电针治疗阿尔茨海默病的作用机制及其与Aβ血脑屏障清除途径相关性的研究

AβAlzheimer disease (AD) is a progressive cerebral neurodegenerative process with cognition impairment. Aβ hypothesis is the most popular pathogenesis for AD. Dysfunction of Aβ elimination across blood-brain barrier(BBB) may promote cerebral Aβ level, resulting in both extracellelar Aβ containing senile plaques(SP),and cerebrovascular amyloidosis(CAA). Aβ elimination across BBB had been a hot topic in the study of AD pathogenesis and therapeutic targets. Acupuncture has a good effect on disorders of central nervous system; it can dredge meridians and collaterals, promote qi and blood circulation, remove blood stasis, and recover cognitive impairment. Its effect on regulating the function and structure of cerebro -vasculature (cerebral collaterals) may promote cerebral Aβ elimination across BBB. The present experiment will take APPV695/717I transgenic mice (Tg mice) as AD animal model,on the basis of understanding the exact process of Aβ deposit,forming SP or cerebral microvascular amyloidosis, by testing its study and memory impairment by water maze, investigating the ingredients, location of Aβ and its transfer receptor(LRP1 and RAGE) in BBB by immunohistochemistry method, Thioflavin S staining,transmission electron microscopy, immunoelectron microscopy and scanning electron microscopy, testing the amount of Aβ and LRP1/RAGE in brain and BBB by ELISA and Western Blotting method, so as to verify Aβ elimination across BBB as the important mechanism of AD. At the same time, Electroacupuncture(EA) intervition on Du 20 and Ki 1 for 3 months (3 times a week),will be do on Tg mice to show EA effect on memory impairment, on Aβ deposit, on Aβ elimination across BBB. The study plan to verify the function of EA on AD, and support the theory of Chinese medicine on dementia's pathology that phlegm and blood stasis obstruct the cerebral collateral,and support the mechanism of EA on dementia that EA may tonify kindy to dredge cerebral collateral.

Aβ级联学说是目前最公认的阿尔茨海默病发病机制，脑血管途径Aβ清除异常导致了脑内Aβ水平升高，成为脑血管壁Aβ沉积和淀粉样斑块沉积的共同发病机制。前期研究提示，电针一定程度上降低了脑组织Aβ水平和脑血管壁Aβ沉积。本研究拟以Aβ沉积形成过程为切入点，以APP转基因鼠为AD动物模型，电针"涌泉、太冲"干预3个月，应用Morris水迷宫检测其学习记忆行为学变化，以免疫组化方法、激光共聚焦技术、透射电镜、免疫电镜技术，观察脑微血管及脑间质Aβ沉积的主要构成成分、沉积部位及转运受体LRP1、RAGE的阳性表达，以ELISA、WB方法检测脑组织可溶性及不可溶性Aβ含量、LRP1、RAGE含量，以阐明阿尔茨海默病的脑血管清除障碍发病机制，及电针对APP转基因鼠学习记忆行为、脑血管壁Aβ沉积和血脑屏障转运受体的影响，为阐明中医AD"痰瘀浊毒阻络"病机及电针"益肾通络法"治疗AD的疗效机理提供实验依据。

以AD发病的Aβ级联学说及微血管清除途径功能异常学说为基础，将中医AD病机“痰瘀浊毒阻络”转化为血脑屏障Aβ转运清除障碍导致微血管壁Aβ沉积和老年斑形成，以研究电针“通络化浊”对脑Aβ的清除效应，及其机制是否与提高脑微血管壁Aβ清除转运受体LRP1相关。.在观察不同月龄APP/PS1转基因鼠脑Aβ沉积病理的基础上，选择老年斑形成初期的6月龄转基因鼠为AD动物模型，电针干预6周后，Morris水迷宫空间学习记忆能力得到改善，Aβ免疫组化、硫黄素S染色的脑微血管壁Aβ沉积及老年斑负荷得到降低，ELISA海马Aβ40、Aβ42水平同时降低，WB皮层LRP1水平增高，提示电针可能通过提升脑LRP1水平，降低脑内可溶性及不可溶性Aβ水平，提升其空间学习记忆能力。同时激光共聚焦技术显示脑微血管壁存在LRP1免疫表达；免疫电镜显示其基膜层有Aβ存在；7月龄与5月龄ELISA结果显示脑皮层LRP1存在增龄性降低。实验结果显示，在一定程度上，电针可提高6月龄AD鼠脑微血管LRP1表达，减少微血管基膜层Aβ，提示电针可能通过提高脑微血管壁LRP1水平而增强脑内Aβ经血脑屏障清除。.为提高检测指标的精准性，又以Aβ沉积形成之前的4月龄AD鼠为模型进行电针研究，脑间质液Aβ微透析技术、ELISA、WB及PCR结果显示，电针可能通过提高海马LRP1mRNA表达，提高海马LRP1蛋白水平，促进可溶性Aβ40、Aβ42清除，从而降低脑Aβ水平。实验显示，电针1周可降低海马脑间质液Aβ42水平。.综上所述，本课题研究从不溶性Aβ、可溶性Aβ、脑间质液Aβ不同角度，证实电针可降低不同月龄APP/PS1转基因鼠脑Aβ水平，并在一定程度上提高LRP1表达，说明电针改善AD空间学习记忆能力与提高Aβ血脑屏障清除、降低脑Aβ水平相关。针刺治疗AD“通络祛浊”法从Aβ级联学说及血管清除途径功能异常学说得以阐释。.但由于脑组织LRP1表达部位及功能的多样性，Aβ转吞受体的多样性，血脑屏障多细胞组成的复杂性，故需要从神经血管单元角度或从血管周隙清除途径，进一步研究电针促进脑Aβ清除的机制。