肠源性内毒素血症在阿尔茨海默病发病中作用及相关机制研究

【作者】 王锋；

【导师】 韩德五； 许瑞龄；

【作者基本信息】 山西医科大学 ， 生理学， 2009， 博士

【摘要】 老年性痴呆是一种慢性进行性精神衰退性疾病,临床表现以痴呆症状最为突出,病理改变以大脑的萎缩和变性为主。临床上主要包括阿尔茨海默型痴呆（Alzheimer’s Disease,AD）、脑血管性痴呆（VascularDementia,VD）和其他混合痴呆等,是世界范围内严重影响老年人健康的常见病、多发病。以往认为欧美国家人群中的痴呆患者以AD为主,亚洲国家则以VD为主。我国几个城市普查60岁以上老年人中,VD为人口的324/10万,AD为328/10万。1990年张明远等与美国几家科研单位合作流行病学调查发现,上海在55岁以上痴呆患者中,AD占64.7%,VD占26.8%。说明在我国至少在某些地区AD在痴呆患者中占主要地位。而AD防治的最大障碍是病因未明。AD在65岁以上的老年人有11%、80岁以上有50%会出现病症。其主要特征为智力的损伤,包括学习记忆、语言、读写、行为,以及对周围环境的识别,最终可导致死亡。其病理改变为弥漫性脑萎缩,以颞叶前、中部及海马、顶叶及前额叶区的萎缩最明显,脑室扩大,脑回变窄、脑沟加宽、脑裂加大;显微镜下可见神经原纤维缠结（NFT）,老年斑（SPs）和颗粒空泡变性比健康老年人明显增多。多年来对AD的病因做了大量的研究,但迄今为止,病因仍不明,确切的致病因素并未找到。因为病因极其复杂,有患者自身的生物学因素,也有多种环境与社会因素的影响。在AD发病机制的众多假说中,炎症机制假说占有重要的地位,大量的研究证明在AD发生过程中脑内有大量的炎性因子,如白介素-1（IL-1）、肿瘤坏死因子（tumor necrosisfactor-α,TNF-α）、白介素-6（IL-6）、转化生长因子β（Transforming Growth factorβ,TGF-β）、环氧化酶-2（Cyclooxygenase-2,COX-2）、补体及急性期反应蛋白的分泌。这些炎性因子通过一系列的信号转导途径,如NF-κB、丝裂原激活蛋白激酶(（mitogen-αctivated proteinkinase,MAPK）途径激活神经小胶质细胞（microglia,MG）和星形细胞（astrocyte,AC）,使之释放更多的炎性因子,同时它们又共同参与了脑局部炎症过程,与淀粉样蛋白（βamyloid,Aβ）和Tau蛋白的沉积紧密相关,这些研究确定了局部炎症在AD的病理生理过程中发挥了重要作用,甚至有的作者将炎症同SP和NFT共同并列为AD的3大主要特征。韩德五教授于1995年首次提出的肝功能衰竭发生机制的肠源性内毒素血症（Intestinalendotoxemia,IETM）假说认为肠源性内毒素激活枯否细胞所分泌的多种活性物质（如细胞因子、炎性介质、自由基等）可引起“继发性肝损伤”,是导致多种急慢性肝病演化为肝功能衰竭发生的重要机制之一。大量的实验及临床研究证明,不但肝脏疾患伴有IETM,烫伤、烧伤、创伤、出血性休克、急性胰腺炎等多种疾病也伴有IETM。在IETM中发挥核心作用的内毒素是革兰氏阴性杆菌细胞壁外膜表层构成成份,是脂多糖（LPS）与蛋白的复合体,是体内巨噬细胞最敏感的激活剂。正常肠道中含有大量的细菌和内毒素,生理状态下由于肠粘膜屏障的保护作用,肠腔中存在的内毒素只有极少量进入外周循环,对机体维持一定的免疫力可能有意义;同时肝脏的枯否细胞也会吞噬清除血液中过多的内毒素,防止IETM的发生。但是,当机体受到严重创伤（包括手术）、烧伤、放化疗和长期传统的肠外营养时,肠粘膜有可能发生通透性增高;或枯否细胞吞噬活性严重下降,对血浆中内毒素清除能力下降时,就有可能导致细菌及内毒素移位,引起内源性感染,甚至多脏器功能衰竭（MOF）。内毒素是到目前为止已知的诱导IL-1、TNF-α等最重要的因子,可以充分激活巨噬细胞,释放大量炎症因子,激活细胞因子级联反应:1、枯否细胞活化后可生成和释放TNF-α、IL-1、IL-6、血小板激活因子（platelet-activatedfactor,PAF）、PGs等细胞因之的分泌,损伤肝细胞;枯否细胞活化后所释放的白三烯,特别是LT-B₄具有强烈的化学趋化作用,吸引并激活中性粒细胞,产生并释放自由基,使细胞膜和细胞器膜发生脂质过氧化而损伤细胞。2、枯否细胞活化后生成和释放的活性氧中间产物（ROI）可引发脂质过氧化反应,损伤生物膜系统、核酸和蛋白质导致肝细胞死亡。3、枯否细胞与肝窦内皮细胞是花生四烯酸特别是PGD₂、PGE₂及血栓素的重要来源,可经对应的受体作用于肝细胞,引起蛋白磷酸化及膜稳定性变化。通过以上机制,IETM会导致全身炎症反应综合症（SIRS）、多器官功能不全（MOD）,甚至多器官功能衰竭（MOF）。综上所述,IETM会导致机体发生严重的SIRS,而局部炎症在AD的病理生理过程中发挥了重要作用。那么同样高发于老年人的IETM与AD二者之间有何联系?IETM会不会是AD发病的一个危险因素?IETM在AD发病过程中有何作用?目前尚未见到相关报道,为此,我们设计了本次课题研究研究计划。本研究由以下三个部分组成:第一部分D-半乳糖和三氯化铝建立的AD大鼠模型的研究实验目的及背景关于AD的发病机制目前有胆碱能学说、基因突变和多态性学说、自身免疫学说、能量代谢障碍学说等。目前,依据各种学说已建立了多种AD动物模型,但每种模型均有一定的局限性,不能全面模拟AD的病理特征。为了建立一种准确可靠的AD模型,我们进行了本次实验,以期为AD研究提供一种新的大鼠模型。大量研究表明,铝元素具有剂量与作用时间依赖性的诱导动物脑内发生病理变化,具有明显的神经毒作用,可引起明显的学习及记忆减退。D-半乳糖能较好的模拟小鼠的衰老表现,使小鼠机体出现与自然衰老相似的代谢紊乱特征,还引起脑神经元的一系列退行性改变,但腹腔注射D-半乳糖和AlCl₃制备阿尔茨海默病大鼠模型的报道还未见到。实验方法1、AD模型制备:模型组腹腔注射D-半乳糖60mg·kg^-1·d^-1（生理盐水配制,60g/L,给药量0.2ml/200g）和AlCl₃ 25mg·kg^-1·d^-1（双蒸水配制,25g/L,给药量0.2ml/200g）,连续90天;空白对照组腹腔注射等量的生理盐水和等量的双蒸水。2、Morris水迷宫行为学实验2.1定位航行实验水池分为N、E、S、W 4个象限。实验前1d将大鼠放入水池（不含平台）自由游泳2 min熟悉水迷宫环境,试验历时5d,每天分上午（n-1）,下午（n-2）两个时间段,每段分别训练4次。训练开始时将平台置于SW象限,每个时间段分别从池壁4个起始点将大鼠面向池壁放入水池,记录每次找到平台的时间（逃避潜伏期,escape latency）和游泳路径。如大鼠在120s内找不到平台,由实验者将其引上平台,潜伏期记为120s,并记录为1次寻找站台错误,每次间隔4min让大鼠休息,再行下次试验。2.2空间探索实验在第5天下午第5次训练时拆除水下平台,然后任选一个入水点将大鼠面向池壁放入水中,测其120s内在各象限的游泳距离及占总距离的百分率和120 s内跨越各象限平台相应位置次数及占总次数的百分率,并记录大鼠120s内搜索平台的路线图。3、大鼠脑组织总蛋白（TP）、组织过氧化物酶（POD）、总抗氧化能力（T-AOC）、胆碱乙酰转移酶（ChAT）、胆碱酯酶（ChE）、一氧化氮（NO）、一氧化氮合酶（NOS）测定:取各组大鼠脑组织,分析天平称湿重,按脑组织:生理盐水0.2g:1.8ml比例制成10%脑组织匀浆,3000r/min,离心15min,取上清液,冻存待测。TP、POD、T-AOC、ChAT、ChE、NOS、NO试剂盒由南京建成生物制品有限公司提供,严格按照操作说明进行操作。4、大鼠脑组织Tau蛋白浓度检测:双抗体夹心式酶联免疫吸附法（ELISA）。5、大鼠脑组织凋亡测定:TUNEL法与流式细胞仪检测。6、大鼠脑组织电镜观察:大鼠脑组织切成1mm³大小的组织块,经2%二甲砷酸钠缓冲戊二醛和1%锇酸双固定,丙酮脱水,包埋,制成超薄切片,在JEM 100CX型透射电镜下观察脑组织的改变。7、大鼠脑组织Aβ1-40检测:免疫组织化学法。8、大鼠脑组织APP、PS1和BACE mRNA检测:两步法半定量逆转录聚合酶链反应（RT-PCR）技术检测。实验结果本部分实验通过给大鼠腹腔注射D-半乳糖和AlCl₃,不但复制出大鼠整体衰老过程、学习记忆力减退、脑组织ChAT活性降低、ChE活性升高所致乙酰胆碱能系统活性降低,脑组织抗氧化能力降低及NO系统活性增高等AD特征性的变化。同时,AD大鼠脑组织内APP、PS1、BACE mRNA表达增强、出现Aβ沉积、Tau蛋白含量增高、脑细胞凋亡、老年斑等AD的特征性病理变化。实验结论腹腔注射D-半乳糖和AlCl₃可以成功制备阿尔茨海默病模型,为AD的研究提供了一种新的大鼠模型。第二部分IETM在AD模型发病中相关机制研究实验目的及背景IETM假说是韩德五教授于1995年首次提出的肝功能衰竭发生的机制。在IETM中发挥核心作用的内毒素可以激活枯否细胞分泌多种活性物质（如细胞因子、炎性介质、自由基等）,可引起“继发性肝损伤”,甚至会导致全身炎症反应综合症（SIRS）、多器官功能不全（MOD）,甚至多器官功能衰竭（MOF）的发生。大量的实验及临床研究证明,在烫伤、烧伤、创伤、出血性休克、急性胰腺炎等多种疾病也有IETM的发生。Goto等的研究表明337名72岁的老年人中有21.5%的老年人血浆内毒素水平高于正常,可见即使在正常人,IETM水平也随着年龄增长在逐渐升高。炎症机制假说在AD发病机制的众多假说中占有重要的地位,大量的研究证明在AD发生过程中脑内有大量的炎性因子,如TNF-α、IL-1、IL-6、TGF-β、COX-2、补体及急性期反应蛋白的分泌。这些炎性因子通过一系列的信号转道途径,如NF-KB、MAPK途径激活神经MG和AC,使之释放更多的炎性因子,同时它们又共同参与了脑局部炎症过程,与Aβ和Tau蛋白的沉积紧密相关,这些研究确定了局部炎症在AD的病理生理过程中发挥了重要作用。在我们所制备的AD模型体内是否也出现了IETM?如果是,IETM与AD二者之间有无内在的联系?IETM是否参与了AD的发病过程?IETM会不会是AD发病的一个危险因素?目前尚未见相关报道,为此我们进行了第二部分实验。实验方法1、血浆LPS、TNF-α、IL-1β及IL-10测定:采用改良过氯酸法检测血浆LPS含量,鲎试剂盒由上海医学化验所提供;放射免疫分析法测定血浆中TNF-α、IL-1β及IL-10含量,试剂盒由解放军总医院东亚免疫研究所提供。2、大鼠肝脏枯否细胞功能状态检测:免疫组织化学SABC染色法检测肝组织溶菌酶LYZ阳性细胞-KC的数量和形态。3、二胺氧化酶（Diamine oxidase,DAO）、谷氨酰胺（Glutamine,Gln）及谷氨酰胺酶（Glutaminase）活性检测:DAO采用黎君友等分光光度计法,谷氨酰胺采用酚一次氯酸法检测,谷氨酰胺酶活性采用荧光法测定。4、肠粘膜常规HE染色,光镜下观察粘膜结构变化。5、大鼠血清S-100β测定:双抗体夹心式酶联免疫吸附法（ELISA）,试剂盒购自上海森雄生物科技有限公司。6、大鼠脑组织ZO-1蛋白检测:免疫印迹法（Western blot）。实验结果腹腔注射D-半乳糖和AlCl₃制备的AD模型大鼠血浆中LPS、TNF-α、IL-1β及IL-10含量均明显升高,与对照组比较有统计学差异（P＜0.01）;肝组织KC数量减少,吞噬LPS功能减弱;肠粘膜及血脑屏障通透性均明显升高,屏障功能明显下降。实验结论腹腔注射D-半乳糖和AlCl₃制备的AD模型大鼠KC吞噬功能、肠粘膜屏障及血脑屏障功能均明显下降,血浆中LPS及TNF-α、IL-1β等炎症因子含量增高,AD大鼠体内出现了IETM。第三部分LPS激活BV-2细胞及其分子机制的研究研究目的及背景脑内炎症在AD、帕金森病等慢性神经变性疾病发病机理上发挥着重要作用。作为脑内固有免疫细胞的MG在AD发病中的急性与慢性神经炎症过程中均担当了重要的角色。MG被激活后产生释放大量的前体炎症因子,可以产生细胞毒性因子（蛋白水解酶、细胞因子、兴奋性氨基酸、毗啶、2,3-二羧酸、补体蛋白、活性氧媒介物、一氧化氮等）,Seabrook等的实验证明脑内炎症和MG有着密切关系,LPS诱导MG激活产生炎性细胞因子TNF-α、IL-1、IL-6,其信号转导途径有PTK途径、磷酸脂酶A₂（PLA₂）途径,还与细胞内核转录因子NF-κB、CREB等的活化有关。有研究表明,Aβ在AD发病中的毒性作用机制可能与其扰乱细胞内钙稳态、产生活性氧和自由基、增加兴奋性毒性等作用有关。我们通过体外培养BV-2细胞来研究在IETM中发挥核心致病作用的LPS对MG的激活作用、MG激活后释放的炎症因子在AD发病中的作用及其具体机制,从而进一步揭示IETM在AD发病中的作用及其机制。实验方法1、BV-2细胞培养:BV-2细胞株购自中国医科院协和医科大学基础医学细胞中心。含体积分数为0.1的胎牛血清的DMEM高糖培养基加青霉素100u/ml、链霉素100u/ml,置37℃、体积分数为0.05的CO₂的培养箱内培养,每隔1d换1次培养液,三四天传代1次。每次实验前细胞在含有10g/L牛血清白蛋白的DMEM培养基中培养48h后使细胞处于同步化状态。2、BV-2细胞分组:BV-2（阴性对照组）;BV-2+Aβ:30min、1h、3h、6h、12h、24h组;BV-2+LPS:30min、1h、3h、6h、12h、24h组。分别加入含有LPS（100ng/ml）、Aβ（20μg/ml）的培养基,培养30min、1h、3h、6h、12h、24h后,进行以下实验项目,每个处理组每个时间点观察6片。3、BV-2细胞形态学观察:取对数生长期BV-2细胞接种于6孔培养板中（密度为2×10⁵/孔）,每组设3个平行孔,倒置显微镜下观察BV-2细胞形态,拍照。4、BV-2细胞吞噬功能定量测定:参考Hirose的方法进行,细胞培养皿（10⁴个细胞/片/皿）中加入聚苯乙烯乳珠0.5×10⁸个,培养30min、1h、3h、6h、12h、24h后,PBS洗三次,洗去未被吞噬聚苯乙烯乳珠,直接倒置显微镜下对细胞所吞噬聚苯乙烯乳珠进行计数,观察,照像。每皿任取5个视野,每个视野随机数20个BV-2进行计数,取细胞吞噬聚苯乙烯乳珠数的平均值代表该皿细胞的吞噬功能。5、BV-2细胞OX-42检测:免疫荧光法。Aβ1-40与LPS分别作用30min、1h、3h、6h、12h、24h后终止细胞培养,PBS清洗3次,丙酮室温固定,3%过氧化氢-甲醇室温下10min,正常山羊封闭血清,室温下孵育20min,滴加一抗5μl（小鼠抗大鼠OX-421:100）,湿盒内37℃孵育2h,二抗（FITC）,室温1h,倒置显微镜观察拍照。每皿任取5个视野,取每个视野OX-42阳性表达细胞数及荧光强度代表该皿细胞的活性。6、BV-2细胞培养上清液TNF-α、IL-1β测定:双抗体夹心式酶联免疫吸附法（ELISA）。7、BV-2细胞凋亡检测:各组细胞用PBS制成细胞悬液,不低于1×10⁶个细胞/管,20%冰乙醇固定,轻轻混匀,PBS洗1次,重悬于500μlPBS后流式细胞仪检测。8、BV-2细胞p38、JNK、NF-κB蛋白表达水平检测:免疫印迹法（Western blot）。9、BV-2细胞[Ca²⁺]i变化检测:按实验要求培养的BV-2,加入终浓度为10μmol/L的Fluo-3/AM,37℃负载40min,置于MRC-1024型共聚焦显微镜下,488nm激发波长,522nm发射波长扫描。细胞游离[Ca²⁺]i动态测定:选定待测BV-2细胞后,先预扫4min获得基线值,继续扫描至4min时分别各自快速加入LPS 100ng/ml和Aβ1-40 20μg/ml观察荧光值的变化,用随机附带的Time-course/Rationmetric软件给出加药前后神经元内荧光值随时间变化的曲线。每皿取5个视野,每个视野随机选取10个BV-2细胞进行荧光值统计。荧光值强度代表[Ca²⁺]i的浓度。实验结果Aβ1-40与LPS均可以激活BV-2细胞,使其形态发生明显变化,吞噬能力增强,OX-42表达增强,TNF-α、IL-1β分泌增多,凋亡率较对照组明显升高（P＜0.01）,p-p38、p-JNK及NF-κB表达水平均明显高于对照组,[Ca²⁺]i明显升高。实验结论LPS可以通过p38、JNK、NF-κB信号转导通路迅速激活BV-2细胞,提高其吞噬能力,促进其分泌炎症因子;而且,LPS对BV-2细胞还有促凋亡作用,并可以使BV-2细胞钙稳态失衡,发生钙超载。LPS对BV-2的这种激活作用与Aβ1-40相似,可见LPS在AD的发病中发挥了与Aβ1-40类似的致病作用。通过以上三部分研究,我们可以得出以下结论:1、腹腔注射D-半乳糖和AlCl₃可以成功制备AD大鼠模型。2、AD模型大鼠血浆中LPS及炎症因子水平升高,枯否细胞吞噬能力、肠粘膜屏障功能及血脑屏障功能均明显下降,体内有IETM的发生。3、LPS可以通过p38、JNK、NF-κB信号转导通路激活BV-2细胞,释放炎症因子;促进BV-2细胞凋亡;使BV-2细胞钙稳态失衡,发生钙超载。从而从多个角度导致AD的发病。综上所述,IETM是AD发生发展的重要影响因子之一。更多还原

【Abstract】 Alzheimer’s disease is the most prevalent form of dementia affecting more than 20 million people worldwide.AD was originally recognised by AloisAlzheimer in 1907 as a separate form of dementia.Furthermore,as a consequence of the worlds aging population,the prevalence of AD is expected to increase.This highlights the importance of research investigating the mechanisms behind the development of the disease.Apart from the recognisable behavioural differences,it is difficult to positively diagnose someone with Alzheimer’s during the early stages of the disease.However the AD brain shows a consistent pathology amongst patients, with amyloid aggregates and neurofibrillary tangles evident in the AD brain.Intense research has identified genes involved in the development of these pathologies,namely the amyloid precursor protein and the presenilins.The increasing knowledge on AD pathogenesis has revealed the complex nature of this disease and highlights the need to elucidate the molecular mechanisms involved.This review discusses the current knowledge of the molecular mechanisms of Alzheimer Disease,focusing on amyloidogenesis,the role of the presenilin genes and the importance of animal models to further elucidate the mechanisms behind the development of the disease.The most common form of AD in the population（approximately 90%） occurs sporadically and is late in onset,usually occurring after 65 years of age.Familial Alzheimer’s disease（FAD）,only accounts for approximately 10%of cases and symptoms usually occur before the age of 65.The mode of inheritance of AD differs for each type.The majority of FAD and sporadic AD cases have a complex inheritance,while only 10%of FAD cases are inherited in an autosomal dominant pattern.In affected individuals the disease causes a progressive and permanent decline in memory and cognitive abilities.The first cognitive area affected is episodic memory.During disease progression,attention,executive functions,semantic memory,language and spatial orientation all begin to deteriorate.However the molecular,cellular and pathological triggers for the onset of the cognitive deterioration are poorly understood.Since this original observation the two main histological features of amyloid plaques and neurofibrillary tangles（NFT） have been described in the AD brain.These features are found to be present in the temporal neocortex and hippocampal regions of the AD brain.The hippocampus resides in the cerebral cortex of the forebrain and is thought to be involved in memory storage.Amyloid plaques and NFTs result from an aberration in deposition of the amyloid beta peptide（Aβpeptide） and the hyperphosphorylated tau protein respectively and these depositions lead to neuronal loss and neurotoxicity in the AD affected brain.Accumulation of Aβpeptides may be the key event in pathogenesis of AD.The exact mechanism by which Aβpeptide deposition induces neurotoxicity is unclear,but it appears the oxidative stress plays an important role.Oxidative stress is extensive in AD and Aβpeptides stimulate oxidative stress by both direct and indirect mechanisms.Aβpeptides by themselves may act as enzymes,as they are capable of directly producing hydrogen peroxide and generating free radicals through metal ion reduction.As well,Aβpeptide can bind to mitochondrial proteins resulting in the generation of free radicals.Furthermore,Aβpeptides generate oxidative stress via neuroinflammation. Considerable evidence has supported that neuroinflammation is associated with AD pathology. The death of neurons observed in AD is partly attributed to the activation of two major brain cell types,astrocytes and microglia that participate in the immune/inflammatory response to Aβdeposition.However,these cells can have both neuroprotective and neurodegenerative functions.Intestinal endotoxemia（IETM） was establish by Dewu Han in 1995,it claims that liver injury induced by various pathogenic factors（such as hepatitis virus,ethanol,drugs and hepatotoxicants,etc.） through their respective special pathogenesis is referred to as "primary liver injury"（PLI）.Liver injury resulted from endotoxin（lipopolysaccharide,LPS） and the activation of Kupffer cells by LPS while intestinal endotoxemia（IETM） occurred during the occurrence and development of hepatitis is named the "secondary liver injury"（SLI）.The after which has lost their own specificities of primary pathogenic factors is ascribed to IETM.The "secondary liver injury" is of important action and impact on development and prognosis of hepatitis.More severe IETM commonly results in excessive inflammatory responses,with serious hepatic necrosis,further severe hepatitis and even induces acute liver failure.Recent research show that IETM can be found in shock,stress,pancreatitis,and so on.In IETM,LPS plays a important role,it can lead inflammatory reaction.All inflammatory reactions are also immune reactions.It is the innate immune system that is first called into action. Later,the adaptive system may also respond.Chronic inflammation signifies that an immune reaction is being sustained.That is because healing has failed to take place.Chronic inflammation may involve the innate immune system,the adaptive immune system,or a combination of the two.AD is the prototypical autotoxic disorder.Classical immunologists had originally declared that it was a sterile,noninflammatory degenerative condition.The conclusion was based on the absence of infiltrating lymphocytes and monocytes which were easily observed in CNS infections and in presumed autoimmune diseases such as multiple sclerosis.This observation vindicated Hortega’s original conclusion in 1919 that microglia were phagocytic cells of mesodermal origin.Moreover,it established that chronic infammafion could exist in the absence of leukocyte infiltration.Two subsequent steps showed that the inflammation was self-damaging.The first was immunohistochemical,in which dystrophic neurites being damaged by the membrane attack complex of complement could be observed in AD tissue.The second was epidemiological,in which those taking anti-inflammatory agents appeared to be spared from AD.If the inflammation observed immunohistochemically had been beneficial instead of harmful,taking anti-inflammatories should have increased the risk of AD,and if they were merely cleaning up debris,then they should have had no effect.These epidemiological findings,which have been replicated in more than 20 studies,clearly show that the inflammation is contributing to the disease pathology,and that long-term consumption of NSAIDs reduces the risk of AD from twoto five fold.Any reservations that might still be held that microglia are the sentinels of the brain that respond to disease pathology should have been dispelled by the recent in vivo movies.When an abnormality was induced by laser damage to a capillary,the microglia immediately changed to an activated morphology.Within minutes,they began migrating to the lesion site where they sealed off the affected area and commenced phagocytosing the extravasated blood.Microglias are the brain representatives of van Furth’s monocyte phagocytic system.Their counterparts,such as Langerhan’s cells of the skin,Kupfer cells of the liver,osteoclasts of bone, and macrophages in many organs,are tissue-based phagocytes which can be presumed to carry out similar functions to microglia throughout the body.While it is only speculation at this stage, it might be anticipated that all cells of the monocyte phagocytic system would respond to pathological challenges in a similar fashion to those observed of brain microglia.Inflammatory cytokines are more powerful mediators and are therefore a better target. Enbrel and Remicade,which block TNF,have had great success in treating rheumatoid arthritis. They are globulins which do not cross the blood-brain barrier and are therefore not suitable for CNS diseases.Nevertheless,they illustrate the promise of small molecules which could reach the brain and block the actions of TNF.Presumably,blockers of IL-1 and IL-6 would also be effective.Blockers of complement activation,especially the membrane attack complex,are also attractive targets for future therapy.As well,blockers of intracellular pathways that promote DNA transcription of inflammatory mediators should be effective.There are numerous reviews that cover the toxic effects of inflammatory mediators and the spectrum of possible agents or pathways to block in order to reduce such effects.In order to explore the releationship between IETM and AD,we carry out this research. Our research includes three parts:1、Research of the AD rat model by intraperitoneal injection with D-Gal and AlCl₃.Methods:We established AD rat model by subjected to 90 days of intraperitoneal injection with D-galactose(60mg·kg^-1·d^-1) and aluminum trichloride(25mg·kg^-1·d^-1) to establish the Alzheimer disease’s model,the TP、POD、T-AOC、CHAT、ACHE、NOS、NO in rat brain were detectd by kits.The learning and memory ability of the rats were observed by Morris water maze. The level of apoptosis,Tau、Aβ1-40 and express of APP、PS1 and BACE of rat brain were explored by TUNEL,ELISA,Immunohistochemistry and RT-PCR.Results:The ability of learning and memory is obviously decreased in the rats treated with D-Galactose and AlCl₃ compared with the normal rats,while the express of APP、PS1 and BACE mRNA is increased.And there is apoptosis,deposition of Aβ1-40 and Tau in AD rat model.Conclusion:Intraperitoneal injection with D-Gal and AlCl₃ can make AD rat model.2、Research about the role of IETM in AD.Methods:We explored the level of LPS,TNF-α、IL-1β、IL-10、DAO、Gln and Glutaminase in plasma of rat,the functional status of KC by immunohistochemistry,the level of S-100βin plasma and ZO-1 in brain by ELISA and Western blot.Results:The level of LPS、TNF-α、IL-1β、IL-10、DAO、Gln and S-100βin AD rat model is raise up besides Glutaminase and ZO-1.Conclusion:The intestinal mucosa barrier and BBB were damaged,phagocytosis of KC was decreased.The level of IETM in AD model was step up.3、The mechanism of IETM influenced AD by use LPS to induce BV-2.Methods:BV-2 was cultured in DMEM with FCS、PCN and STM.Set group as:BV-2+Aβ: 30min、1h、3h、6h、12h、24h;BV-2+LPS:30min、1h、3h、6h、12h、24h.Observe the morphous in inverted microscope.Detect the phagocytosis of BV-2 with polystyrene and expression of OX-42 with immunofluorescence.The level of TNF-α、IL-1βin clear supernatant was detectd by ELISA,the expression of p38、JNK、NF-κB was detect by Western blot.Apoptosis was detectd by flow cytometry and[Ca²⁺]I was detect by Confocal Laser Scanning Microscope.Results:LPS and Aβ1-40 can induce BV-2,promote it phagocytosis、up-regulation the expression of OX-42 and secretion of TNF-α、IL-1β,apoptosis and[Ca²⁺]i was increased.The expression of p-p38,p-JNK and NF-κB increased too.Conclusion:LPS can activate BV-2 to release TNF-α、IL-1β.Latter lead to Inflammatory reaction through signal transduction,such as p38、JNK、NF-κB,finally induce AD. Conclusion1、Intraperitoneal injection with D-Gal and AlCl₃ can eatablish AD rat model.2、The level of IETM in AD model was step up.The intestinal mucosa barrier and BBB were damaged,phagocytosis of KC was decreased.3、LPS can activate BV-2 to release TNF-α、IL-1β.Latter lead to Inflammatory reaction through signal transduction,such as p38、JNK、NF-κB;LPS can induce BV-2 apoptosis and calcium overload.Through these channels LPS finally induce AD.In conclusion,there is IETM in AD,which play a important role.更多还原