多种因素共同作用的一种神经退行性疾病,以认知障碍为主要表征,病理表现有大脑前额叶皮层及海马区神经元细胞凋亡及减少,以及神经原纤维结,老年斑等。阿尔茨海默症的发病机制多种多样,主要有基因学说、Ap毒性学说、Tau蛋白异常修饰学说、自由基损伤学说、细胞凋亡学说、代谢紊乱学说和胆碱能紊乱等。3-羟基丁酸(3-hydroxybutyric acid)是人体脂动员后产生的酮体的主要构成物质之一,大脑不能储存糖原作为能量储备,当糖原匮乏,大脑细胞将会启动异常脂动员及消耗脑内蛋白质作为能源补偿,从而导致脑内代谢紊乱,细胞异常死亡,导致认知障碍和其他病征。酮体可以作为能量半成品直接被大脑所应用,尤其3-羟基丁酸为中性不带电荷的小分子物质,易于穿透血脑屏障,及时补偿大脑能量；3-羟基丁酸甲酉旨(3-hydroxy butyric acid methyl ester, HBME)是3-羟基丁酸的甲酯化物质,和3-羟基丁酸一样具有良好的血脑屏障通透性,前期研究显示3-羟基丁酸及其甲酯化物具有加强代谢效率、减少自由基伤害、促进细胞生长、保护神经细胞并具有一定的改善正常小鼠学习记忆能力等多种功能。本论文以淀粉样前体蛋白(amyloid precursor protein, APP)过表达单转基因小鼠(APPsw e)为表征单纯性由于Aβ堆积引起的病理性痴呆的痴呆模型小鼠,以快速老化小鼠SAMP8及其近亲同源抗快速老化小鼠SAMR1为阴性对照为表征增龄性的认知障碍的痴呆模型小鼠；主要研究3-羟基丁酸甲酯对这两种痴呆模型小鼠的学习认知能力的影响；探讨3-羟基丁酸甲酯对这两种痴呆模型小鼠学习认知能力影响的可能作用机制。主要结果包括：(1)通过对正常小鼠进行急性毒性实验、蓄积毒性实验、亚急性毒性实验、Ames实验和哺乳动物细胞体外细胞微核实验对三羟基丁酸甲酯进行毒理安全性评价。实验结果表明：3-羟基丁酸甲酯无急性毒性、蓄积毒性、致突变及损伤染色体毒性,按相关外源化学物质毒理安全性评价指标体系评判,为安全无毒物质。(2)以APP过表达单转基因小鼠及快速老化小鼠为两种痴呆模型小鼠,分别设置空白对照组及实验组,空白组灌胃给予生理盐水,实验组灌胃给予不同浓度(20、40、60mg/kg/d)3-羟基丁酸甲酯。10周后,采用Morris水迷宫进行行为学实验。实验结果表明,3-羟基丁酸甲酯明显改善两种痴呆模型组小鼠的学习、记忆能力,同时对于两种老年痴呆模型组小鼠长时程记忆也表现出显著的提升效果,提升效果呈现剂量依赖关系,在高浓度剂量组提升效果最为显著。(3)上述两种痴呆模型小鼠在结束Morris水迷宫行为学实验后,处死取脑,采用蛋白免疫技术(Western Blot Assay),对其与学习记忆相关的海马及前额叶上皮组织进行相关蛋白：环磷腺苷效应元件结合蛋白(cAMP-response element binding protein, CREB)及磷酸化CREB、硫氧还蛋白(thioredoxin, Trx)、细胞周期蛋白依赖性激酶-5(cyclin-dependent kinase-5,Cdk5)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)半胱氨酸天冬氨酸酶-3前体(pro-caspase-3)及半胱氨酸天冬氨酸酶-9前体(pro-caspase-9)的检测分析。结果显示,不同浓度3-羟基丁酸甲酯均能上调两种痴呆模型小鼠海马区及前额叶皮层组织中的CREB及磷酸化CREB、Trx、Cdk5及Bcl-2的蛋白表达量,两种痴呆模型小鼠海马区及前额叶皮层组织中的pro-caspase-3、 pro-caspase-9表达无下降。实验结果表明,3-羟基丁酸甲酯对两种痴呆模型学习记忆能力的改善其机制可能通过调控CREB/Cdk5的信号通路；利用Trx减少自由基伤害；并且通过Bcl-2表达的上调稳定住凋亡因子Caspase-3及Caspase-9的无活性前体,阻止其切割,抑制神经细胞凋亡途径；保护和修复神经元细胞的结构与功能,从而促进及改善两种痴呆模型小鼠学习记忆能力。改善作用在两种痴呆模型小鼠的两个脑区都表现出3-羟基丁酸甲酯的剂量依赖性,在高浓度组效果显著,表明3-羟基丁酸甲酯加强代谢效率促进细胞生长、保护神经细胞。综上所述,3-羟基丁酸甲酯安全无毒,对两种痴呆模型小鼠的学习记忆能力有明显的改善作用,其改善机制可能是通过加强代谢效率促进细胞生长、保护神经细胞,调控学习记忆相关信号通路,启动自由基清除及抑制神经细胞凋亡途径。本论文为3-羟基丁酸甲酯作为一种可能的抗老年痴呆及延缓衰老药物的开发提供基础理论研究。更多还原

【Abstract】 Alzheimer’s disease is also known as senile dementia with high incidence in people over the age of 50. It is a neurodegenerative disease caused by environmental, genetic and other factors and mainly characterized by cognitive impairments. Its pathological manife-stations include neuron apoptosis and reduction in the prefrontal cortex and hippocampal area, neurofibrillary tangles, elderly spots, etc. A variety of hypotheses has been put for-ward on the pathogenesis of Alzheimer’s disease, including theories on genes, AP toxicity, abnormal modification of Tau protein, free radical damage, the disequilibrium of calcium homeostasis, apoptosis, metabolic disorders, and cholinergic theory, etc.3-hydroxy butyrate (3-hydroxybutyric acid) is one of the main composition of ke-tone bodies produced after human lipid mobilization. The brain cannot store glycogen as energy storage, but in the case of glycogen scarcity, the brain cells will start the abnormal lipid mobilization and protein consumption as energy compensation resulting in brain metabolism disorders, abnormal cell death, leading to cognitive impairments and other symptoms. Ketone bodies can be used as the semi-finished energy product directly by the brain. Being an uncharged neutral small molecule,3-hydroxybutyrate is easy to penetrate the blood brain barrier and thus timely compensate brain power.3-hydroxy butyric acid methyl ester (3-hydroxy butyricacid methyl ester, HBME) is the methyl ester substance of 3-hydroxy butyric acid. Like 3-hydroxybutyric acid, HBME has good blood brain barrier permeability. Previous studies have shown that, in normal mice,3-hydroxybutyric acid and its methyl ester can enhance metabolic efficiency, reduce free radical damage, promote cell growth, protect nerve cells and has a certain improvement in learning mem-ory ability and a variety of functions.In this thesis, the transgenic mice (APPswe) with amyloid precursor protein (APP) single gene overexpression were used as the dementia model because its characteristic pathological dementia is caused only by the A beta accumulation. Senescence accelerated mice (SAMP8) were also used as the age-related dementia model for characterization of cognitive impairment and its close relative homology senescence resistant mice (SAMR1) were used as the negative control. The impact of HBME on learning cognitive ability and the possible mechanism were studied in these two models.The main results include:(1) through the experiment of acute toxicity, accumulative toxicity test in normal mice, subacute toxicity test,Ames test and in vitro mammalian cell micronucleus test for toxicological safety evaluation of three hydroxymethyl butyrate. The experimental results show that:3-hydroxybutyrateno acute toxicity, cumulative toxicity,mutagenicity and toxicity of chromosomal damage, according to the relevant exogenous chemi-cals toxicological safety evaluation of evaluation index system for the safe, non-toxic substances.(2) to APP over expression of single transgenic mice and senescence accelerated mouse for two kinds of dementia model mice, are respectively arranged in blank con-trol group and experimental group, blank groupwere given saline, the experimental group were administered intragastrically with different concentrations (20,40,60 mg/kg/d) 3-hydroxy butyric acid methyl ester. After 10 weeks, the behavioral experi-ments using the Morris water maze. The experimental results show that,3-hy-droxy methyl butyrate obviously improve the learning and memory ability, two kinds of dementia model mice, at the same time for two kinds of senile dementia model mice in long term memory also showed remarkable promotion effect, improve the effect showed a dose dependent relationship, in high concentration dose group to enhance the effect of the most significant.(3) The above two kinds of dementia model mice at the end of the Morris water maze behavior after the experiment, the rats were sacrificed and the brain, by pro-tein immunization technique (Western Blot Assay),carries on the related protein in learn-ing and memory related and superior frontal epithelial tis sue:CREB(cAMP-response element binding protein, CREB) and phosphorylated CREB, thioredoxin (thioredoxin, Trx),cyclindependent kinase-5 (cyclin-dependent kinase-5, Cdk5), B lymphocyte tumor-2 gene (B-cell lym-phoma-2, Bcl-2) of caspase-3 pre-cursor (pro-caspase-3) and caspase-3 precursor-9 (pro-caspase-9) the detection and anal-ysis. The results show, different concentrations of 3-hydroxy methyl buty- rate were up-regulated expression of two kinds of dementia model mice in hippocam-pus and frontal cortex tissue CREB andphosphorylation of CREB, Trx, Cdk5 and Bcl-2 protein expression of pro-caspase-9, pro-caspase-3, two kinds of dementia model mice hippocampus and frontal cortex tissue without falling. The experimental results show that,3-hydroxy methyl butyrateon two kinds of dementia model of learning and memory ability and improve itsmechanism may be through the signal transduction path-way of CREB/Cdk5; by using Trx to reduce free radical damage; and through Bcl-2 expression upregulation stabilize an inactive precursor apoptosis factor Caspase-3 and Caspase-9, to prevent the cutting, inhibit nerve cell apoptosis pathway; structure and function to protect and repair neurons, so as to promote and improve two kind of dementia model of learning and memory of mice.Improve the role in two brain re-gions of two kinds of dementia model mice showed 3-hydroxybutyrate dose depen-dent, in the effect of high concentration group was obviously, indicated that 3-hydroxy methyl butyrateenhanced metabolic efficiency promoting the growth of cells, protect nerve cells.In summary,3-hydroxy methyl butyrate is safe and non-toxic, significant improve-ment on learning and memory ability of two kinds of dementia model mice, its mechanism may be through the strengthening of improvedmetabolic efficien-cy promoting the growth of cells, protect nerve cells, regulation of learning and memory signal transduction pathway, initiated free radical scavenging and inhibiting neuronal apoptosis pathway.This thesis provided the basic theory research with 3-hydroxybutyric acid methyl ester as a possible anti senile dementia and delay the developmentof aging medicine.更多还原