基于“肝肾相关”理论从Ca2+-CaM/CaMKⅡ-CREB通路探讨黑逍遥散干预阿尔茨海默病的作用

Traditional Chinese medicine holds the belief that occurrence of AD is mainly caused by kidney vacuity and essence depletion and disorder of qi and blood. According to the key theory of "liver controlling dispersion and strategy and storing soul, the theoryhomogeny of liver and kidne", we made a study of AD based on the correlation of liver and kidney and focus from liver treatment , acquiring partial validation. Ca2+-CaM/CaMK II signal pathway is closely tied to the function of brain, so research focused on the hippocampus, and less in cerebral cortex and basal forebrain, especially on AD. This study, which is based on the theory of the correlation of liver and kidney and combined with previous work, was implemented to establish the theoretical hypothesis that key molecules of Ca2+-CaM/CaMK II-CREB signal pathway play role in cascade response among the different microenvironments of AD mouse’s cerebral cortex, hippocampus and basal forebrain, and to observe the possible interfering action of heixiaoyaosan decoction. Therefore, as an object in AD mice, the study was designed to explore the expression laws of Ca2+-CaM/CaMK II-CREB signal pathway in different areas of brain by employing methods of immunohistochemistry, qPCR and Western blot detection, and to observe the response dialog among Tau, APP, PSEN, ApoE ,NEP and Aβstarch peptide gene. After using heixiaoyaosan decoction to intervene ,by correlation analysis, we intend to reveal the mechanisms of AD and interfering effects of Heixiaoyaosan decoction and to provide theoretical support for clinical application from the point of Ca2+-CaM/CaMKII- CREB signal pathway.

中医认为肾虚精亏、气血失调为AD主要病机之一，本课题组依据“肝主疏泄、主谋虑而藏魂；肝肾同源”理论，开展了基于肝肾相关并重点从肝论治AD及黑逍遥散干预研究工作，取得部分验证。因Ca2+-CaM/CaMKⅡ-CREB通路与脑功能关系密切且既往研究多集中在脑海马区，而对脑皮层和基底前脑研究较少，尤其在AD领域更少，故本研究结合前期工作建立“Ca2+-CaM/CaMKⅡ-CREB通路关键分子在AD小鼠大脑皮层、海马区和基底前脑微环境中的级联响应及黑逍遥散的可能作用”工作假说，以AD小鼠为对象，采用免疫组化、qPCR和WB技术检测Ca2+-CaM/CaMKⅡ-CREB关键分子在不同脑区域中的表达以及与Tau、APP、PSEN、ApoE、NEP、Aβ淀粉肽之间对话响应，通过相关性分析，拟从Ca2+-CaM/CaMKⅡ-CREB通路关键分子角度揭示AD发生机制以及黑逍遥散干预效应，为临床应用提供理论。

随着社会人口老龄化，阿尔兹海默病（Alzheimer's disease，AD）正成为一种严重影响中老年人健康及其生活质量的重大疾病。本课题依据“肝主疏泄、主谋虑而藏魂；肝肾同源”理论，应用“微观”和“宏观”相结合的新型研究方法，将藏象理论、脏腑病证和现代医学细胞分子生物学相结合，开展了基于肝肾相关论治AD及黑逍遥散干预治疗研究工作。本研究结合前期工作建立“Ca2+-CaM/CaMKⅡ-CREB通路关键分子在AD小鼠大脑不同脑区微环境中的级联响应及黑逍遥散的可能作用”工作假说。为证实该假说，我们以AD模型小鼠为研究对象，采用Morris水迷宫检测治疗前后小鼠行为学的变化，应用铀铅双染色后透射电镜观察小鼠海马和皮层组织的病理变化；采用免疫组化、qPCR和WB、激光共聚焦等技术检测Ca2+-CaM/CaMKⅡ-CREB关键分子在不同脑区域中的表达以及与ApoE、Aβ淀粉肽之间对话响应，同时观察黑逍遥散及其不同剂量从Ca2+-CaM/CaMKⅡ-CREB通路关键分子角度揭示AD发生机制以及干预效应。研究表明，水迷宫检测各治疗组小鼠逃避潜伏期均显著减短，跨原平台和有效区域次数显著增加，透射电镜观察治疗组小鼠大部分海马神经元结构结构正常，激光共聚焦示治疗组小鼠海马组织神经元细胞[Ca2+]i浓度显著降低，治疗后小鼠海马和皮层组织中APP、PS1、ApoE mRNA相对表达量显著降低，APP，PERK 蛋白的表达量均明显降低，CREB1、p-CREB1阳性表达显著增强，CaMKⅡα 蛋白表达量显著升高，p-CaMKⅡα蛋白表达量显著降低。通过相关性分析，研究证实了大脑皮层、海马区中Ca2+-CaM/CaMKⅡ-CREB 信号通路关键分子与[Ca2+]i、APP、ApoE、NEP mRNA动态水平的相关性；阐明了黑逍遥散对AD的治疗作用以及干预Ca2+-CaM/CaMKⅡ-CREB信号通路是其主要作用靶点，为进一步研究防治AD提供新思路和药物作用的新靶点,也为临床应用提供理论。