【作者】 徐佳；

【导师】 王丽萍；

【作者基本信息】 吉林大学 ， 细胞生物学， 2019， 博士

【摘要】 阿尔茨海默症（Alzheimer’s disease,AD）,俗称老年痴呆,是一种典型的神经系统退行性疾病。据2018年阿尔茨海默症报告统计,目前全球范围内有4700万人患有阿尔茨海默症。AD患者以大脑皮层及海马区的β淀粉样蛋白（Amyloidβ,Aβ）斑块沉积、tau蛋白过度磷酸化形成的神经纤维缠结（Neurofibrillary tangles,NFT）以及神经元丢失为主要病理特征,其临床表现为认知功能下降,记忆力减退、语言及行为障碍,AD严重的影响患者的身心健康以及生活质量,在世界范围内引起广泛关注。当前研究表明,淀粉样前体蛋白（β-Amyloid precursor protein,APP）的错误剪切与Aβ过度沉积能够导致机体氧化还原稳态失衡,使神经元损伤,进而引发认知障碍;同时,氧化压力水平的提高可进一步促进APP的错误切割和Aβ斑块沉积的形成。两者相互作用,是导致AD病发的重要因素。因此,调节生物体内氧化还原稳态并减少Aβ沉积是抗AD药物研究的重要方向和热点内容。次血红素六肽（Deuterohemin-AlaHisThrValGluLys,DhHP-6）是基于天然微过氧化物酶（Microperoxidase-11,MP-11）设计的含血红素新型过氧化物酶模拟肽,具有较强的清除自由基活性,且具有更好的水溶性、稳定性,易于合成。前期研究表明,DhHP-6在生物体内表现出具有良好的抗氧化作用,DhHP-6可延长野生型线虫Bristol N2的寿命达19.3%,并保护N2线虫抵抗热激和百草枯诱导的氧化损伤。因而,基于氧化应激与自由基损伤学说,本文主要探究次血红素六肽抗阿尔茨海默症的作用效果及其作用机制。本研究首先以Aβ_1-42转基因秀丽隐杆线虫CL4176为模型,初步探究DhHP-6对AD模型线虫的保护作用。结果表明DhHP-6能显著延长CL4176线虫的寿命,延缓Aβ过表达伴随的瘫痪表型发生,降低线虫体内Aβ斑块的沉积。此外,我们对DhHP-6给药的CL4176线虫进行转录组学分析和realtime PCR基因表达量测定,发现DhHP-6可通过调控线粒体氧化应激相关基因发挥对CL4176线虫的保护作用。在动物实验中,我们选取APPswe/PS1dE9双转基因AD小鼠为动物模型,探究DhHP-6对AD模型小鼠认知能力及脑部病理变化的改善效果。水迷宫、Y-迷宫实验结果表明,DhHP-6能显著改善AD小鼠的空间学习能力和短期记忆能力;筑巢实验、三箱社交实验、新物体识别实验、避暗实验、旷场和自主活动实验结果表明,DhHP-6能显著提高AD小鼠的认知水平、社交能力、新物体识别以能力、趋避行为及空间探索能力,并减轻AD小鼠的焦躁行为,改善海马区功能损伤;更重要的是,DhHP-6能降低AD小鼠脑部Aβ斑块沉积,减少脑组织和血浆中可溶性Aβ_1-40和Aβ_1-42含量,减少AD小鼠脑部细胞凋亡,改善小胶质细胞和星形胶质细胞形态,降低脑组织炎症因子（IL-6,IL-1β,TNF-α,IL-17A）水平,提高脑组织抗氧化酶的活力,恢复线粒体形态。此外,DhHP-6对于AD小鼠内脏组织无毒性作用。为进一步探究DhHP-6对AD的作用机制,我们选择小鼠海马区神经元细胞系HT22细胞构建了APP_595/596-HT22,并进一步评估DhHP-6对APP_595/596-HT22的神经保护作用。结果表明,DhHP-6能显著提高APP_595/596-HT22细胞的抗氧化酶类活性,降低炎症因子水平。此外,通过western blot检测细胞内抗氧化相关蛋白的表达水平,发现DhHP-6可通过Nrf-2/HO-1信号通路发挥抗氧化保护作用。综上所述,DhHP-6对AD具有较好的作用效果,在生物体内可通过Nrf-2/HO-1信号通路发挥抗氧化及神经保护作用,为抗AD的新药研发奠定基础。更多还原

【Abstract】 Alzheimer’s disease（AD）,commonly known as dementia,is regarded as a typical neurodegenerative brain disease.There are approximately 47 million people with senile dementia globally.AD is characterized by the deposition of amyloid,neurofibrillary tangles（NFT）,loss of neurons,progressive deterioration of memory and cognition,and a gradual worsening of behavioral disturbances,which threatens human health seriously,also brings heavy burden to the patients,family and society in worldwide.Current studies have shown that proteolytic cleavage of the amyloidβprecursor protein（AβPP）byβ-andγ-secretase could lead to the production and accumulation of Aβ₁–₄₀0 and Aβ₁–₄₂.β-amyloid as a kind of pathogenic factors will self-assemble into various amyloid species,which leads to the imbalance of redox homeostasis,neuron damage and cognitive impairment.Moreover,excessive free radicals promote the wrong cleavage of APP and Aβplaque deposition even further.These two factors promote each other and form a vicious circle.Therefore,regulating the redox homeostasis in vivo and reducing Aβdeposition are important directions and hot topic of anti-AD drug researches.Deuterohemin-AlaHisThrValGluLys（DhHP-6）as a novel peroxidase mimetic peptide,is designed on the natural microperoxidase-11（MP-11）with heme-porphyrin,and it has strong free radical scavenging activity.Furthermore,it has better water solubility,stability and is easy to synthesize.Previous studies showed that DhHP-6 could extend the lifespan of C.elegans（Bristol N2）for 19.3%,protect it against heat-and paraquat-induced damage.Therefore,it is crucial to explore the mechanism of DhHP-6 against Alzheimer’s disease based on free radical damage hypothesis.In this paper,we chose Aβ_1-42 transgenic nematode as AD model to study the protective effect of DhHP-6 against Alzheimer’s disease.Results showed that DhHP-6 prolonged the lifespan,delayed the paralysis,and reduced the deposition of Aβplaque significantly in Aβ_1-42 transgenic C.elegans（CL4176）.More importantly,we found DhHP-6 played an important role in regulating the expression of mitochondria-associated genes based on transcriptomics analysis and realtime PCR assay.Further,we selected APPswe/PS1dE9 double transgenic AD mice as a model to explore the improvement effect of DhHP-6 on cognitive ability and brain lesion.Results indicated that DhHP-6significantly improved the spatial learning ability and short-term memory ability of AD mice by Morris water maze and Y-maze,and significantly improved the cognitive level,social ability,new object recognition,approach-avoidance behavior,spatial exploration ability and neurotrosis in the hippocampus of AD mice through nesting,three-chamber social,new object recognition,step-through task,open field and autonomic activities experiments.More importantly,DhHP-6inhibited the cell apoptosis by reducing the Aβplaque deposition in brain,and reduced the levels of soluble Aβ_1-40 and Aβ_1-42 in brain tissue and plasma.Besides,it also improved the morphology of microglia and astrocytes,reduced inflammatory factors,increased the activity of antioxidant enzyme and improved the morphology of mitochondria.In addition,DhHP-6 was safe and nontoxic for the viscera of AD mice.To further explore the mechanism of DhHP-6 on AD,we constructed APP_595/596-HT22transfection nerve cell based on mouse hippocampal neuron cell line HT22.Results showed that DhHP-6 had neuroprotective effect on the overexpression of APP induced cytotoxicity,and which significantly improved the activity of antioxidant enzymes and reduced the level of extracellular inflammatory factors in APP_595/596-HT22 cell.Moreover,DhHP-6 exerted antioxidant function and effect through Nrf-2/HO-1 signaling pathway in APP_595/596-HT22 cell by western blot assay.In summary,DhHP-6 exerted antioxidant and neuroprotective effect on AD through the Nrf-2/HO-1 signaling pathway in vivo,which could provide new ideas and set the foundation for the development of new drugs against Alzheimer’s disease.更多还原