Aβ致痴呆老龄大鼠模型海马MARCKS表达变化机制及中药调节作用研究

【作者】 苏芮；

【导师】 范吉平； 韩振蕴；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2010， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease, AD)是目前导致痴呆最常见的原因,其发病机制尚不清楚,并且缺乏有效的治疗方法。富含丙氨酸的豆蔻酰化蛋白激酶C的作用底物(myristoylated alanine-rich C-kinase substrate, MARCKS)是在大脑中表达丰富的联系神经元表面信号与树突棘可塑性的重要膜蛋白,与学习记忆功能密切相关。β-淀粉样蛋白(βamyloid, Aβ)在神经元外沉积形成的老年斑是AD的特征性病理表现,同时它又是导致其它病理表现和痴呆症状的原因,AD除了老年斑和神经纤维缠结,树突棘可塑性降低也是AD的重要病理表现之一。树突棘可塑性决定于细胞骨架肌动蛋白的运动,该过程受细胞膜表面4,5二磷酸磷脂酰肌醇(Phosphatidylinositol 4,5-bisphosphate, PIP2)的控制。PIP2可以激活多种肌动蛋白结合蛋白,导致细胞骨架肌动蛋白的运动,进而使树突棘可塑性发生变化。MARCKS通过其效应区(effector domain, ED)分别与PIP2和纤维状肌动蛋白(fibrous actin, F-actin)结合。MARCKS在蛋白激酶C(protein kinase C, PKC)磷酸化或Ca2+的作用下,离开细胞膜,并释放PIP2和f-actin,使肌动蛋白结合蛋白被PIP2激活,并导致f-actin依赖性树突棘可塑性变化。故本研究以MARCKS为切入点,探索具有神经元毒性作用的Aβ(1-40)作用下老龄大鼠海马MARCKS的表达变化,并进一步探讨MARCKS变化导致的树突棘可塑性变化以及中药复方苁蓉益智胶囊对这一变化的作用,探讨中药治疗AD的新途径。目的：1.建立侧脑室一次性注射Aβ(1-40)至痴呆老龄大鼠模型,并评价该模型作为老年痴呆动物模型的有效性。2.研究Aβ(1-40)对痴呆老龄大鼠海马MARCKS表达的影响,进一步探索MARCKS介导的树突棘可塑性的变化以及复方苁蓉益智胶囊对Aβ致痴呆老龄大鼠海马MARCKS表达,及其介导的树突棘可塑性变化的作用机制。3.根据本研究结果,深入探讨AD的中医发病机制。方法：1对18-20月龄的老龄大鼠一次性侧脑室注射聚集态Aβ(1-40)建立模型,通过Morris水迷宫检测模型大鼠学习记忆功能,通过免疫组化检测海马Aβ(1-40)含量,通过病理检测,观察其海马病理变化,评价其表面效度；根据乙酰胆碱酯酶抑制剂安理申改善该模型学习记忆功能的有效性评价其预测效度；根据其导致痴呆的机理评价其结构效度。2将大鼠随机分为年轻组、老龄组、假手术组、模型组、安理申组以及中药组,模型组、安理申组以及中药组一次性侧脑室注射聚集态Aβ(1-40)建立老龄大鼠痴呆模型。中药和安理申组分别予口服复方苁蓉益智胶囊和安理申溶液14d。通过行为学、ELISA、RT-PCR和westernblotting等检测方法,观察模型大鼠学习记忆功能、Aβ(1-40)、MARCKS mRNA和树突棘可塑性标记蛋白drebrin含量的变化。结果：1该模型的表面效度成立：该模型可以模拟AD样记忆障碍、海马Ap含量增多以及神经元丢失等病理表现；预测效度成立：乙酰胆碱酯酶抑制剂可以改善该模型的学习记忆功能,减少模型大鼠海马Ap的含量,减少其海马神经元的变性和凋亡；结构效度成立：Ap(1-40)导致的病理变化符合淀粉样蛋白级联假说。2行为学检测表明模型组的学习记忆功能明显低于年轻组,安理申组和中药组大鼠的学习记忆功能明显高于模型组；模型组海马MARCKS mRNA比年轻组、老龄组及假手术组均明显升高(P<0.01)；模型组海马drebrin含量较年轻组、老龄组及假手术组均明显降低(P<0.01)；中药组海马MARCKS mRNA比模型组明显降低(P<0.01),安理申组海马MARCKS mRNA没有明显变化；中药组和安理申组海马drebrin含量与模型组相比无明显差别。结论：1侧脑室注射Ap(1-40)致痴呆老龄大鼠模型是用于研究AD发病机制以及新疗法的有效工具,尤其适合与Aβ相关的病理机制以及针对Aβ神经毒性治疗方法的研究。2侧脑室注射Ap(1-40)可以明显增加老龄大鼠海马MARCKS mRNA的表达,降低树突棘可塑性,并且导致老龄大鼠学习记忆障碍。根据MARCKS与树突棘可塑性之间的关系,推断MARCKS升高,导致与树突棘可塑性变化的游离态PIP2减少,很可能是Ap(1-40)致神经元可塑性降低的关键环节。基于“毒损脑络”理论研制的具有益肾补肝,解毒通络作用的复方苁蓉益智胶囊能够降低脑室注射Ap(1-40)致痴呆老龄大鼠海马MARCKS mRNA的表达,从而改善痴呆老龄大鼠学习记忆功能。其是否能够通过调节MARCKS的含量来增加树突棘可塑性,还需要进一步的证明。3.根据“毒损脑络”理论,对AD中医病机的探讨,发现Ap由人体产生,并可以导致神经元丢失,具有败坏形体的特征,属于中医“内生毒邪”的范畴。进一步思考“络”的含义,发现随年龄的增长,富含脂质的神经元细胞膜最易受到氧化损伤,并且细胞膜是联系细胞表面信号和细胞内功能变化的途径,是人体气机的重要通路,属于“络”的范畴。所以“毒损脑络”是AD的重要病机。更多还原

【Abstract】 Alzheimer’s disease (AD) is the most common cause of dementia. Mechanisms of its pathogenesis are not clear. And there is no effective treatment for it. P-amyloid protein(Aβ).deposition around neuron is a core pathology, which can cause the symptom of dementia, as well as other pathologies. In addition to senile plaque and neurofibrillary tangle, decreasing dendritic spine plasticity is also one of the key pathologies of AD. Spine plasticity is decided by the dynamic of Actin cytoskeleton. This procession is mediated by the actin binding protein activation by Phosphatidylinositol 4,5-bisphosphate (PIP2). Myristoylated alanine-rich C-kinase substrate (MARCKS), is a rich and important membrane protien in brain, which connect neuron signal with dendritic spine plasticity. MARCKS can bind to PIP2 and f-actin separately with its effecter domain. When phorsphorlated by PKC or interacted with Ca2+, MARCKS can leave membrane, at the same time, PIP2 and f-actin can be released. F-actin binding proteins can be activated, which can induce the f-actin depended dendritic spine plasticity. So, This PhD project aims to clarify the mechanism of the MARCKS alteration and the dendritic spine plasticity alteration mediated by it in AD animal model, as well as the mechanism of Chinese medicine effects.Objective:1. Establish and assess the validity of dementia model induced by AP protein intracranial injection, which is used for the research of Alzheimer’s disease.2. Observe the alteration of MARCKS mRNA and drebrin, which is a marker protein of dendritic spine plasticity, in hippocampus of old dementia rats induced by P-amyloid(Aβ)(1-40). Observe the effect of Fu Fang Cong Rong Yi Zhi capsule on the alteration of MARCKS mRNA and drebrin level in hippocampus of the dementia animal model.4. Implore AD pathology in Chinese medicine on the base of this research.Methods:1. Establish the dementia old rat model induced byβ-amyloid protein intracerebroventrical injection. Face validity was assessed by behavior test, pathological; predictive validity was assessed by the effect of cholinesterase inhibitor on learning and membrane function improvement; construct validity was assessed by mechanism of the mechanism of Aβinducing dementia.2. Animals were assigned randomly to young group, old group, sham operation group, model group, donepezil group and Chinese medicine group. Model, west and Chinese medicine group received Aβ(1-40) intracerebroventrical injection to build dementia animal model, and then Chinese and west medicine group received Fu Fang Cong Rong Yi Zhi capsule and donepezil for 14 days, respectively. Observe changes of behavior, Aβ(1-40), MARCKS mRNA and drebrin by morris test, RT-PCR, ELISA test and westernblotting respectively.Results:1. Face validity was supported:the model could mimic the memory impairments of Alzheimer’s disease, Predictive validity was supported:cholinesterase inhibitor could improve the learning and memory function of the model; Construct validity was supported:The pathological changes induced by Aβ(1-40) were correspondent with the amyloid hypothesis of AD.2. Learning and memory function of model group was decreased, compared with young group. Learning and memory function of Chinese and donepezil group were better than model group. MARCKS mRNA of model group was increased significantly, compared with young group, old group and sham operation group (P<0.01). Drebrin level of model group is decreased significantly, compared with young group, old group and sham operation group(P<0.01). MARCKS mRNA of Chinese medicine was increased significantly (P<0.01), campared with model group. There is no difference between drebrin level of Chinese medicine group, donepezil group and that of model group.Conclusion:1. This model could be used as an effective protocol for us to understand the neurobiology of AD and new therapy for it.2. Aβ(1-40) can increase the level of MARCKS m RNA, decrease the level of drebrin in old rat hippocampus, as well as decrease the learning and memory function of old rats. So MARKS may play a key role in the process of neuroplasticity reduction caused by Aβ(1-40). Fu Fang Cong Rong Yi Zhi capsule with the effect of benefiting kidney and liver, clearing toxin and improving blood vessel, which is based on the hypothesis "toxin damaging luo of brain" can reduce the level of MARCKS mRNA in hippocampus of old dementia rats caused by Aβ(1-40), through which it can improve the learning and memory function of the dementia old rats. Whether this medicine can increase the dendritic spine plasticity through this pathway, needs to be researched more deeply.3. Implore the pathology of AD in Chinese medicine, it is discovered that Aβhad the character of damaging of brain and marrow internal toxin, so it belongs to internal toxin, internal toxin damaging brain and marrow is also the pathology of the disease. Membrane is the most easily damaged by oxidation. Membrane is the important pathway for Qi circulation, which can be belonged to the definition of "luo" in Chinese medicine. The Chinese medicine hypothesis of "toxin damaging brain vessel" is an important pathology of AD.更多还原