【作者】 张振清；

【导师】 柴锡庆；

【作者基本信息】 河北医科大学 ， 神经病学， 2014， 博士

【摘要】 阿尔兹海默病(Alzheimer’s disease，AD)是一种起病隐袭且进行性发展的神经系统退行性疾病，临床上以进行性记忆障碍、失语、失用、失认、视空间损害、执行功能障碍及人格和行为改变为特征。AD的发病机制复杂，至今未有定论，目前人们提出几种假说，如胆碱能神经异常学说、淀粉样蛋白级联学说、自由基与凋亡学说、tau蛋白异常修饰学说、线粒体代谢障碍学说、兴奋性氨基酸毒性学说及基因突变学说等。但以上任何一种学说均不能完全阐明AD的发病机制。经过大量深入的研究，人们发现氧化应激、细胞凋亡、线粒体功能障碍与AD的发病有密切关系。AD的主要病理特征是大脑皮质弥漫性萎缩，大量的神经元丧失，细胞内神经纤维缠结和细胞外淀粉样斑块沉积，淀粉样斑块的主要成分是β-淀粉样蛋白（Aβ），它是从淀粉样前体蛋白（amyloid precursor protein,APP）由β-分泌酶和γ-分泌酶通过连续的蛋白水解裂解衍生的。研究表明，β-淀粉样蛋白(β-amyloid, Aβ)在脑内的异常代谢、沉积是AD发病的核心环节，各种原因导致的Aβ生成和清除的代谢失衡，引起Aβ在脑组织中的异常积聚，进而触发了与AD病理生理、生化相关的级联反应。在Aβ的持续刺激下，脑实质内小胶质细胞活化、活性氧(reative oxygen species, ROS)生成增多。众多有害自由基的产生及凋亡因子活化，激活细胞凋亡途径，并诱导氧化应激损伤细胞，如DNA、蛋白质和脂类。然而，Aβ诱导的细胞凋亡具体的信号转导通路并没有被完全阐明。以上各个因素都直接或间接致使中枢神经系统受损。越来越多的研究证实，中枢神经细胞功能紊乱和凋亡可能由神经毒性所致，而这一神经毒性可能恰恰是由于Aβ沉积导致，从而导致痴呆的发生。由此推论脑内氧化应激、细胞凋亡在AD的发病机制中起重要作用，抗氧化、抗凋亡已成为防治AD重要方法之一。近年来大量研究已经证实AD发病与脑组织细胞凋亡有关。P53基因作为凋亡通路中的关键蛋白，与基因组的稳定性及细胞周期密切相关。正常情况下，P53基因在机体内环境的稳定和组织重建过程中发挥重要作用，过度表达和累积则会导致一系列的病理生理变化。免疫组织细胞学研究已证实AD大脑的老年斑内和神经纤维缠结中存在大量P53蛋白。那么，它在AD患者中枢神经组织细胞内的表达是否会随病程的延长而表达升高，增高的P53与AD患者的认知能力是否存在某种潜在联系，同时又是哪些因素促进其在患者体内过度表达，本文将就上述问题一一展开分析讨论。高原红景天属于景天科红景天属(Rhodiola)，广泛存在于我国高原地区，为我国传统藏药之一，对许多疾病都有独特功效。现代研究证实红景天具有较强的抗氧化损伤及清除氧化自由基的能力，可以促进细胞代谢及增强细胞活力。近几年来，红景天在提高脑细胞功能、增强记忆力方面的作用逐渐受到重视。我们课题组动物实验亦表明，红景天在痴呆相关疾病的治疗上可能具有潜在的应用前景。而红景天的主要有效单体成分红景天苷，能否对抗Aβ产生的中枢神经细胞损伤，能否可以改善Aβ所致的认知功能障碍以及通过哪种具体分子机制发挥作用至今尚无系统深入的研究。接受Aβ海马内注射的大鼠及接受Aβ刺激的SH-SY5Y细胞，是相对经典的AD动物模型和细胞模型，由于上述两种模型在体内体外较好地模拟AD的病理过程，与其他实验模型相比，更为切实的反应了AD的发病进程。本实验研究通过建立Aβ诱导的AD细胞及动物模型，从行为能力的改变、病理生理代谢产物的生成、生物酶学活性的变化、信号蛋白表达的激活及基因转录翻译调控等诸多层面，系统研究了红景天苷对Aβ1-40所致AD细胞及动物模型内在信号转导机制及认知功能障碍的干预作用。第一部分研究红景天苷对Aβ1-40导致的AD大鼠模型认知功能障碍的干预作用目的：体内实验部分，建立Aβ1-40诱导的AD大鼠模型，检测红景天苷对Aβ1-40所致AD大鼠认知功能障碍的干预作用。方法：大鼠海马内注射Aβ1-40制备AD模型，术后分别灌胃治疗，每日1次给予已定剂量、浓度的红景天苷(50mg·kg-1·d-1)，连续21天。自第17天开始，采用Morris水迷宫测试大鼠学习记忆能力，连续5天。数据以xˉ±s表示，用SPSS16.0统计分析软件进行数据处理并分析，组间资料应用重复测量方差分析，计数资料应用非参数秩和检验，以P<0.05判定为有显著性差异。结果：监测各组大鼠在Morris水迷宫测试实验中寻找到平台并爬上平台的寻台潜伏期。在测试的5天内所有大鼠的寻台潜伏期都表现为缩短趋势。AD模型组与假手术组相比，AD模型组寻台潜伏期明显延长(P＜0.05)，结果提示给予Aβ海马内注射后导致学习及记忆功能受损。红景天苷(50mg·kg-1·d-1)治疗组寻台潜伏期均值较模型组明显下降，与AD模型组相比明显缩短(P＜0.05)。自第五天的定位航行轨迹显示：假手术组大鼠游泳线路清晰、目标明确，能够在较短时间及距离内找到平台。海马内给予Aβ注射的大鼠寻找目标明显减弱，游泳路线无序、繁乱、盲目，需要经历较长距离及时间才能找到平台。而红景天苷治疗组大鼠找到平台前的游泳距离缩短，寻找目标的能力得以改善。监测撤去平台后各组大鼠在原平台象限活动时间与总时间比值及跨平台次数，与假手术组相比，AD模型组大鼠在原平台所处象限搜索时间所占比值明显降低(P＜0.05)，搜索能力测试中跨过原平台所在位置的次数也显著减少(P＜0.05)，红景天苷治疗组(50mg·kg-1·d-1)在原平台所在象限搜索时间所占比值及跨平台次数均较模型组显著升高和增加(P＜0.05)，但未恢复至正常水平。结论：在接受Aβ1-40双侧海马内注射的大鼠，其记忆学习能力显著下降，给予已定量红景天苷治疗的AD大鼠，学习记忆能力的下降逆转。说明课题组已成功建立Aβ1-40双侧海马内注射所诱导的AD实验大鼠模型。提示红景天苷对Aβ1-40诱导的AD实验模型大鼠认知功能障碍具有显著改善作用。第二部分红景天苷对Aβ1-40所致AD模型大鼠氧化应激及凋亡的影响目的：氧化应激反应是不同刺激因素所诱发神经细胞损伤及多种中枢神经功能退行性紊乱疾病的共同通路。Aβ1-40能通过不同途径激发脑组织细胞内的氧化应激反应，进而产生大量ROS。本部分实验研究通过检测红景天苷对Aβ1-40所导致AD实验模型大鼠海马组织细胞内总ROS生成、血清及海马组织细胞内SOD活性、血清及海马组织细胞内MDA含量来深入研究探讨红景天苷对Aβ1-40所致AD模型大鼠脑内氧化应激损伤的影响。方法：Aβ1-40海马内注射制备AD实验大鼠模型，术后给予已定量红景天苷(50mg·kg-1·d-1)灌胃治疗，每日1次共21天。建立模型成功后，采用DCFH-DA作为荧光检测探针，采用流式细胞技术测定AD实验模型大鼠海马组织细胞内总ROS生成，氧化酶法测定AD大鼠模型血清及海马组织细胞内SOD活性，采用硫代巴比妥酸法检测大鼠血清及海马组织细胞内MDA的含量。数据用ˉx±s表示，用SPSS16.0统计分析软件进行统计学数据分析，用方差分析及LSD进行组间比较和两组间比较，以P<0.05为有显著性差异。结果：（1）本部分实验研究采用流式细胞技术（FCM）检测各组大鼠海马组织细胞的ROS含量。与假手术组相比，AD模型组大鼠海马组织细胞内ROS含量明显增加(P＜0.05)。给予红景天苷（50mg·kg-1·d-1）治疗21天后，AD模型大鼠海马组织细胞内总ROS含量受到显著抑制(P＜0.05)，虽然未回到正常水平，但与假手术组相比(P＞0.05)。（2）按实验要求测定AD模型大鼠血清SOD活性。与假手术组相比，AD模型组大鼠血清内SOD活性显著降低(P＜0.05)，给予红景天苷（50mg·kg-1·d-1）治疗21天后，大鼠血清SOD活性较AD模型组显著升高(P＜0.05)，虽然未回到正常水平，但与假手术组相比(P＞0.05)，海马组织细胞内SOD活性检测结果与血清学检测结果趋势相同。（3）与假手术组相比，AD模型组大鼠血清MDA含量显著升高(P＜0.05)，给予红景天苷（50mg·kg-1·d-1）治疗21天后，血清MDA含量较AD模型组显著降低(P＜0.05)，虽然未回到正常水平，但与假手术组相比(P＞0.05)，海马组织细胞内MDA含量检测结果与血清学检测结果趋势相同。(4)实验研究通过检测AD实验模型大鼠海马组织细胞的凋亡情况。与假手术组相比，AD模型组大鼠海马组织细胞凋亡率显著升高(P＜0.05)，给予红景天苷（50mg·kg-1·d-1）治疗21天后，实验结果证实经药物干预治疗，AD模型大鼠海马组织细胞凋亡较AD模型组显著降低(P＜0.05)，但与假手术组相比(P＞0.05)，仍未回到正常水平，结论：红景天苷可能通过有效抑制Aβ1-40所导致的AD大鼠模型海马组织细胞内的总ROS生成、同时提高血清及海马组织细胞内SOD活性并减少血清及海马组织细胞内MDA含量，从局部和整体双向验证红景天苷可以提高AD模型大鼠的抗氧化应激能力并减轻神经细胞因氧化应激所造成的损伤。第三部分红景天苷对Aβ1-40所致AD模型大鼠海马NADPH氧化酶-ROS通路的影响目的：NADPH氧化酶是组织细胞内ROS产物的主要来源。本部分实验通过观察NADPH氧化酶家族成员亚基的的表达与激活，对红景天苷是否可以抑制AD模型大鼠海马组织细胞内ROS产生的上游通路的潜在机制进行探讨。方法：采用Aβ1-40海马内注射制备AD大鼠模型,术后给予已定量的红景天苷(50mg·kg-1·d-1)灌胃治疗，每日1次共21天。建立模型成功后，采用RT-PCR技术和Western blot技术检测大鼠海马组织中gp91phox及其他亚基在mRNA和蛋白水平的表达。数据用ˉx±s表示，用SPSS16.0统计分析软件进行统计学数据分析，采用方差分析及LSD进行组间比较及两组间比较，以P<0.05为有统计学差异。结果：(1) Western Blot结果显示，Aβ1-40可导致AD模型大鼠海马组织细胞内p22phox、p67phox、gp9lphox及p47phox蛋白表达明显升高。AD模型组上述各蛋白的含量分别是假手术组的2.16、2.57、3.02及2.71倍(P＜0.05)。而给予已定量红景天苷干预治疗后，大鼠海马组织p22phox、p67phox、gp9lphox及p47phox蛋白表达较AD模型组分别降低了24.1%、26.3%、31.1%及34.2%(P＜0.05)。(2) RT-PCR检测。研究结果显示，与假手术组相比，AD模型组大鼠海马组织细胞内p22phox、p67phox、gp9lphox及p47phoxmRNA表达显著升高。AD模型组较假手术组mRNA水平分别升高了84.1%、96.3%、101.1%及104.2%(P＜0.05)。而给予已定量红景天苷干预治疗后，能够显著抑制Aβ1-40的这种诱导效应。红景天苷干预组较AD模型组分别降低了34.3%、36.1%、33.4%及30.1%(P＜0.01)。结论：红景天苷可抑制NADPH氧化酶家族成员的亚基进而抑制ROS的产生，此过程可能是通过抑制其家族成员亚基的表达和抑制其亚基激活来实现，或者是两个作用相互协同作用来抑制NADPH氧化酶活化。红景天苷通过对NADPH氧化酶-ROS通路的有效抑制，明显减轻了Aβ1-40所诱导的AD实验模型大鼠脑组织细胞内氧化应激反应。第四部分红景天苷对Aβ1-40所致AD模型大鼠海马ROS-P53线粒体凋亡通路的影响目的：为了进一步明确ROS导致细胞凋亡率增加的具体机制，我们检测了ROS生成后可能导致细胞凋亡的相关蛋白P53的表达。同时验证在P53基因基础上红景天苷对Bcl-2、Bax及线粒体凋亡通路的影响方法：采用Aβ1-40海马内注射制备AD大鼠模型,术后给予已定量的红景天苷(50mg·kg-1·d-1)灌胃治疗，每日1次共21天。建立模型成功后，采用Western-blot检测AD模型大鼠海马组织细胞中细胞凋亡的相关蛋白P53的表达。同时验证在P53基因基础上红景天苷对Bcl-2、Bax及线粒体凋亡通路蛋白的表达，数据用ˉx±s表示，用SPSS16.0统计分析软件进行统计学数据分析，采用方差分析及LSD进行组间比较及两组间比较，以P<0.05为有统计学差异。结果：(1) Western Blot结果显示，Aβ1-40可致使AD模型大鼠海马组织细胞核内P53表达，并使其表达显著升高。AD模型组最高细胞核内P53蛋白的含量可达假手术组的5-6倍(P＜0.05)。而给予已定量红景天苷干预治疗后，AD模型大鼠海马组织细胞核内P53蛋白表达与AD模型组相比降低近50%(P＜0.05)。(2)与假手术组相比，AD实验模型组大鼠海马组织细胞Bax表达显著升高；而AD实验模型组大鼠海马组织细胞Bcl-2的表达明显下调。AD实验模型组较假手术组Bax升高151%(P＜0.05)；AD实验模型组较假手术组Bcl-2下调近70%(P＜0.05)。而给予已定量红景天苷干预治疗后，能够显著抑制Aβ1-40的上述诱导效应。Bax及Bcl-2在红景天苷干预组较AD模型组分别降低和升高了40%及55%(P＜0.05)。(3)与假手术组相比，AD实验模型组大鼠海马组织细胞内Caspase-9及Caspase-3表达显著升高；AD实验模型组较假手术组Caspase-9及Caspase-3升高151%及124%(P＜0.05)，在给予已定量红景天苷干预治疗后，能够显著抑制Aβ1-40的上述诱导效应。Caspase-9及Caspase-3在红景天苷干预组较AD模型组分别降低了40%及55%(P＜0.05)。结论：红景天苷抑制NADPH氧化酶产生ROS进而抑制P53入核活化从而抑制caspase-9和caspase-3表达，说明红景天苷通过对NADPH氧化酶-ROS-P53-线粒体凋亡途径抑制，减轻了Aβ1-40诱导的AD脑组织细胞内氧化应激及凋亡的发生。第五部分红景天苷通过PI3K/Akt/Nrf-2/HO-1信号通路减少P53在细胞核内的表达抑制细胞凋亡目的：探讨红景天苷保护神经细胞防止β-淀粉样蛋白损伤的机制。从而为海马内注射β-淀粉样蛋白诱导的AD模型提供了有力的证据。因为接受β-淀粉样蛋白刺激的SH-SY5Y细胞和海马内注射β-淀粉样蛋白大鼠是经典的AD细胞及动物模型，已被广泛用于研究。所以我们研究发现，红景天苷在体外实验中能有效防止SH-SY5Y细胞由于β淀粉样蛋白所诱导的细胞凋亡，从而保护神经细胞免受氧化损伤，其机制可能是通过上调HO-1的表达，降低神经细胞P53的核表达来实现。来逆转β-淀粉样蛋白诱导的神经细胞P53的核表达实现。方法：Western blot检测蛋白表达；总蛋白（30μg）对每个样品用10%SDS-PAGE分离，并PVDF膜转移过夜，和抗体的特异性结合前采用5%脱脂奶粉或BSA的磷酸盐缓冲盐水封闭。然后检测HO-1、P53、Akt蛋白和Bax、Bcl-2蛋白，活性状态的caspase-9/3或Nrf-2抗体（1:1000）4℃过夜。洗涤三次后，用磷酸盐缓冲盐水（0.1%Tween-20的pH7.4）中和，ARE-荧光素酶活性测定法：SH-SY5Y细胞接种于6孔板中以1×105cells/ml的密度，孵育过夜。在每个样品中，荧光素酶报告质粒构建携带该ARE启动子和β-半乳糖苷酶的表达载体质粒2μg，使用转染试剂以每加入10μl2μg的DNA的比例共转染。根据由制造商提供的方法测定。简而言之，将细胞用冷PBS洗涤，并收获被动裂解缓冲液中。离心后，用于测定荧光素酶活性，取上清液20μl由光度计测定。TUNEL染色：末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）法在SH-SY5Y细胞被用来通过检查DNA片段化处理之后的细胞，以评估细胞死亡。简言之，将细胞培养于6孔细胞培养板并如上所述进行处理。处理后，将细胞用PBS洗涤，然后沉淀到显微镜载玻片。残余的PBS中，然后取出，并用95%乙醇固定细胞。用4%不含甲醇的甲醛的PBS中进行第二次的固定。载玻片再次用PBS洗涤，并通过加入荧光素12-dUTP标记的DNA中的带切口端部在凋亡细胞中检测到片段化DNA。玻片在37℃温育1小时，并终止反应用2×SSC。将载玻片在PBS中洗涤，然后用荧光显微镜在400×可视化，和绿色的荧光率与DNA片段化。实验分别分三次重复完成，并TUNEL阳性细胞的百分比进行测定。数据用xˉ±s表示，用SPSS16.0统计分析软件进行统计学数据分析，采用方差分析及LSD进行组间比较及两组间比较，以P<0.05为有统计学差异。结果：(1) Aβ1-40可显著降低细胞活力而红景天苷可逆转此现象，(2)红景天苷提高细胞存活率这一现象可被ZnPPIX所抑制。由此表明红景天苷对HO-1的诱导性表达和增加细胞生存能力起着关键性作用，(3)红景天苷通过Nrf-2的激活诱导HO-1表达，同时红景天苷抑制氧化应激诱导的细胞凋亡，而P53活化剂-RITA可以诱导细胞凋亡。结论：P53蛋白直接诱导细胞凋亡，从而加重认知功能障碍。因此，P53可视为氧化应激在AD的早期标志。我们发现，在AD脑组织及细胞实验中，红景天苷可激活PI3k/AKT途径激活Nrf-2及HO-1表达，从而著降低P53的核表达，从而抑制细胞凋亡及认知功能障碍的发生。更多还原

【Abstract】 Alzheimer’s disease (AD) is a insidious progressive neurodegenerativedisease. In clinnical，it is characterised by progressive loss of memory,cognitive dysfunction, agnosia，apraxia，aphasia，execution dysfunction, visionspace damages and personality behavior changes which generalized dementia.The pathogenesis of AD is multitude and complex and not fully understoodyet. Researchers proposed various hypotheses with respect to etiology of AD,such as the cholinergic hypothesis, amyloid hypothesis, the free radicals andapoptosis hypothesis, the tau proteins hypothesis, dysmetabolism hypothesis,the excitatory amino acids hypothesis, the genic mutation hypothesis, et al.However, none of them can fully explain the pathogenesis of AD. With thedevelopment of research, people have come to realize that the oxidative stress,apoptosis and Mitochondrial dysfunction which are general reactions in thebody have inseparable connection with AD.Plenty of evidence show that abnormal metabolism of β-amyloid (Aβ)peptides and accumulation of excessive Aβ play critical roles in progress ofAD. The stimulation by Aβ persistently can activate microglia, generatereactive oxygen species (ROS), then produce harmful free radicals andactivate apoptosis molecules, These reactions exert direct or indirect injuriesof nervous system. Increasing evidence demonstrate that Aβ deposition causeda series of neurotoxic effect which cause neuron disfunction, death and furtherlead to dementia. Oxidative stress in the brain and apoptosis played significantroles in this course. The therapy from anti-oxidative and anti-apoptosis hasbecome one of the most important strategies for the prevention of AD.In recent years, many studies have confirmed that the incidence of AD isclosely related to tissue and cell apoptosis. P53is a key protein in theapoptosis pathway, the normal and basal expression of P53is closely related to the stability of the genome and cell cycle. Under the normal conditions, italso plays an important role in the body’s internal environment of stability andreconstruction process of the organization, the overexpression andaccumulation of P53will lead to a series of pathophysiological changes. Theimmunohistochemical studies have confirmed that the plaques andneurofibrillary tangles in the brain of AD contain a lot of P53protein. So, withthe course of the disease, its expression in the central nervous system of ADwhether reinforcing or not, whether there is a potential link between increasedP53and cognitive abilities in patients with AD or not, and what factorspromote its overexpression, this article will discuss one by one.Plateau rhodiola is a perennial plant of Rhodiola family. It was atraditional famous Tibetan medicine. Researches show that rhodiola havespecial effects in preventing oxidative damage, scavenging free radicals,improving cell metabolism and enhancing cell vitality. In the past few years,growing attention has been received on its benefits of enhancing the brainfunction and improving memory. Animal experiments also showed thatrhodiola may have good prospects in the treatment of dementia and relatedfields. However, salidroside as the main effective component of rhodiola, itseffect and mechanism on againsting damages of axoneures by Aβ, improvingcognitive deficit of Alzheimer’s disease is still not clear and need systematicand profound researches.The rat received hippocampal injection of Aβ and the SH-SY5Y cellswere stinulated by Aβ are mature models for AD. These models is closer tothe real pathological processes of AD than the others, because of its fulllyresponsed for the courses of Aβ deposition in the brain. This study establishedAβ induced rats and cell models of AD. Systematical investigations were firstundertaken from the aspects of behavior cognitive change, generation ofphysiopathologic metabolism product, enzymology changes in activity,protein expression, transcriptional regulation and so on. The aim to explore thesignal transduction mechanisms and the effects of salidroside on cognitivedysfunction in Alzheimer’s disease model rats induced by Aβ1-40. Part1The effects of salidroside on cognitive dysfunction of AD modelratsObjective: To establish Aβ1-40induced rat models of AD and observe theeffects of salidroside on cognitive dysfunction of this model.Methods: Aβ1-40was injected into bilateral hippocampus to create ADmodel. Rats were administered by gavage with salidroside in settled dose anddensity (50mg·kg-1·d-1) everyday for21days. Morris water maze testingsystem was undertaken since17th day to observe the change of learning andmemory abilities in rats，and continuous5days. Data were presented asˉx±sand analyzed with multi-variate test of repetitive measure ANOVA usingSPSS16.0statistical program. Enumeration data were analyzed with Rank sumtest. A level of P<0.05was considered statistically significant.Results: The analysis of the place navigation trial showed that the escapelatency decreased from Day1to Day5in all groups. The AD model ratsdisplayed longer escape latencies than the rats of sham control group(P＜0.05).The animals which were treated with salidroside displayed significantly lowerescape latencies than those in AD model group(P＜0.05). Representativenavigation paths at day5of training demonstrated that spatial learningacquisition was impaired in the animals of AD model group relative toanimals of salidroside-treated group. In the spatial probe trial, The AD modelrats spent significantly less time in the quadrant where the platform washidden than animals in sham control group(P＜0.05). The number of crossingsto the previous location of the platform was decreased in AD model grouprelative to animals in negtive control group(P＜0.05). Animals in salidroside(50mg·kg-1·d-1) group spent more time in the target quadrant and showedstatistically more platform-passing times than animals in AD model group(P＜0.05).Conclusions: In rats with bilateral hippocampal injections by Aβ1-40, thememory, learning ability are significant declined, the therapy with quantitativesalidroside intervention of AD rats and the declined ability of learning and memory reversed.The behavioural data obtained in the Morris water maze testdemonstrate that salidroside is able to protect animals from the memoryimpairments induced by hippocampal injection of Aβ1-40.Part2The effects of salidroside on anti-oxidative activities of AD modelratsObjective: Oxidative stress can be seen as a neuronal cell injury inducedby different stimuli and a variety of degenerative disorders of central nervousfunction common pathways. In a variety of ways, Aβ1-40can stimulateintracellular oxidative stress in brain tissue reaction, and a large number ofROS produce consequently. The aim of this part is to observe the effects ofsalidroside on the generation of total ROS, the superoxide dismutase (SOD)activity, the malondialdehyde (MDA) level and the in serum and hippocampusof AD model rats to discuss the effect of salidroside on ROS in AD models,then the apoptosis rate was checked.Methods: Aβ1-40was injected into bilateral hippocampus to create ADmodel. Rats were administered by gavage with salidroside in settled dose anddensity (50mg·kg-1·d-1) everyday for21days. The generation of total ROS inhippocampus was determined by flow cytomertry technology usingDCFH-DA, the superoxide dismutase (SOD) activity and the malondialdehyde(MDA) level in serum and hippocampus were determined by separatebiochemical kit, last the apoptosis rate was checked by TUNEL. Data werepresented as xˉ±s and analyzed with ANOVA and LSD using SPSS16.0statistical program. A level of (P＜0.05) was considered statisticallysignificant.Results:(1) This experimental study examined the total ROS productsin the hippocampus cellular of AD model rats. To detect ROS levels in eachgroup of cells by flow cytometry (FCM). Compared with the sham controlgroup, the ROS levels of hippocampus intracellular in AD model groupincreased significantly (P<0.05). AD model rats were treated with salidroside （50mg·kg-1·d-1）for21days, the ROS levels were significantly inhibited(P<0.05), although not back to normal levels, compared with negative controlgroup (P>0.05).(2) According to the experimental requests, to determineSOD activity (content) in serum of AD model rats. Compared with thecontrol group, the serum SOD activity in AD model group decreasedsignificantly (P<0.05), giving Salidroside (50mg·kg-1·d-1) for21days, serumSOD activity was significantly higher compared with the AD model group(P<0.05), although not back to normal levels, but compared with the negativecontrol group (P>0.05), SOD activity in the hippocampus cells the same as theserological results.(3) Compared with the negative control group, serumMDA in AD model group was significantly higher (P<0.05), givingsalidroside (50mg·kg-1·d-1) for21days, serum MDA content decreasedsignificantly compared with the AD model group (P<0.05), although not backto the normal level, but compared with the sham control group (P>0.05),MDA content in hippocampus cells have the same trend with the serologicalresults.(4) To determine apoptosis in hippocampus cells of AD model rats.Compared with the sham control group, the apoptosis rate in hippocampalcells of AD model rats were significantly higher (P<0.05), giving salidroside(50mg·kg-1·d-1) for21days, the apoptosis in hippocampus cells decreasedsignificantly compared with the AD model group (P<0.05), although not backto normal levels, but compared with the sham control group (P>0.05).Conclusion: Salidroside can effectively inhibit ROS generate inhippocampal tissue cells of AD model rats,while increase SODactivity(content) in serum and hippocampal tissue cells, and to reduce theMDA levels in serum and hippocampus tissue cells, thus, it was validatedfrom local and global mutual that salidroside can increase the ability ofoxidative stress in AD model rats and reduce damage to nerve cells caused byoxidative stress. Part3The impacts of salidroside on NADPH oxidase-ROS pathway inhippocampus of AD model rats induce by Aβ1-40Objective: NADPH oxidase is a major source of ROS products in cells.The aim of this part is to discuss the underlying mechanisms that whethersalidroside inhibited ROS upstream passage or not by observing the subunitexpression and activation in NADPH oxidase family members.Methods: Aβ1-40was injected into hippocampus to create AD model.Rats were administered by gavage with salidroside in settled dose and density(50mg·kg-1·d-1) everyday for21days. After establishing a successful model,using RT-PCR and Western blot to detect the expressions of gp91phox andother subunits reside in hippocampal tissues in the mRNA and protein Level.Data were presented asˉx±s and analyzed with multi-variate test of repetitivemeasure ANOVA using SPSS16.0statistical program. Enumeration data wereanalyzed with Rank sum test. A level of P<0.05was considered statisticallysignificant.Results:(1) Western Blot showed：Aβ1-40may induce p22phox, p67phoxgp9lphox and p47phox protein expression was significantly increased inhippocampus cells of AD model rats. Protein content above-mentioned in ADmodel group were2.16,2.57,3.02and2.71times than the sham control group(P<0.05). But after given quantitative salidroside, the protein expression ofp22phox, p67phox gp9lphox p47phox in hippocampal tissue were lower by24.1%,26.3%,31.1%and34.2%than the AD model group(P<0.05).(2)RT-PCR testing showed that: compared with the sham control group, theexpression of p22phox, p67phox gp9lphox and p47phox mRNA significantlyincreased in AD model rats hippocampus cells. The mRNA levels in ADmodel group were increased by84.1%,96.3%,101.1%and104.2%than thenegative control group (P<0.05). But after given quantitative salidroside, itcan significantly inhibit the effect induced by Aβ1-40. Salidroside interventiongroup decreased by34.3%,36.1%,33.4%and30.1%than AD model group(P<0.01).Conclusion: Salidroside inhibits NADPH oxidase family membersthereby inhibiting ROS production, this process may be caused by inhibitingthe expression and activation of family members subunit, or inhibiting NADPH oxidase activation by mutually synergy. Salidroside throughsuppressed NADPH oxidase-ROS pathway effectively, reduced oxidativestress response significantly in brain cells of AD rat model induced by Aβ1-40.Part4The impacts of salidroside on ROS-P53mitochondrial apoptoticpathway in hippocampus of AD model rats induce by Aβ1-40Objective: To further search the specific mechanism of apoptosis rateafter ROS, we examined the expression of apoptosis-related protein P53afterROS generation. Also verify the impact salidroside on Bcl-2, Bax andmitochondrial apoptotic pathway based on P53gene.Methods: Aβ1-40was injected into hippocampus to create AD model.Rats were administered by gavage with salidroside in settled dose and density(50mg·kg-1·d-1) everyday for21days. After establishing the successful models,Western-blot was used to detect the expressions of apoptosis-related proteinsP53. Also verify that the expression salidroside on Bcl-2, Bax andmitochondrial apoptotic pathway basised on P53gene. Data were presented asxˉ±s and analyzed with multi-variate test of repetitive measure ANOVA usingSPSS16.0statistical program. Enumeration data were analyzed with Rank sumtest. A level of P<0.05was considered statistically significant.Results:(1) Western Blot showed: Aβ1-40can cause nucleus P53expression in hippocampus cells of AD model rats, and it was significantlyhigher expression. In AD model group, the highest in the nucleus of P53protein up to5-6times than the sham control group(P<0.05). After givenquantitative salidroside, the nuclear P53protein expression in hippocampus ofAD model rats decreased nearly50%compared with the AD model group(P<0.05).(2)Compared with the sham control group, the Bax expression inhippocampal cells of AD model rats significantly increased; while Bcl-2expression in hippocampal tissue cells of AD model rats was significantlyreduced. The Bax increased151%than the sham control group (P<0.05); TheBcl-2in AD model rats down nearly70%than the sham control group (P<0.05). After given quantitative salidroside, it can significantly inhibiteffects induced by Aβ1-40. Bax and Bcl-2in the salidroside intervention groupdecreased and increased by40%and55%than AD model group (P<0.05).(3)Compared with the sham control group, the caspase-9and caspase-3expression was significantly increased in hippocampus cells of AD model rats;The caspase-9and caspase-3in AD model group increased by151%and124%than the sham control group (P<0.05). After given the quantitativesalidroside, it can significantly inhibit the effects induced by Aβ1-40. caspase-9and caspase-3in the intervention group with salidroside decreased by40%and55%compared with AD model group (P<0.05).Conclusion: The salidroside can inhibit NADPH oxidase to generateROS, thereby inhibiting P53tranfer into the nucleus to activation, thusinhibiting the expression of caspase-9and caspase-3, it indicated thatsalidroside can inhibit the NADPH oxidase-ROS-P53-mitochondrial apoptosispathway, reducing the oxidative stress and apoptosis in AD brain tissueinduced by Aβ1-40.Part5Salidroside reduces p53during Aβ1-40-induced neurotoxicity byinduction of heme oxygenase-1through PI3K/Akt/Nrf-2signal pathwaysObjective: The objective of this study was to investigate how salidrosideprotects neurocytes from Aβ1-40. Animal models of AD induced by injection ofAβ1-40into hippocampus provide numberous evidences for cellular apoptosis.Thus, SH-SY5Y cells stimulated by Aβ1-40and rat received hippocampalinjection of Aβ1-40AD models were used for research in vitro and vivo,respectively. We found that salidroside in vitro experiment could preventAβ1-40-induced apoptosis of SH-SY5Y cells and protect brain in vivo fromoxidative damage and also reduce p53nucleus expression in betaamyloid-induced neurocytes injury through upregulation of HO-1.Methods: Total protein (30μg) for each sample was separated by10%SDS-PAGE and transferred overnight to PVDF membranes, and the nonspecific binding of antibodies was blocked by5%nonfat dried milk orBSA in phosphate-buffered saline. Membranes were then probed with HO-1,p53, t-Akt, p-Akt, Bax, Bcl-2, cleaved caspase-9/3or Nrf-2antibody (1:1000)overnight at4C. After three washes with phosphate-buffered saline(0.1%Tween-20PH7.4). The protein bands were visualized using an enhancedchemiluminescence Western blotting detection kit and the results wereanalyzed using imaging densitometer. ARE-luciferase activity assay:SH-SY5Y cells were plated in24well plates at a density of1×104/cells andincubated overnight.2μg of the luciferase reporter plasmid with AREpromoter and the β-galactosidase vector plasmid were co-transfected to eachsample, using transfection reagents at the proportion of2μg DNA per10μl. Tocorrect for the transfection efficiency, phRL-SV40-β-galactosidase was used.2hours later, the luciferase activity was detected according to the methodprovided by the manufacturer. In brief, cells were washed with PBS in4C andharvested with lysis buffer then centrifugated, supernatant (20μl) was used forthe detection of the luciferase activity, which was measured by a luminometer.TUNEL staining: The terminal deoxynucleotidyl transferase dUTP-mediatednicked end labeling (TUNEL) assay in SH-SY5Y cells was used to assess celldeath by examining DNA fragmentation following treatment of cells. Briefly,cells were grown on6well cell culture plates and treated as described above.After treatment, cells were washed with phosphate-buffered saline (PBS), theslides were immersed in4%formaldehyde in PBS at4C and then in3%H2O2and in0.2%Triton X-100for5-10min at room temperature. Equilibrationbuffer (100μl) was added, and the slides were incubated at room temperaturefor10min. TdT reaction mix (50μl) was then added, and the cells wereincubated for60min at37C. The slides were then immersed in2×SSC for15min. Propidium iodide (PI) was added and incubated for15min to stain allcells. The localized green fluorescence of apoptotic cells was detected againsta red background by fluorescence microscopy. For the quantification ofTUNEL-positive (apoptotic) cells, a minimum of200cells was counted pergroup, and the percentage of the positively labeled cells was calculated. All analyses were performed using the SPSS16.0for Windows, statisticaldifferences were checked by ANOVAs correction or Student’s t-tests formultiple comparisons. P﹤0.05were set as statistically significant. All datawere checked for normality and homogeneity of variance previously and wereexpressed as mean±standard error.Results:(1) Aβ1-40can inhibit the cell viability,and salidroside can induceHO-1protein expression through PI3K/Akt signals involvement in inductionof HO-1,(2)The effect of salidroside can be reversed by ZnPPIX,(3)Salidroside induces HO-1by Nrf-2activation,Salidroside inhibits oxidativestress-induced apoptosis,Salidroside attenuates cognitive deficits and inhibitsp53nucleus translocation induced by Aβ1-40, P53activator-RITA inducesapoptosisConclusion: In summary, we demonstrated that salidroside protects braincells from Aβ1-40via induction of HO-1. The expression of HO-1bysalidroside was dependent on PI3K/Akt pathway and Nrf-2translocation.Salidroside also attenuated cognitive deficits, LDH release, and P53release inAD rat brain by ZnPPIX-sensitive manner (Fig.7). Thus, salidroside may bean important novel therapeutic agent for treatment of AD.更多还原