【作者】 王昭君；

【导师】 祁金顺；

【作者基本信息】 山西医科大学 ， 生理学， 2014， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease, AD)即老年性痴呆(senile dementia),是一种以认知和精神状况改变为主要表现的神经系统退行性疾病,主要临床表现有智力下降、人格改变、进行性学习、记忆和注意力丧失。AD的主要病理学特征为：脑内出现高密度的老年斑(senile plaque, SP)、神经原纤维缠结(neurofibrillary tangles, NFTs)和神经元丧失等。随着社会人口逐渐老龄化,AD发病率也随之增加,严重威胁老年人的健康和生活质量,给社会带来巨大的人力及财力负担。然而,AD的病因和发病机制目前尚无定论,虽然普遍认为老年斑中的主要成分β-淀粉样蛋白(P-amyloid protein, Aβ)与AD的发病有关,但迄今为止仍缺乏有效清除Aβ或拮抗Aβ神经毒性作用的治疗性药物,攻克AD仍是医学乃至生命科学面临的一项艰巨而紧迫的任务。Humanin(HN)的发现为AD和其它记忆损伤疾病的治疗开辟了一条新的道路。日本东京KEIO大学医学院的Hashimoto教授及其同事根据AD患者的枕叶在发病过程中完好无损这一现象,推测在AD发病过程中,枕叶的神经元一定启动了某种基因的表达。以此为出发点,通过对从AD患者脑的枕叶提取的cDNA文库的表达进行功能监测,他们于2001年发现了一个编码24肽的基因,并将其命名为Humanin (HN)。离体实验证明,这种多肽能有效、特异地抑制多种FAD基因和Ap衍生物诱发的神经元的凋亡,因此被称为神经生存肽。目前,HN的作用机制仍不清楚,但这一发现至少为治疗AD带来了新的启示,人们期待通过对该多肽进行修饰,开发出一种具有美好前景的治疗AD的药物。令人欣喜的是,HN除存在于人染色体外,科学家们已在大鼠、小鼠、猴子、线虫等多种动物体内得到HN的对应物质。caricasole等从大鼠的cDNA文库中鉴别出HN的类似碱基片断,编码一条由38个氨基酸残基组成的多肽(C.末端比HN长14个氨基酸残基),并将其定名为Rattin (RN)。Rattin的发现为利用大鼠模型进行HN实验研究提供了极大便利。本研究在脑内注射Ap制备AD大鼠模型的基础上,对大鼠特异性的HN衍生物——Rattin的神经保护作用进行了系统的行为学、电生理和分子生物学机制研究,旨在为AD的预防和治疗提供一个新的、有效的策略。本研究进行了如下三部分工作：(1)鉴于Morris水迷宫是经典的评价动物空间学习和记忆能力的可靠方法,本研究通过Morris水迷宫行为学试验,观察了双侧大鼠海马内注射Rattin对Aβ31-35所致认知功能伤害的有效逆转效应；(2)鉴于AD患者认知功能下降与Ap伤害突触传递和突触可塑性有关,且海马LTP已被视为学习记忆机制的细胞学模型,本研究利用在体海马CAl区场电位记录手段,在成功诱导兴奋性突触后电位(EPSP)的基础上,观察了Rattin处理对Aβ31-35所致海马LTP压抑的逆转效应；(3)考虑到HN的酪氨酸蛋白激酶机制,本研究以原代培养的大鼠海马神经元为模型,观察了Rattin是否对Aβ31-35片段引起的神经元损伤具有保护作用,并使用Real-time PCR技术观察了酪氨酸蛋白激酶信号通路中MAPK、IP3K和JAK/STAT3三条不同途径在Aβ31-35所致细胞损伤以及Rattin神经保护作用中的贡献,在了解到Rattin可能是通过JAK/STAT3信号通路发挥的神经保护作用后,我们利用流式蛋白定量技术进一步检测了磷酸化STAT3的蛋白表达变化。最后,利用激光共聚焦Ca2+荧光成像技术,观察了Rattin是否对Aβ31-35片段引起的钙超载具有预防和拮抗作用,并利用JAK特异性拮抗剂AG490进一步明确了Rattin保护作用的机制。第一部分Rattin保护大鼠空间学习和记忆功能免受Aβ31-35片段引起的损伤目的：为了阐明Rattin在大鼠脑内的神经保护作用,本研究采用了经典的行为学方法---Morris水迷宫技术观察了双侧海马内注射Rattin对Aβ31-35诱导的大鼠空间学习和记忆功能损伤的影响。方法：选用无视觉和运动障碍的雄性SD大鼠(200-300g),随机分为对照组、Aβ31-35组、高浓度Rattin组以及不同浓度Rattin和Aβ31-35联合注射组,.每组10只。将麻醉后大鼠在脑立体定位仪下,应用微量注射器将Rattin和/或Aβ31-35注入双侧海马内,待动物清醒并恢复两周后,进行Morris水迷宫试验。试验包括定位航行、空间探索和可见平台三部分实验,主要观察药物处理对大鼠逃避潜伏期、游泳距离、大鼠在目标象限(即平台所在象限)游泳所占时间、距离百分比的影响。同时,测定大鼠游泳速度和视力,以排除大鼠的运动和视力障碍对检测指标的影响。结果：(1)Aβ31-35损伤了大鼠空间学习和记忆功能。首先利用连续五天的定位航行试验评估大鼠获取空间信息的学习能力,第六天撤除水下平台后通过空间探索试验检测大鼠的空间记忆能力。结果显示,随着训练日期的延长,各组大鼠寻找水下隐藏平台的平均逃避潜伏期和距离都逐渐减小。但与对照组相比,Aβ31-35组(n=10)大鼠的空间学习能力明显下降,寻找隐藏平台的逃避潜伏期和距离均延长。在训练的第2-5天平均逃避潜伏期分别为55.32±1.68秒(P<0.01),33.54±1.41秒(P<0.01),24.17±0.55秒(P<0.01),和18.38±0.51秒(P<0.01),明显长于对照组(n=10)的28.61±0.99秒,20.61±0.97秒,16.50±0.45秒和14.47±0.52秒。同样,在训练的第2-5天Aβ31-35组大鼠的平均逃避距离延长,分别为685.19±18.68厘米(P<0.01),491.34±13.60厘米(P<0.01),393.80±9.33厘米(P<0.01)和262.05±6.59厘米(P<0.01),明显长于同一时间点对照组的540.11±14.45厘米,349.33±16.30厘米,281.56±13.15厘米和208.92±3.55厘米。空间探索试验中,Aβ31-35组大鼠在目标象限中游泳时间和距离占总时间和总距离的百分比明显减少,分别从对照组的47.63±1.43%和45.09±1.41%下降至29.35±1.09%(P<0.01)和29.26土1.18%(P<0.01)。这些结果提示：双侧海马内注射Aβ31-35严重损伤了大鼠的空间学习和记忆功能。(2) Rattin有效防止了Aβ31-35所致的大鼠空间学习记忆损伤。首先发现,在隐藏平台试验中单独使用即使是高浓度的Rattin (2nmol)都不影响大鼠的逃避潜伏期和距离,目标象限内游泳时间和距离的百分比也没有改变。然而,不同浓度的Rattin (0.02、0.2、2nmol)可以剂量依赖性阻止Aβ31-35所致的空间学习和记忆缺陷。与单独使用Aβ31-35组比较,低浓度Rattin (0.02nmol)+Aβ32-35组(n=10)没有明显改变大鼠的平均逃避潜伏期和距离。但在0.2nmol Rattin+Aβ31-35组(n=10),训练第2-5天的平均逃避潜伏期分别下降至42.11±0.72秒(P<0.01),27.36±0.29秒(P<0.01),20.18±0.50秒(P<0.01),和15.48±0.27秒(P<0.01)；平均逃避距离分别下降至626.58±6.52厘米(P<0.01),427.67±10.77厘米(P<0.01),323.52±4.00厘米(P<0.01),和226.01±2.94厘米(P<0.01)。在2nmol Rattin+Aβ31-35组(n=10)平均逃避潜伏期进一步降至33.15±0.72秒(P<0.01),26.10±0.71秒(P<0.01),18.66±0.55秒(P<0.01)和14.92±0.19秒(P<0.01)；平均逃避距离下降至585.26±9.20厘米(P<0.01),381.80±6.54厘米(P<0.01),306.31±4.34厘米(P<0.01)和208.52±2.15厘米(P<0.01)。与低浓度Rattin (0.02nmol)相比,高浓度Rattin (0.2nmol和2nmol)+Aβ31-35组的平均逃避潜伏期和距离明显降低。在空间探索试验中,不同浓度的Rattin预处理剂量依赖性阻止了Aβ31-35所致的记忆缺失,使大鼠在目标区域的游泳总时间和总距离百分比明显增加,其中在0.2nmol和2nmol Rattin组时间百分比分别增加至36.60±1.67%(P<0.01)和42.71±1.58%(P<0.01),显著高于单独使用Aβ31-35组的29.35±1.09%；这两组的距离百分比分别增加至33.99±1.68%(P<0.05)和39.99±1.81%(P<0.01),高于单独使用Aβ31-35组的29.26±1.18%。上述结果提示单独使用Rattin不影响大鼠的空间认知功能,但不同浓度的Rattin预处理可以浓度依赖性防止Aβ31-35所致空间学习和记忆的损伤。(3)空间探索实验之后进行的可视平台实验显示,各组大鼠逃避潜伏期没有显著差异,到达可视平台的平均时间几乎均为14秒左右。连续5天学习期间,各组大鼠游泳速度也没有统计学差异(P>0.05),平均游泳速度约为19cm/s。这些结果提示,Aβ31-35所致空间学习和记忆的损伤和Rattin的保护作用不是由于大鼠的视力和运动能力发生变化引起的。结论：双侧海马内注射.Aβ31-35能够明显损害大鼠的空间学习和记忆能力,Rattin本身对大鼠的学习、记忆能力没有影响,但是可以剂量依赖性地拮抗Aβ31-35引起的大鼠空间学习记忆能力的损伤。提示脑内HN及其衍生物的高表达或使用外源性合成肽有可能对AD认知功能的下降起到一定的预防和治疗作用。第二部分Rattin保护在体大鼠海马长时程增强免受Aβ31-35引起的损伤目的：利用电生理学手段,在大鼠海马记录场兴奋性突触后电位(field excitatory postsynaptic potential,fEPSP)的基础上,观察了双侧海马内注射Aβ31-35和Rattin以及二者联合应用对大鼠海马CA1区在体长时程增强(LTP)的影响,旨在探讨Rattin拮抗Aβ31-35神经毒性作用的电生理学机制。方法：在Morris水迷宫试验之后,将麻醉大鼠固定在脑立体定位仪上,在三维推进装置的引导下,将绑定的同心圆双极刺激电极和单极记录电极精确插入到大鼠海马内Schaffer侧枝和CA1区。给予高频刺激(high frequency stimulation,HFS)后,在海马CA1区记录fEPSP,观察各种药物对基础fEPSP和LTP的影响。结果:(1)Aβ31-35抑制了在体大鼠海马CA1区的长时程增强效应。在给予海马内Schaffer侧枝三组高频刺激后,对照组(n=6)的fEPSP平均振幅从最初设定的对照值100%突然增加至187.67±7.24%,并且在高频刺激后一小时仍然维持在150%以上,表明在这种在体实验条件下可成功诱导长时程增强效应。与对照组相比较,注射Aβ31-35(10nmol, n=6)后明显抑制了海马长时程增强效应。高频刺激后0、15、30、60分钟时,fEPSP平均振幅分别从对照组的187.67±7.24%,167.01±1.66%,161.21±2.31%和150.53±3.01%降至165.96±4.11%(P<0.01),133.83±3.25%(P<0.01),126.11±3.94%(P<0.01)和112.14±1.94%(P<0.01)。(2) Rattin注射剂量依赖性地拮抗了Aβ31-35对海马长时程增强效应抑制作用。首先观察到,单独注射Rattin (10nmol, n=6)没有显著改变fEPSP平均幅度。在联合应用不同浓度的Rattin (0.02nmol、0.2nmol和2nmol)和Aβ31-35(10nmol)组中,低浓度(0.02nmol) Rattin没有改变Aβ31-35对LTP的抑制效应。fEPSP振幅在四个不同时间点的百分比分别为165.33±4.08%(P>0.05),138.63±2.43%(P>0.05),125.95±2.33%(P>0.05)和114.01±2.13%(P>0.05),与单独使用Aβ31-35相比有一定增加但是并没有显著性差异。较高浓度(0.2nmol和2nmol)的Rattin可以显著拮抗10nmol的Aβ31-35对LTP的抑制作用(n=6),在0.2nmol Rattin+Aβ31-35组,fEPSP平均振幅在相同时间点分别增加至166.40±5.95%(P>0.05),143.24±4.03%(P<0.05),135.25±3.61%(P>0.05)和124.43±1.54%(P<0.01)；在2nmol Rattin+Aβ31-35组,fEPSP平均振幅在相同时间点增加至172.10±5.21%(P>0.05),145.42±4.45%(P<0.05),136.44±4.23%(P<0.05)和131.48±1.50%(P<0.01),显著高于单独使用Aβ31-35组。该结果提示,较高浓度的Rattin (0.2nmol和2nmol)能有效保护大鼠海马CAl区的突触可塑性。(3) Rattin和Aβ31-35不影响海马的双脉冲易化(PPF)。为了澄清突触前效应是否参与了Aβ31-35和Rattin对突触可塑性的作用,在所有各组HFS之前都检测了海马CA1区的PPF。在Schaffer侧枝给于成对脉冲刺激后,PPF规则性出现,且第二个fEPSP振幅高于第一个fEPSP。在对照组、Rattin和不同浓度的Rattin加Aβ31-35组中PPF比值分别为170.05±5.20%,169.53±3.60%,173.18±2.68%,173.89±2.88%,169.68±3.78%和171.68±3.43%,各组间没有任何显著性差异(P>0.05)。上述结果提示Rattin和Aβ31-35不影响大鼠海马CA1区突触前神经递质的释放。结论：Rattin可以剂量依赖性拮抗Aβ31-35引起的在体大鼠海马CA1区LTP的抑制。Rattin对LTP的保护效应与其行为学的结果保持了高度的一致性,这从电生理角度在解释了Rattin改善大鼠认知功能损伤的可能机制。第三部分Rattin保护海马神经元拮抗Aβ31-35的分子机制研究目的：进一步调查Rattin是否可以抑制Aβ31-35对细胞的毒性作用,并探讨其保护作用是否通过酪氨酸激酶信号通路中的某些途径发挥效应,同时观察Rattin对Aβ31-35诱导的钙超载是否有保护作用。方法：以原代培养的大鼠海马神经元为模型,观察Aβ31-35片段引起的神经元损伤以及Rattin的保护效应,并使用Real-time PCR技术观察酪氨酸蛋白激酶信号通路中MAPK、IP3K和JAK/STAT3三条不同途径在Aβ31-35细胞损伤过程中的作用以及Rattin的神经保护作用；于此同时,采用激光共聚焦Ca2+荧光成像技术,观察Aβ31-35对原代培养的大鼠海马神经元细胞内Ca2+浓度(intracellular calcium concentration,[Ca2+]i)的影响,研究Rattin对Aβ31-35诱导的钙超载的保护作用,进一步利用JAK特异性拮抗剂AG490明确Rattin保护作用的机制。结果：(1)Aβ31-35和Rattin预处理对海马神经元存活率的影响。与对照组相比,Aβ31-35组(11=6)的细胞活性百分比显著减少(42.5±3.3%,P<0.01)；单独给予Rattin,即使在高浓度下(1001μM),对细胞活力也没有影响,细胞存活率仍然为98.3±2.9%(P>0.05)；但Rattin可以剂量依赖性地抑制Aβ31-35介导的细胞毒性作用,与单纯Aβ31-35组相比,低浓度Rattin (1μM)+AP31-35组(n=6)的细胞存活率没有显著性(45.8±2.77%),高浓度Rattin(10μM,100μM)+Aβ31-35组则明显增加,细胞存活率分别为62.23±3.0%(P<0.01)和96.5±4.6%(P<0.01)；给予特异性酪氨酸蛋白激酶抑制剂后,在Genistein+Rattin+Aβ31-35共处理组细胞存活率为44.07±3.65%(P<0.01),较Rattin+Aβ31-35组明显下降。上述结果提示,Rattin可以剂量依赖性拮抗Aβ31-35引起的海马神经元损伤,这种神经保护作用可能与上调酪氨酸蛋白激酶信号通路有关。(2) Rattin和Aβ31-35对酪氨酸蛋白激酶相关信号通路的影响。为了阐明Rattin神经保护作用的分子机制,本实验采用荧光实时定量PCR技术检测了原代海马神经元中MAPK、IP3K和STAT3mRNA的基因表达变化。在单纯Aβ31-35处理组(n=6),STAT3mRNA的表达(0.7226±0.05393)较空白对照组(1.0090±0.07034)显著性下调(P<0.01)；相反,p38MAPK和IP3K mRNA表达水平分别增加到1.2890±0.04055和1.3223±0.0554,较对照组的0.9657±0.05092和0.9647±0.07828显著上调(P<0.01)。与Aβ31-35组相比,100μM Rattin+20μMAβ31-35共处理组中STAT3mRNA的表达水平上调为1.0188±0.11248(P<0.01),而p38MAPK和IP3K mRNA基本未变(1.3647±0.0783和1.2752±0.03528,P>0.05)。这一结果提示,STAT3mRNA基因表达的上调可能与Rattin的神经保护机制有关。(3) Rattin和Aβ31-35对磷酸化STAT3蛋白表达的影响。为了验证Rattin神经保护作用的分子机制确实是通过JAK-STAT3信号通路实现的,本实验采用流式蛋白定量技术检测了原代海马神经元中磷酸化STAT3的蛋白表达变化。在单纯Aβ31-35处理组(11=6),p-STAT3的表达(17.5533±2.8854pg/ml, P<0.01)较空白对照组(43.2467±3.3689pg/ml)显著性下调；与Aβ31-35组相比,100μMRattin+20μM AP31.35共处理组中p-STAT3的表达水平上调为29.8133±2.3655pg/ml (P<0.01)。这一结果提示,p-STAT3蛋白表达的上调可能与Rattin的神经保护机制有关。(4) Rating和Aβ31-35对原代海马神经元细胞内[Ca2+]的影响。将静息状态下(记录后2分钟内)的相对荧光强度设定为1,对照组荧光强度在近30min内没有明显变化,散点图几乎近于水平直线。给予Aβ31-35(n=20)之后,大部分神经元的荧光强度逐渐且显著增加,在记录时间范围内(>20分钟)始终保持持续的高水平,给药后20分钟时[Ca2+]i的相对荧光强度为192.1±2.71%,与对照组相比有显著性差异(P<0.01)。在Rattin(100μM)+Aβ31-35组,Aβ31-35引起的[Ca2+]i水平升高现象被Rattin显著性逆转(120.15±3.6%,n=20,P<0.01)。进一步,给予100μM AG490(JAK抑制剂)预处理30min之后,Rattin的保护效应明显下降。与Rattin+Aβ31-35组相比AG490预处理组的相对荧光强度明显升高(184.07±2.98%,n=20,P<0.01)。提示AG490可以阻断Rattin拮抗Aβ31-35引起的[Ca2+]i超载,说明Rattin的神经保护作用可能与JAK/STAT3途径激活有关。结论：Rattin可以剂量依赖性拮抗Aβ31-35引起的海马神经元损伤,这种神经保护作用与酪氨酸蛋白激酶信号通路中的JAK/STAT3途径,而非MAPK和IP3K途径的激活有关；Aβ31-35的神经毒性与细胞内钙超载有关,Rattiri可以通过激活JAK/STAT3信号通路减轻Aβ31-35引起的细胞内钙超载。因此,激活脑内JAK/STAT3相关信号通路可能将有利于Ap相关的认知障碍的预防和治疗。更多还原

【Abstract】 Alzheimer’s disease (AD) is an irreversible neurodegenerative disease, which occurs in the central nervous system, and characterized by progressive cognitive dysfunction, including loss of learning and memory, and dementia finally. The main pathological features of AD consist of high density of senile plaque (SP) in the brain, neurofibrillary tangles (NFTs) and loss of neurons. However, the etiology and pathogenesis of AD are not clear. The accumulation of amyloid-β (Aβ) in brain is thought to be causative for the progression of AD. Consequently, it is critical to clear the aggregated AP or block the AP toxicity in the brain for the prevention and clinical treatment of AD.Humanin (HN) and its derivatives have been thought to have potential therapeutic application in AD. Hashimoto et al. found that the occipital lobe did not change in the whole process of AD in patients. So they speculated that some genes in occipital lobe neurons must have been activated in the process of AD. HN is discovered as a24-amino acid peptide, and the cDNA was identified from an AD patient’s occipital lobe of brain in2001. HN can effectively protect neuronal cells against almost all AD-related insults, such as various FAD genes, anti-APP antibody, and neurotoxic Aβ in vitro. However, the underlying molecular mechanisms of HN’s neuroprotective roles remain unclear. Furthermore, it was found that HN also existed in rat, mouse, monkey and nematode. Caricasole et al. cloned a homologue gene of HN in the rat named as Rattin, which encodes a peptide of38amino acids (14residues longer than Humanin), with73%identity in the conserved region to HN. The availability of Rattin facilitates the studies in rats aimed at elucidating the mechanism of action of HN-like peptides and the study of their pharmacological properties in vivo. Therefore, on the basis of preparing AD rat model with Aβ injection, the present study investigated the neuroprotecitve effects and mechanisms of Rattin against Aβ-induced impairments in behavioral, electrophysiological and molecular levels.We carried out the research in three parts as follows:(1) considering that MWM test is the classic method to evaluate the ability of spatial learning and memory of rats, we investigated the effects of Rattin on the Aβ31-35-induced impairment of spatial learning and memory of rats by bilateral intrahippocampal injection and using MWM test;(2) in view of the close correlations between spatial cognitive behavior and hippocampal LTP, which is widely accepted as one of the cellular models of learning and memory, the present study investigated the effects of Rattin on the Aβ31-35-induced impairment in in vivo LTP in rat hippocampal CA1region by recording field excitatory postsynaptic potentials (fEPSPs);(3) the protective effects and mechanisms of Rattin on Aβ31-35-induced neurotoxicity in primary cultured rat hippocampal neurons were observed, and three different tyrosine protein kinase signal pathway (MAPK, IP3K and JAK/STAT3) and intracellular calcium concentration ([Ca2+]i were checked by using real-time PCR and laser confocal image techniques. Part ⅠThe Neuroprotection of Rattin against Neurotoxic Amyloid β Protein in Spatial Learning and Memory of RatsObjective:The deposition of amyloid β protein (AP) is thought to be responsible for the loss of memory in Alzheimer’s disease (AD), and Aβ31-35should be a shorter active sequence for the neurotoxicity of Aβ. Rattin, a rat homolog of humanin (HN), shares the ability with HN to protect neurons against amyloid β protein (Aβ)-induced cellular toxicity but with much more effectiveness than HN. Significantly, Rattin facilitates the studies of HN-like peptides in rat model and avoids the interspecific differences that directly using HN in rats. However, it is still unclear whether Rattin can prevent against the Aβ-induced cognitive deficits. In the present study, we investigated the effects of Rattin and Aβ31-35on the spatial learning and memory of rats by using Morris Water Maze (MWM) test.Methods:Sprague-Dawley (SD) rats (200-300g) were divided randomly into six groups:Control, AP31.35, Rattin(2nmol), and Rattin (0.02,0.2,2nmol)+Aβ31-35group (n=10, per group). Rats were anesthetized with urethane and placed in the stereotaxic apparatus for surgery and injection. MWM tests (Hidden platform test, probe trials, visible platform test) were performed2weeks after drugs injection. The escape latency (s), distance traveled (cm) and swimming speed (cm/s) were calculated in acquisition phase (hidden platform tests), and the percentage of the total time and the distances in the different quadrants was recorded in probe trials. To exclude the possibility that the results above such as the change in escape latency were due to the impairment of visual or motor ability of rats, the escapelatencies of rats were tested again in visible platform condition after probe trials, and the average swim speeds of rats in all groups during5days of successive learning were also calculated.Results:(1) Aβ31-35impaired the spatial learning and memory of rats in MWM test. In MWM test, the learning ability of rats to acquire spatial information was first assessed by five consecutive days of hidden platform test. Subsequently, the spatial memory was tested by probe trials on day six. As expected, the average escape latency and distance of rats in searching for the hidden underwater platform decreased with the increase of training days in each group. However, the spatial learning ability of rats in the Aβ31-35group (n=10) was significantly affected, with longer escape latency and distance in searching for the underwater platform. The average escape latencies were55.32±1.68seconds (P<0.01),33.54±1.41seconds (P<0.01),24.17±0.55seconds (P<0.01), and18.38±0.51seconds (P<0.01) on training days2-5, respectively, significantly larger than the values of28.61±0.99seconds,20.61±0.97seconds,16.50±0.45seconds, and14.47±0.52seconds in control group (n=10). Similarly, the average escape distances of the rats in Aβ31-35group increased on training days2-5, being685.19±18.68cm (P<0.01),491.34±13.60cm (P<0.01),393.80±9.33cm (P<0.01) and262.05±6.59cm (P<0.01) respectively, significantly larger than the values of540.11±14.45cm,349.33±16.30cm,281.56±13.15cm and208.92±3.55cm in the control group on the same training days. In the memory test with probe trials, the percentage of total time and distance in the target quadrant significantly decreased in the Aβ31-35group, from47.63±1.43%and45.09±1.41%in control group decreased to29.35±1.09%(P<0.01) and29.26±1.18%(P<0.01), respectively. These results indicate that bilateral intrahippocampal injection of Aβ31-35seriously impaired the spatial learning and memory of rats.(2) Rattin prevented against Aβ31-35-induced deficits in spatial learning and memory of rats. To investigate the neuroprotective roles of Rattin against Aβ31-35, the effects of Rattin alone on the spatial learning and memory of rats were observed first. We found that Rattin alone, even at a high concentration (2nmol), did not affect the escape latency and the escape distance of rats in the hidden platform tests. Similarly, the percentage of swimming distance in target quadrant was not changed by Rattin alone, compared with the control group. In addition, pretreatment with different concentrations (0.02,0.2,2nmol) of Rattin dose-dependently prevented the Aβ31-35induced deficits in spatial learning on almost all training days. Compared with Aβ31-35alone group, the average escape latency and distance in low concentration (0.02nmol) Rattin plus Aβ31-35group (n=10) were not significantly changed. However, in0.2nmol Rattin plus Aβ31-35group (n=10), the average escape latency decreased to42.11±0.72seconds (P<0.01),27.36±0.29seconds (P<0.01),20.18±0.50seconds (P <0.01), and15.48±0.27seconds (P<0.01); the mean escape distances decreased to626.58±6.52cm (P<0.01),427.67±10.77cm (P<0.01),323.52±4.00cm (P<0.01), and226.01±2.94cm (P<0.01) on training days2-5, respectively. In2nmol Rattin plus Aβ31-35group (n=10), the average escape latency further decreased to33.15±0.72seconds (P<0.01),26.10±0.71seconds (P<0.01),18.66±0.55seconds (P<0.01),14.92±0.19seconds (P<0.01); the mean escape distances decreased to585.26±9.20cm (P<0.01),381.80±6.54cm (P<0.01),306.31±4.34cm (P<0.01), and208.52±2.15cm (P<0.01) on training days2-5, respectively. Also, compared with the lower concentration of Rattin (0.02nmol), higher concentrations (0.2nmol and2nmol) of Rattin showed enhanced protective effects, with significant decreases in the average escape latency and distance in coapplication groups. In the probe trials, pretreatment with different concentrations of Rattin dose-dependently prevented the Aβ31-35-induced memory deficit. The percentage of total time and total distance for rats spent in the target quadrant significantly increased in higher concentrations of Rattin plus Aβ31-35groups, in which the time percentages increased to36.60±1.67%and42.71±1.58%in0.2nmol and2nmol Rattin groups respectively, significantly larger than the value of29.35±1.09%in AP31.35alone group (P<0.05); the distance percentages increased to33.99±1.68%(P<0.05) and39.99±1.81%(P<0.01) in the two Rattin groups, respectively, larger than the value of29.26±1.18%in Aβ31-35alone group. The results indicate that Rattin alone do not affect the spatial cognition of rats, but pretreatment with Rattin dose-dependently prevent against AP31-35induced deficits in spatial learning and memory.(3) Neither Rattin nor Aβ31-35did affect the vision and motor ability of rats. To exclude the possibility that the results above such as the change in escape latency were due to the impairment of visual or motor ability of rats, the escape latencies of rats were tested again in visible platform condition after probe trials, and the average swim speeds of rats in all groups during5days of successive learning were also compared. There was no difference in the escape latency among all groups in the visible platform test, and the average time reaching to the visible platform was approximately14s. In addition, there was no significant statistical difference (P>0.05) in swimming speeds among all groups in5days of successive learning acquisition, with an approximately19cm/s of average swim speed. These results indicated that the vision and the motor ability of rats were not affected by Rattin or Aβ31-35in the MWM tests.Conclusion:These findings show that bilateral intrahippocampal injection of AP31.35could impair the spatial learning and memory, while Rattin could dose-dependently prevent the Aβ31-35-induced decline in spatial cognitive behavior of rats. Therefore, the present study strongly suggests that application of exogenous Rattin or up-regulation of endogenous HN in the brain might be beneficial to the prevention and treatment of Aβ-related cognitive deficits such as in AD. Part ⅡRattin Protects against Aβ31-35-Induced Impairment of Hippocampal Long Term Potentiation in Rat Hippocampal CA1Region in vivoObjective:Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the elderly leading to progressive loss of memory and cognitive deficits. Amyloid β protein (Aβ) is thought to be responsible for loss of memory in AD, and Aβ31-35should be a shorter active sequence responsible for the neurotoxicity of Aβ. In the behavior study above, we found that Rattin has a protective effect against Aβ31-35-induced behavior impairment. However, its underlying mechanisms are almost unclear. As an electrophysiological neuronal model of synapse plasticity, hippocampal long term potentiation (LTP) has been widely used for the research of cellular basis of learning and memory. Therefore, in the present study, we investigated the effects of Rattin and Aβ31-35on the hippocampal LTP of rats by using in vivo hippocampal field potential recording.Methods:In vivo electrophysiological recording of LTP in hippocampal CA1region of rats was performed after finishing the MWM test. Rats were anesthetized with urethane and placed in the stereotaxic apparatus for surgery and electrophysiological recording. A pair of parallel bound stimulating/recording electrodes was inserted into the hippocampus. The tip of the monopolar recording electrode was positioned at the stratum radiatum in the CA1region and the tip of bipolar stimulating electrode was inserted into the hippocampal Schaffer-collateral region. LTP was induced by using a high frequency stimulus (HFS), and fEPSPs were monitored for a further1h to observe the induction and maintenance of LTP.Results:(1) Aβ31-35suppressed in vivo hippocampal LTP in the CA1region of rats. The change in fEPSP amplitude was used to represent the synaptic efficacy in the CA1region. Immediately after delivering three sets of HFS, the average amplitude of fEPSPs in control group (n=6) increased abruptly to187.67±7.24%from the initial control value set as100%, remaining at150%1hour after HFS, indicating a successful induction of LTP in this in vivo experimental condition. Compared with control, Aβ31-35(10nmol, n=6) injection significantly suppressed the hippocampal LTP. The average standardized fEPSP amplitude in Aβ31-35group decreased to165.96±4.11%(P<0.01),133.83±3.25%(P<0.01),126.11±3.94%(P<0.01), and112.14±1.94%(P<0.01) from187.67±7.24%,167.01±1.66%,161.21±2.31%and150.53±3.01%in control group at0min,15min,30min, and60min after HFS, respectively. However, injection of Rattin alone did not change the fEPSP amplitude at the same four time points post-HFS (10nmol, n=6), compared with the control group.(2) Rattin partly and dose-dependently prevented the Aβ31-35-induced depression of hippocampal LTP. Furthermore, we investigated the effects of Rattin on the Aβ31-35-induced impairment of LTP by co-application of different concentrations of Rattin (0.02nmol,0.2nmol, and2nmol) and Aβ31-35(10nmol).0.2nmol and2nmol, but not0.02nmol, of Rattin significantly prevented10nmol Aβ31-35-induced suppression of LTP (n=6, per group). In0.02nmol Rattin plus Aβ31-35group, the percentage of fEPSP amplitude was165.33±4.08%(P>0.05),138.63±2.43%(P>0.05),125.95±2.33%(P>0.05), and114.01±2.13%(P>0.05) at the four time points, respectively, with an increase but without significant difference compared with Aβ31-35alone group. In0.2nmol Rattin plus AP31-35group, the average fEPSP amplitude increased to166.40±5.95%(P>0.05),143.24±4.03%(P<0.05), 135.25±3.61%(P>0.05), and124.43±1.54%(P<0.01) at the same time points. In particular, in2nmol Rattin plus Aβ31-35group, the average fEPSP amplitudes further increased to172.10±5.21%(P>0.05),145.42±4.45%(P<0.05),136.44±4.23%(P<0.05), and131.48±1.50%(P<0.01) at the same four time points post-HFS, respectively, significantly larger than the values in Aβ31-35alone group. The results indicate that higher concentrations (0.2nmol and2nmol) of Rattin effectively protected hippocampal synaptic plasticity in the CA1region of rats.(3) Neither Rattin nor Aβ31.35did affect the hippocampal PPF. To clarify whether the presynaptic mechanism was involved in the effects of Aβ31.35and Rattin on synaptic plasticity, PPF in the hippocampal CA1region was examined in all groups immediately before HFS. After paired pulses were applied to the Schaffer collaterals, the PPF always appeared, with the second fEPSP larger than the first one. The PPF ratio values were170.05±5.20%,169.53±3.60%,173.18±2.68%,173.89±2.88%,169.68±3.78%, and171.68±3.43%in control, Aβ31-35, Rattin, and different concentrations of Rattin plus Aβ31-35groups, respectively, without any significant statistical difference (P>0.05). These results indicate that Rattin and AP31-35do not affect the presynaptic neurotransmitter release in the hippocampal CA1region of rats.Conclusion:Rattin effectively prevente against Aβ31-35-induced LTP suppression in dose dependent manner. These findings may partly explain the cellular mechanism of Rattin in improving spatial learning and memory, suggesting that Rattin might be one of the promising candidates for the treatment of AD in the future. Part ⅢEffects of Rattin on Aβ31-35-Induced Neurotoxocity and Molecular Mechanisms in Cultured Primary Rat Hippocampal NeuronsObjective:To further investigate whether Rattin can inhibit Aβ31-35toxic effect on cultured primary rat hippocampal neurons, and which tyrosine kinase signaling pathway is involved in the protective role of Rattin, we observed the effects of Rattin on Aβ31-35-induced neuronal death and calcium influx in cultured primary rat hippocampal neurons.Methods:CCK-8assay, real-time PCR, flow cytometry and calcium image techniques were used to observe the effects of Rattin on Aβ31-35-induced neurotoxocity, molecular signaling pathways and [Ca2+]i on cultured primary rat hippocampal neurons.Results:(1) Rattin prevented Aβ31-35-induced decline in cell viability. The growth of cultured hippocampal neurons were observed at different days after plating, and the mature cells were used for further experiments7-10days after plating. CCK-8cell viability assay was used to measure the toxicity of the pre-incubated Aβ31-35(20μM) with or without added Rattin in different concentration (1,10,100μM) on primary cultured hippocampal neurons. As shown in Fig.2, the percentage of cell viability in the Aβ31-35group (n=6) significantly decreased to42.5±3.3%from100%of control (P<0.01). Meanwhile, we found that Rattin alone, even at a high concentration (100μM), had no effect, the value of cell viability being98.3±2.9%(P<0.05) compared with control group. Interestingly, Rattin inhibited cytotoxicity induced by Aβ31-35in a dose-dependent manner. Compared with Aβ31-35alone group, the percentage of cell viability in low concentration (1μM) Rattin plus Aβ31-35group (n=6) were not significantly changed (45.8±2.77%). However, the percentage of cell viability significantly increased in higher concentrations of Rattin plus AP31.35groups, in which the cell viability percentages increased to62.23±3.0%and96.5±4.6%in10μM and100μM Rattin groups, respectively, significantly larger than the Aβ31-35alone group (P<0.05). The values in Genistein plus Rattin and Aβ31-35group (n=6) was44.07±3.65%, similar to that in Aβ31-35alone group.(2) Rattin did not affect Aβ31-35-induced increase in MAPK and IP3K mRNA, but effectively blocked Aβ31-35-induced down-regulation of STAT3mRNA in cultured primary rat hippocampal neurons. To clarify the probable molecular mechanism underlying the neuroprotective roles of Rattin against Aβ in spatial cognition and synaptic plasticity, the expression levels of MAPK, IP3K, and STAT3mRNA in the hippocampus of rats were measured by using real-time PCR technique. As shown in Fig.3, the expression of STAT3mRNA in20nmol Aβ31-35group (n=6) was significantly down-regulated, from1.0090±0.07034in control group (n=6) decreased to0.7226±0.05393(P<0.01). On the contrary, p38MAPK and IP3K mRNA in10nmol Aβ31-35group significantly increased to1.2890±0.04055and1.3223±0.0554from0.9657±0.05092and0.9647±0.07828in the control group (P<0.01). Interestingly, compared with Aβ31-35alone group, the relative mRNA levels for STAT3in the co-application of100μM Rattin and20μM Aβ31-35group increased to1.0188±0.11248(P<0.01), while the expression of p38MAPK and IP3K mRNA were not changed, and the values were1.3647±0.0783and1.2752±0.03528, respectively. The results indicate that the activation of STAT3mRNA expression may be involved in the neuroprotective mechanism of Rattin.(3) Rattin effectively blocked Aβ31-35-induced down-regulation of STAT3mRNA in cultured primary rat hippocampal neurons. To clarify the probable molecular mechanism underlying the neuroprotective roles of Rattin against Aβ31-35in spatial cognition and synaptic plasticity, the expression levels of p-STAT3in the hippocampus of rats were measured by using BD Cytometric Bead Array (CBA) Phospho Stat3(Y705) Flex Set kit and BD CBA Cell Signaling Master Buffer Kit (BD Biosciences) with a FACSCalibur flow cytometer (BD Biosciences). As shown in Fig.4, the expression of p-STAT3in20nmol Aβ31-35group (n=6) was significantly down-regulated, from43.2467±3.3689pg/ml in control group (n=6) decreased to17.5533±2.8854pg/ml (P<0.01). On the contrary, compared with Aβ31-35alone group, the levels for p-STAT3in the co-application of100μM Rattin and20μM Aβ31-35group increased to29.8133±2.3655pg/ml (P<0.01). The results indicate that the activation of p-STAT3expression may be involved in the neuroprotective mechanism of Rattin.(4) Pretreatment with Rattin significantly protected against Aβ31-35-induced elevation of [Ca2+]i, which could be abolished by JAK inhibitor. The mechanism of Aβ-induced neurotoxicity involves in the perturbation of Ca2+homeostasis. Firstly, we investigated the change of [Ca2+]; level in rat primary cultured hippocampal neuron by applying20μM Aβ31-35using laser scanning confocal fluorescent imaging technique. As shown in Fig.4A, the relative fluorescent intensity at resting condition in the control group (n=20) was very stable, nearly being a straight horizontal line. After application of Aβ31.35(n=20), the fluorescent intensity in most of neurons gradually and persistently increased during all of recording time. Fig.4B showed the relative fluorescent intensity values of [Ca2+]i in different experimental groups at20min after application of Aβ31-35. Obviously, Aβ31-35increased the relative fluorescent intensity of [Ca2+]i, being192.1±2.71%, significantly larger than the value in control group (P<0.01). This result indicates that Aβ31-35can increase [Ca2+]i which might be responsible for the neurotoxicity of Aβ seen in cultured hippocampal neurons. Further, we investigated the effects of pretreatment with Rattin on AP31-35induced [Ca2+]i elevation. As shown in the Fig.5, Aβ31-35-induced elevation of [Ca2+]i level was mostly reversed by Rattin (100μM) and the relative fluorescent intensity decreased to120.15±3.6%(n=20, P<0.01). These results indicated that Rattin can protect against Aβ31-35-induced intracellular calcium overloading. To investigate the molecular mechanism of the protection of Rattin against Aβ31-35, we also observed the effects of Rattin on Aβ31-35induced [Ca2+]i elevation in the presence of100μM AG490, a JAK inhibitor. The result showed that pretreatment with AG490for30min, essentially blocked the protective effect of Rattin against Aβ31-35induced [Ca2+]i elevation. Compared with co-application of Rattin and Aβ31-35, the relative fluorescent intensity in co-application of AG490(100μM), Rattin (10μM) plus Aβ31-35group increased to184.07±2.98%(n=20, P<0.01). These results indicated that the protective roles of Rattin against Aβ31-35induced [Ca2+]i elevation are closely associated with the activation of JAK.Conclusion:Rattin has neuroprotection against Aβ31-35-induced neurotoxicity in a dose-dependent manner, and the underlying protective mechanism of Rattin might be mediated via JAK/STAT3of protein tyrosine kinase signal pathways. Meanwhile, the results also indicate that the mechanism of Rattin is probably associated with maintaining intracellular calcium homeostasis. Therefore, application of Rattin or activation of its signaling pathways in the brain might be beneficial to the prevention and treatment of AP-related cognitive deficits.更多还原