【作者】 何苗；

【导师】 魏敏杰；

【作者基本信息】 中国医科大学 ， 药理学， 2008， 博士

【摘要】 目的阿尔茨海默病（Alzheimer’s disease,AD）是一种发生于中老年的以进行性认知障碍和记忆能力下降为主的退行性神经病变。其典型的病理改变是神经细胞间出现大量以β-淀粉样肽（β-Amyloid,Aβ）为核心的老年斑（senileplaques,SP）、神经元的胞体中出现神经原纤维缠结（neurofibrillary tangles,NFT）等。AD的发病机制目前尚未完全阐明,但自由基损伤学说和细胞凋亡学说备受人们的关注。自由基形成和氧化应激增强,进一步引起神经细胞凋亡,是导致AD的功能退化和神经变性的基础。p75神经营养因子受体（P75 neurotrophin receptor,P75NTR）是神经营养素的低亲和力受体,与AD神经元细胞凋亡有关。p75NTR诱导凋亡的信号通路目前仍不十分清楚,但已广泛公认的是其对c-jun氨基末端激酶（c-jun N-terminal kinase,JNK）的激活是介导神经元细胞凋亡的关键步骤,并进一步通过激活促凋亡蛋白P53、Bad等的表达,促进或引起细胞凋亡。此外,JNK还能通过诱导细胞色素C的释放进一步活化Caspase-3而导致细胞凋亡。研究并建立可靠的AD动物模型对于探明AD的病因、发病机制及防治药物的研发均有重要的意义。到目前为止用于AD研究的动物模型很多,其中给小鼠慢性注射D-半乳糖（D-galactose,D-gal）已成为一种常见的制备方法,广泛用于AD等神经退行性疾病的药理学研究。此外,转基因模型是一种病因模型,过量表达人源性突变AD相关基因的转基因鼠,因其具有明确的病因,并能反映AD的部分病理与功能变化,有助于我们了解AD的发病机制,也逐渐成为研究AD的整体模型。防治AD的抗氧化剂及抗凋亡药物的研究与开发成为当今药理学研究领域的重要课题之一。（-）表没食子儿茶素没食子酸酯（epigallocatechin-3-gallate,EGCG）是绿茶的一种主要多酚成分,研究证实绿茶有效的铁螯和、抗氧化、抗炎、抗癌及神经保护等作用的发挥主要依赖于EGCG。Levites等通过研究人类成神经细胞瘤SHSY5Y细胞和大鼠成纤维细胞瘤PC12细胞,发现EGCG能够减少具有神经毒性作用的Aβ的产生。因此,本研究拟通过D-半乳糖皮下注射建立的AD小鼠模型及遗传性的APP/PS1转基因小鼠,采用Niss1、TUNEL染色、免疫组化、Westernblot等方法研究D-半乳糖对小鼠认知行为、病理改变、抗氧化能力、细胞凋亡、Aβ（1-40）、淀粉样前体蛋白（Amyloid precursor Protein,APP）、促凋亡蛋白caspase-3及P75-JNK-P53凋亡相关通路的影响,以及9月龄的APP/PS1转基因小鼠上述指标的改变,同时进一步探讨EGCG对两种AD小鼠模型的神经保护作用及其作用机制。材料与方法D-半乳糖诱导的AD模型鼠实验:取昆明系小鼠96只,随机分成6组,每组16只,即空白对照组（Control）、模型组1（D-gal+ddH₂O）、模型组2（D-gal+oil）、VE阳性对照组（D-gal+280IU/kg VE）、EGCG低剂量处理组（D-gal+2 mg/kgEGCG）、EGCG高剂量处理组（D-gal+6 mg/kg EGCG）。每日给两模型组及VE阳性对照组、EGCG低剂量及高剂量处理组小鼠按150 mg/kg皮下注射3%的D-半乳糖一次,空白对照组小鼠每天皮下注射等体积的生理盐水一次,连续注射6周。第三周开始给VE阳性对照组、EGCG低剂量及高剂量处理组小鼠每日分别按280IU/kg灌胃5.6%VE、按2 mg/kg灌胃0.04%的EGCG、按6 mg/kg灌胃0.12%的EGCG一次,空白对照组及模型组1小鼠灌胃等量的双蒸水一次,模型组2灌胃等量的大豆油一次,连续给药4周。最后一周对小鼠进行水迷宫、避暗及自主活动等行为学检测,其间继续给药。行为学结束后,快速取一部分小鼠海马入-80℃中冻存,分别进行SOD、GSH-Px酶活性、MDA含量的测定及Western blot印迹分析APP、caspase-3、P75、JNK2及P53等蛋白表达水平。另一部分小鼠进行脑灌流固定,分别进行HE染色、Niss1染色、TUNEL染色及Aβ（1-40）、caspase-3、APP等免疫组化的检测。APP/PS1转基因小鼠实验取C57小鼠10只及APP/PS1小鼠20只,随机分成3组:C57对照组（Control）、模型组（APP/PS1）、实验组（APP/PS1+2 mg/kgEGCG）。每日给实验组APP/PS1转基因小鼠按2 mg/kg灌胃0.04%的EGCG一次,给对照组及模型组小鼠灌胃等量的双蒸水一次,连续灌胃4周。最后两日对小鼠进行避暗实验的行为学检测,其间继续给药。行为学结束后,快速取一部分鼠海马入-80℃中冻存,进行Western blot印迹分析P75、JNK2及P53等蛋白表达水平。另一部分小鼠进行脑灌流固定,进行HE染色及Aβ（1-40）免疫组化的检测。实验结果1、EGCG对D-半乳糖诱导的AD小鼠记忆及判断能力的影响水迷宫、避暗、自主活动等行为学结果显示D-半乳糖（150 mg/kg）皮下注射6周后,小鼠出现明显的学习记忆及判断能力的下降,但活动能力未受影响;EGCG（2 mg/kg和6 mg/kg）和VE（280IU/kg）均明显的改善了D-半乳糖诱导的AD模型小鼠的学习记忆障碍。2、EGCG对D-半乳糖诱导的AD模型鼠脑组织病理改变的影响HE染色及Niss1染色结果显示,D-半乳糖（150mg/kg）皮下注射6周后,小鼠大脑皮层及海马区神经元数量明显减少,细胞排列松散,尼氏体分界不清,可见坏死的神经元;EGCG（2 mg/kg和6 mg/kg）和VE（280IU/kg）均明显的减轻了D-半乳糖引起的神经元损伤程度。3、EGCG对D-半乳糖诱导的AD模型鼠脑内Aβ（1-40）蛋白表达的影响Aβ（1-40）蛋白免疫组化染色结果显示:D-半乳糖（150 mg/kg）皮下注射6周后,小鼠大脑皮层及海马区的Aβ（1-40）蛋白表达水平显著增高;EGCG（2 mg/kg和6mg/kg）和VE（280IU/kg）均能明显抑制小鼠大脑皮层及海马区的Aβ（1-40）蛋白表达水平。4、EGCG对D-半乳糖诱导的AD小鼠海马区SOD、GSH-Px酶活性及MDA含量的影响酶学结果显示D-半乳糖（150 mg/kg）皮下注射6周后,小鼠海马内SOD、GSH-Px酶活性明显降低,MDA含量明显升高;EGCG（2 mg/kg和6 mg/kg）及VE（280IU/kg）均能明显的提高小鼠海马内SOD、GSH-Px酶活性,并明显的降低MDA含量。5、EGCG对D-半乳糖诱导的AD模型鼠脑区神经元细胞凋亡的影响TUNEL法染色结果显示D-半乳糖（1 50 mg/kg）诱导的AD模型鼠大脑皮层及海马区可见有较多的凋亡细胞,且细胞凋亡指数与空白对照组比明显增多;EGCG（2 mg/kg和6 mg/kg）及VE（280IU/kg）均能明显的减少D-半乳糖诱导的神经元细胞的凋亡。6、EGCG对D-半乳糖诱导的AD模型鼠脑内caspase-3蛋白表达的影响Caspase-3免疫组化染色及Western blot印迹分析结果显示D-半乳糖（150mg/kg）诱导了小鼠大脑皮层及海马区的caspase-3蛋白表达水平明显增高;EGCG（2 mg/kg和6 mg/kg）明显的抑制了D-半乳糖诱导的小鼠大脑皮层及海马区的caspase-3蛋白表达水平的增高;VE（280IU/kg）无明显的抑制作用。7、EGCG对D-半乳糖诱导的AD模型鼠脑内APP蛋白表达的影响APP免疫组化染色及Western blot印迹分析结果显示D-半乳糖（150 mg/kg）诱导了小鼠大脑皮层及海马区的APP蛋白表达水平明显增高;EGCG（2 mg/kg和6mg/kg）及VE（280IU/kg）均明显的抑制了D-半乳糖诱导的小鼠大脑皮层及海马区的APP蛋白表达水平的增高。8、EGCG对D-半乳糖诱导的AD模型鼠海马内P75NTR、JNK2及P53蛋白表达的影响Western blot印迹分析结果显示D-半乳糖（150 mg/kg）明显的促进了小鼠海马内P75蛋白的剪切,增加了核内转移的p75ICD的表达水平,同时也明显的诱导了小鼠海马区JNK2及P53蛋白表达水平增加;EGCG（2 mg/kg和6 mg/kg）可明显的抑制D-半乳糖诱导的p75ICD、JNK2及P53蛋白表达水平的增加;VE（280IU/kg）未见明显的抑制作用。9、EGCG对APP/PS1转基因小鼠记忆及判断能力的影响避暗实验结果显示APP/PS1转基因小鼠与C57小鼠比出现了明显的记忆障碍;EGCG（2 mg/kg）可明显的改善APP/PS1转基因小鼠的记忆障碍。10、EGCG对APP/PS1转基因小鼠脑内Aβ（1-40）蛋白表达的影响Aβ（1-40）蛋白免疫组化染色结果显示:APP/PS1转基因小鼠大脑皮层及海马区Aβ（1-40）蛋白表达水平与C57小鼠比明显增高;EGCG（2 mg/kg）可明显的降低APP/PS1转基因小鼠大脑皮层及海马区Aβ（1-40）蛋白表达水平。11、EGCG对APP/PS1转基因小鼠海马内P75NTR、JNK2及P53蛋白表达的影响Western blot印迹分析结果显示APP/PS1转基因小鼠海马内p75ICD、JNK2及P53蛋白表达水平与C57小鼠比明显增高;EGCG（2 mg/kg）可明显的抑制APP/PS1转基因小鼠海马区的p75ICD、JNK2及P53蛋白表达水平。结论1、EGCG能够改善D-半乳糖诱导的AD模型小鼠及APP/PS1转基因小鼠的学习记忆障碍。2、EGCG能够提高D-半乳糖诱导的AD模型小鼠海马内的抗氧化酶活性,发挥有效的抗氧化作用。3、EGCG通过抑制D-半乳糖诱导的AD模型小鼠大脑皮层及海马内的APP及Aβ（1-40）蛋白水平,对神经细胞发挥一定的保护作用。4、EGCG通过抑制D-半乳糖诱导的AD模型小鼠及APP/PS1转基因小鼠海马内p75ICD、JNK2及P53凋亡通路相关蛋白表达水平,进一步抑制caspase-3蛋白活性,对神经细胞发挥有效的抗凋亡作用。更多还原

【Abstract】 ObjectiveAlzheimer’s disease（AD） is a progressive neurodegenerative disease,with the characteristic of progressive memory loss,cognitive deterioration and behavioral disorders.AD is pathologically characterized by deposition ofβ-amyloid（Aβ） peptides as senile plaques and neurofibrillary tangles in the brain.The precise mechanisms of AD are not yet clearly understood,but Free Radical Stress Theory and Cell Apoptosis Theory are paid close attention to.Oxidative stress and reactive oxygen species（ROS） have been proposed to be major cause of functional disorder and neurodegeneration in AD.P75 neurotrophin receptor is the low affinity receptor of neurotrophins and it is closely relative to neuronal apoptosis in AD.The apoptosis signaling pathway P75NTR involved in is not yet clearly understood,but it is well known that P75NTR can activate c-jun N-terminal kinase（JNK） and pro-apoptotic protein p53 and Bad,and induce cell apoptosis.In addition,JNK can also promote cytochrome c release and activation of caspase-3.It is very important to study and make suitable animal models for evaluating the cause,pathogenesy of AD and developing drugs to cure AD.So far different animal models have been developed to study the etiology,evolution and new therapeutic alternatives for the illnesses,among which the mice continuously injected with D-galactose（D-gal） have been extensively used for pharmacological research on AD. Transgenic mouse models have been created with mutations in genes related to AD. Moreover,double transgenics,such as APP/PS1 transgenic mice,provide a valuable model for evaluating the pathogenesy of AD.（-）-Epigallocatechin-3-gallate（EGCG）,which is classified the catechin family and is one of the major polyphenol constituents of green tea.It has been reported that EGCG possess potent iron-chelating,antioxidant,anti-inflammatory,anticancer and neuroprotective activities.EGCG has been shown to have neuroprotective effects by elevatingα-secretase activity of amyloid precursor protein（APP） to soluble APP-alpha （sAPP-α） and reducing Amyloid beta（Aβ）-induced neurotoxicity in human SH-SY5Y neuroblastoma and rat pheochromocytoma PC12 cells.In the present study,we used AD mouse model induced by D-gal（150mg/kg.d,6 weeks,sc.） and APP/PS1 transgenic mice,and observed whether EGCG（2 mg/kg.d or 6 mg/kg.d,4 weeks,ig） had the potent antioxidant and anti-apoptotic neuroprotective effects on the AD mice and APP/PS1 transgenic by behavioral and pathological testing,measurements of the activities of total superoxide dismutase（T-SOD） and glutathione peroxidase（GSH-Px）, contents of malondialdehyde（MDA） and activation and expression of pro-apoptotic protein caspase-3,P75,JNK and P53 in the hippocampus of mice by immunohistochemical staining and Western blot analysis.Materials and MethodsNinety-six healthy Kunming mice were randomly divided into six groups（n=16 each group）:control group,D-gal+ddH₂O group,D-gal+oil group,D-gal+VE （280IU/kg） group,D-gal+EGCG（2 mg/kg） group and D-gal+EGCG（6mg/kg） group.The mice of D-gal+ddH₂O group,D-gal+oil group,D-gal+VE（280IU/kg） group,D-gal+EGCG（2 mg/kg.d） group and D-gal+EGCG（6 mg/kg.d） group were subcutaneously injected with 3%D-gal at the dose of 150 mg/kg body weight once daily for 6 weeks,while those of control group were treated with same volume normal saline.From the third week,the mice of D-gal+VE（280IU/kg） group,D-gal+EGCG （2 mg/kg.d） group and D-gal+EGCG（6 mg/kg.d） group were intragastricly given with 5.6%VE at the dose of 280IU/kg and EGCG at the dose of 2 mg/kg.d or 6 mg/kg.d respectively after injection of D-gal.The mice of control group,D-gal+ddH₂O group and D-gal+oil group were administered with same volume vehicle distilled water and soybean oil respectively.After finishing all treatments,animals were evaluated by behavioral testing,and then immediately sacrificed to dissect hippocampus and stored at-80℃for the examinations of activities of SOD and GSH-Px,contents of MDA, and activation and expression of pro-apoptotic protein caspase-3,P75,JNK and P53 in the hippocampus of mice by Western blot analysis.Other mice（n=6） were transcardially perfused with normal saline followed by 4%paraformaldehyde solution. The hippocampus were removed,post-fixed in 4%paraformaldehyde and used for HE, Nissl and TUNEL staining and immunohistochemical staining of Aβ（1-40）,caspase-3 and APP.Ten C57 mice and twenty APP/PS1 mice were randomly divided into three groups （n=10 each group）:control group,APP/PS1 group and APP/PS1+2 mg/kg EGCG group.The mice of APP/PS1+EGCG（2 mg/kg） group were intragastricly given with EGCG at the dose of 2 mg/kg.d for 4 weeks.The mice of control group and APP/PS1 group were administered with same volume vehicle distilled water.After finishing all treatments,animals were evaluated by behavioral testing,and then immediately sacrificed to dissect hippocampus and stored at-80℃for the examinations of expression of pro-apoptotic protein P75,JNK and P53 in the hippocampus of mice by Western blot analysis.Other mice（n=4） were transcardially perfused with normal saline followed by 4%paraformaldehyde solution.The hippocampus were removed, post-fixed in 4%paraformaldehyde and used for HE and immunohistochemical staining of Aβ（1-40）.Results1.The effects of EGCG on learning and memory impairment in AD model mice induced by D-gal.The results by water maze,step-through and spontaneous activity test showed D-gal （150 mg/kg.d,6 weeks,sc.） could significantly impair learning and memory of mice, but could not affect their locomotor activity.And EGCG（2 mg/kg.d or 6 mg/kg.d,4 weeks,ig） and VE（280IU/kg,4 weeks,ig） could evidently improve the learning and memory impairment in AD model mice induced by D-gal.2.The histopathological effects of EGCG on the brains of AD model mice induced by D-gal.The results by HE and Nissl staining showed the number of neuron was significantly decreased,the arrangement of neurons in cortex and hippocampus of D-gal model mice was sparse and Nissl body was decreased and dissolved.EGCG（2 mg/kg.d or 6 mg/kg.d,4 weeks,ig） and VE（280IU/kg,4 weeks,ig） could evidently release neuronal injury induced by D-gal.3.The effects of EGCG on express of Aβ（1-40） in brains of AD model mice induced by D-galThe results by immunohistochemical staining of Aβ（l-40） showed D-gal （150 mg/kg.d,6 weeks,sc.） induced an obvious increase of Aβ（1-40） in the cortex and hippocampus of mice,and EGCG（2 mg/kg.d or 6 mg/kg.d,4 weeks,ig） and VE （280IU/kg,4 weeks,ig） significantly reversed the effect.4.The effects of EGCG on activities of SOD,GSH-Px and contents of MDA in the hippocampus of AD model mice induced by D-gal.The results by biological analysis showed D-gal（150 mg/kg.d,6 weeks,sc.） decreased activities of SOD and GSH-Px,increased contents of MDA in mouse hippocampus,and EGCG（2 mg/kg.d or 6 mg/kg.d,4 weeks,ig） and VE（280IU/kg,4 weeks,ig） significantly elevated the activities of SOD and GSH-Px,decreased the contents of MDA in hippocampus of AD model mice induced by D-gal.5.The effects of EGCG on neuronal apoptosis in brains of AD model mice induced by D-gal.TUNEL staining results showed TUNEL positive neurons were highly widespread in the cerebral cortex and hippocampus in AD model mice induced by D-gal and the cell apoptosis index was obviously increased in the hippocampus of mice in model group,compared with control group.EGCG（2 mg/kg.d or 6 mg/kg.d,4 weeks,ig） and VE（280IU/kg,4 weeks,ig） significantly decreased cell apoptosis index in the hippocampus of mice induced by D-gal.6.The effects of EGCG on activation of caspase-3 in the brains of AD model mice induced by D-gal.The results by immunohistochemical staining and western blot analysis of caspase-3 showed D-gal（150 mg/kg.d,6 weeks,sc.） induced significantly increased in the activation of caspase-3 in the cerebral cortex and hippocampus of mice.EGCG（2 mg/kg.d or 6 mg/kg.d,4 weeks,ig） significantly reduced the activation of caspase-3 in the cerebral cortex and hippocampus of AD model mice induced by D-gal,but VE （280IU/kg,4 weeks,ig） was not observed the effect.7.The effects of EGCG on express of APP in the brains of AD model mice induced by D-gal. The results by immunohistochemical staining and western blot analysis of APP showed D-gal（150mg/kg.d,6 weeks,sc.） induced significantly increased in the express of APP in the cerebral cortex and hippocampus of mice.EGCG（2 mg/kg.d or 6 mg/kg.d,4 weeks,ig） and VE（280IU/kg,4 weeks,ig） significantly reduced the express of APP in the cerebral cortex and hippocampus of AD model mice induced by D-gal.8.The effects of EGCG on express of P75NTR,JNK2 and P53 in the hippocampus of AD model mice induced by D-gal.The results by western blot analysis showed D-gal（150 mg/kg.d,6 weeks,sc.） significantly promoted the shearing of P75 in the hippocampus of mice and increased the displacement of p75ICD to the nucleus,and D-gal（150 mg/kg.d,6 weeks,sc.） also significantly increased the express of JNK2 and P53 in the hippocampus of mice. EGCG（2 mg/kg.d or 6 mg/kg.d,4 weeks,ig） significantly decreased the express of p75ICD,JNK2 and P53 in the hippocampus of AD model mice induced by D-gal,and VE（280IU/kg,4 weeks,ig） was not found obviously inhibitory effect.9.The effects of EGCG on learning and memory of APP/PS1 transgenic mice.The results by step-through test showed APP/PS1 transgenic mice had obvious memory impairment compared with C57 mice.EGCG（2 mg/kg.d,4 weeks,ig） could significantly improve the memory impairment of APP/PS1 transgenic mice.10.The effects of EGCG on the express ofβ-Amyloid（1-40） in brains of APP/PS1 transgenic mice.The results by immunohistochemical staining ofβ-Amyloid（1-40） showed an obvious increase in the express ofβ-Amyloid（1-40） in the cortex and hippocampus of APP/PS1 transgenic mice,and EGCG（2 mg/kg.d,4 weeks,ig） significantly decreased the express ofβ-Amyloid（1-40） in the cortex and hippocampus of APP/PS1 transgenic mice.11.The effects of EGCG on the express of P75NTR,JNK2 and P53 in the hippocampus of APP/PS1 transgenic mice.The results by western blot analysis showed the express of p75ICD,JNK2 and P53 in the hippocampus of APP/PS1 transgenic mice were significantly increased, compared with C57 mice.EGCG（2 mg/kg.d,4 weeks,ig） significantly decreased the express of p75ICD,JNK2 and P53 in the hippocampus of APP/PS1 transgenic mice.Conclusion1.EGCG can improve the learning and memory impairment of mice AD model induced by D-gal and APP/PS1 transgenic mice.2.EGCG has a potent antioxidant effect by elevating the activities of SOD and GSH-Px and decreasing the contents of MD A in hippocampus of AD model induced by D-gal.3.EGCG has a protective effect on AD model mice induced by D-gal by decreasing the express of APP and P-Amyloid（l-40） in the cerebral cortex and hippocampus of mice.4.EGCG has a potent anti-apoptotic effect by reducing the express of p75ICD, JNK2 and P53,and decreasing the activation of caspase-3 in the hippocampus of AD model induced by D-gal and APP/PS1 transgenic mice.更多还原