人参皂苷Rd对阿尔茨海默病模型的神经保护作用及机制探讨
发布时间：2019-06-21 05:07
【摘要】：阿尔茨海默病(Alzheimer disease, AD)多在老年时期起病，起病隐秘，病程缓慢时间长且不可逆，是一组目前病因并不明确的脑部原发性退行性变性疾病。临床上以学习记忆认知能力进行性衰退为主，常伴有一定程度的语言、精神和人格等方面的异常。而其基本病理特点表现为Aβ沉积形成的老年斑、磷酸化Tau蛋白沉积形成的神经纤维缠结以及大量胆碱能神经元的丢失等。由于AD病人发病影响因素较多，病因病理机制非常复杂，故目前治疗并无明显突破，缺乏有效安全的治疗药物与手段。 课题组人员前期已经发现人参皂苷Rd能够抑制AD模型鼠海马内炎症反应的发生，抑制培养的海马神经元氧化应激损伤而保护神经元。故本课题以此为研究基础，从体内、体外、纯体外三方面观察Rd对实验性AD模型的神经保护作用并对其机制作初步探讨，为开发安全有效的防治AD新药提供基础研究依据。 研究目的：探讨人参皂苷Rd对实验性AD模型的学习记忆、病理特征的影响及对其保护机制的初步探讨，为寻找防治AD药物以及研究Rd作用靶点提供一定的实验基础。 研究方法：（1）研究对象：急性脑损伤模型：成年清洁级SD大鼠双侧海马CA1区立体定向微量注射Aβ1-40；慢性AD动物模型：APP转基因小鼠；体外细胞模型：Aβ25-35干预的皮层神经元。（2）对急性脑损伤模型大鼠和APP转基因小鼠进行Morris水迷宫检测观察Rd对AD模型动物的学习记忆行为能力的影响，Nissl染色观察其组织形态学变化；对Aβ25-35干预的皮层神经元进行MTT染色观察Rd对其存活率的影响和进行免疫荧光染色观察细胞形态学变化。（3）Western blot方法检测磷酸化Tau蛋白主要磷酸化位点的表达，观察Rd对AD模型内病理特征的影响，，同时对调控磷酸化Tau蛋白的关键激酶和磷酸酶的表达也进行了观察。初步探讨Rd对AD模型动物的保护作用机制。（4）体外纯化学实验进一步排除各种体内体外环境的影响，Western blot方法检测化学反应后磷酸化Tau蛋白主要磷酸化位点表达的变化，进一步探讨Rd对AD模型动物的保护的作用机制及其作用靶点。 实验结果：（1）Rd（5，10，30mg/kg）对Aβ1-40急性脑损伤大鼠的学习记忆功能均有改善:能够缩短海马微量注射Aβ1-40大鼠水迷宫逃避潜伏期的时间、增加模型鼠穿越原平台次数，减少模型鼠海马CA1区神经元的丢失；Rd（10mg/kg）可明显改善APP转基因小鼠的学习记忆功能：缩短APP转基因小鼠水迷宫逃避潜伏期的时间、增加模型鼠穿越原平台次数；Rd（2.5，5μM）能提高体外培养皮层神经元的活性；（2）Rd能够减少各模型组磷酸化Tau蛋白的表达，抑制GSK-3β的表达，提高PP-2A的活性；（3）体外纯化学实验结果表明Rd能够减少化学反应后各磷酸化位点Tau蛋白的表达。 实验结论：人参皂苷Rd增强了实验性AD模型动物的学习记忆能力，提高了AD细胞模型的存活率。其机制可能为Rd能抑制GSK-3β的表达与活性，提高PP-2A的活性，从而减少过度磷酸化Tau蛋白的形成与沉积，发挥其对AD模型的神经保护作用。
[Abstract]:Alzheimer's disease (AD) is a kind of primary degenerative disease of the brain which is not clear in the old age. It is mainly characterized by the progressive decline of the cognitive ability of learning and memory, often accompanied by some degree of abnormality in the aspects of language, spirit and personality. The basic pathological features of the present invention are age spots formed by A-type deposition, the entanglement of the nerve fibers formed by the phosphorylation of the Tau protein, and the loss of a large number of cholinergic neurons. Due to the high incidence of AD patients, the pathogenesis and the pathological mechanism are very complex, so the current treatment has no obvious breakthrough, and the effective and safe treatment medicine and the method are lacking. The early stage of the research group has found that the ginseng soap and the Rd can inhibit the occurrence of inflammatory reaction in the hippocampus of the AD model, inhibit the oxidative stress damage of the cultured hippocampal neurons, and protect the nerves. In this paper, the neuroprotective effect of Rd on experimental AD (AD) model and its mechanism were studied in vivo, in vitro and in vitro, and a basic study was provided for the development of a safe and effective anti-AD new drug. The purpose of this study was to study the effects of ginseng soap and the effect on the learning and memory and the pathological characteristics of the experimental AD model and the preliminary discussion of its protective mechanism, and to provide some practical measures for finding the target of preventing and treating AD and studying the target of Rd. Foundation of study: (1) Study object: (1) Study object: model of acute brain injury: three-dimensional directional microinjection of A-1-40, chronic AD animal model: APP transgenic mouse; in vitro cell model: A-25-35 intervention Cortical neurons. (2) The effect of Rd on learning and memory capacity of AD model animals was observed by Morris water maze test in rats and APP transgenic mice of acute brain injury model. The effect of Rd on the survival rate and the immunofluorescent staining of the cortical neurons in A-25-35 were observed by MTT staining. (3) The expression of the main phosphorylation sites of the phosphorylated Tau protein was detected by Western blot, and the effect of Rd on the pathological characteristics of the AD model was observed. A preliminary study was made to protect the animal from AD model. (4) The effect of Rd on the expression of the main phosphorylation sites of the phosphorylated Tau protein after chemical reaction was further determined by the in vitro pure chemical experiment, and the mechanism of the protective effect of Rd on the animal in AD model was further discussed. The results showed that (1) Rd (5,10,30 mg/ kg) improved the learning and memory function of A-1-40 rats with acute brain injury: the time to avoid the incubation period of the water maze of the rats with A-1-40 in the hippocampus was shortened, the number of the model mice crossing the original platform was increased, and the hippocampal CA1 of the model rats was reduced. The loss of zone neurons; Rd (10 mg/ kg) can improve the learning and memory function of the APP transgenic mice: shorten the time of the water maze of the APP transgenic mice to avoid the incubation period, and increase the number of the model mice crossing the original platform; and Rd (2.5,5. mu.M) can improve the in vitro culture skin. and (2) Rd can reduce the expression of phosphorylated Tau protein in each model group, inhibit the expression of GSK-3 antigen, and improve the activity of PP-2A; (3) in vitro pure chemical experiment results show that Rd can reduce the phosphorylation sites T after chemical reaction The results of the experiment: The ginseng soap and the Rd enhance the learning and memory ability of the experimental AD model animals and improve the A. The survival rate of the D-cell model can be increased by the mechanism of which the expression and the activity of the GSK-3 antigen can be inhibited by Rd, and the activity of the PP-2A is improved, so that the formation and the deposition of the over-phosphorylated Tau protein can be reduced, and the response to the AD
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R285.5;R749.16
本文编号：2503811
[Abstract]:Alzheimer's disease (AD) is a kind of primary degenerative disease of the brain which is not clear in the old age. It is mainly characterized by the progressive decline of the cognitive ability of learning and memory, often accompanied by some degree of abnormality in the aspects of language, spirit and personality. The basic pathological features of the present invention are age spots formed by A-type deposition, the entanglement of the nerve fibers formed by the phosphorylation of the Tau protein, and the loss of a large number of cholinergic neurons. Due to the high incidence of AD patients, the pathogenesis and the pathological mechanism are very complex, so the current treatment has no obvious breakthrough, and the effective and safe treatment medicine and the method are lacking. The early stage of the research group has found that the ginseng soap and the Rd can inhibit the occurrence of inflammatory reaction in the hippocampus of the AD model, inhibit the oxidative stress damage of the cultured hippocampal neurons, and protect the nerves. In this paper, the neuroprotective effect of Rd on experimental AD (AD) model and its mechanism were studied in vivo, in vitro and in vitro, and a basic study was provided for the development of a safe and effective anti-AD new drug. The purpose of this study was to study the effects of ginseng soap and the effect on the learning and memory and the pathological characteristics of the experimental AD model and the preliminary discussion of its protective mechanism, and to provide some practical measures for finding the target of preventing and treating AD and studying the target of Rd. Foundation of study: (1) Study object: (1) Study object: model of acute brain injury: three-dimensional directional microinjection of A-1-40, chronic AD animal model: APP transgenic mouse; in vitro cell model: A-25-35 intervention Cortical neurons. (2) The effect of Rd on learning and memory capacity of AD model animals was observed by Morris water maze test in rats and APP transgenic mice of acute brain injury model. The effect of Rd on the survival rate and the immunofluorescent staining of the cortical neurons in A-25-35 were observed by MTT staining. (3) The expression of the main phosphorylation sites of the phosphorylated Tau protein was detected by Western blot, and the effect of Rd on the pathological characteristics of the AD model was observed. A preliminary study was made to protect the animal from AD model. (4) The effect of Rd on the expression of the main phosphorylation sites of the phosphorylated Tau protein after chemical reaction was further determined by the in vitro pure chemical experiment, and the mechanism of the protective effect of Rd on the animal in AD model was further discussed. The results showed that (1) Rd (5,10,30 mg/ kg) improved the learning and memory function of A-1-40 rats with acute brain injury: the time to avoid the incubation period of the water maze of the rats with A-1-40 in the hippocampus was shortened, the number of the model mice crossing the original platform was increased, and the hippocampal CA1 of the model rats was reduced. The loss of zone neurons; Rd (10 mg/ kg) can improve the learning and memory function of the APP transgenic mice: shorten the time of the water maze of the APP transgenic mice to avoid the incubation period, and increase the number of the model mice crossing the original platform; and Rd (2.5,5. mu.M) can improve the in vitro culture skin. and (2) Rd can reduce the expression of phosphorylated Tau protein in each model group, inhibit the expression of GSK-3 antigen, and improve the activity of PP-2A; (3) in vitro pure chemical experiment results show that Rd can reduce the phosphorylation sites T after chemical reaction The results of the experiment: The ginseng soap and the Rd enhance the learning and memory ability of the experimental AD model animals and improve the A. The survival rate of the D-cell model can be increased by the mechanism of which the expression and the activity of the GSK-3 antigen can be inhibited by Rd, and the activity of the PP-2A is improved, so that the formation and the deposition of the over-phosphorylated Tau protein can be reduced, and the response to the AD
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R285.5;R749.16
【引证文献】
相关博士学位论文 前1条
1 孔令提;人参皂苷元的药代动力学研究[D];北京协和医学院;2013年
本文编号：2503811
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