基于星形胶质细胞介导的胶质-血管耦合探讨益气通络方药影响BBB防治 AD的机制

Alzheimer’s disease (AD) is the most common type of senile dementia, a neurodegenerative disease characterized by progressive memory loss and cognitive impairment in the elderly.The pathogenesis of AD is perplexing, but traditional Chinese medicine think the key pathogenisis of AD is blood stasis and poision damaged brain collaterals. So enriches qi, promotes blood circulation, and dredges meridians are the treatment. Blood-brain barrier (BBB) dysfunction participate in amyloid- βcascade . Astrocyte mediated glialvascular coupling induce BBB properties through the astoglial endfeet surrounding the brain capillaries and its paracrine effect. an importance of physical contact in addition to secreted factors. In AD, activated Ast are found in proximity to A β plaques, and Ast changes ere earlier than BBB dysfunction . However, the contribution of Aβ-induced astrocytic changes to BBB function is less clear.. Bu-yang-huan-wu-tang (BYHWT) is a famous traditional Chinese medicine formula that has been used clinically in Asia to treat AD. Many studies show that BYHWT can inhibit severe diffuse reactive astrogliosis , improve astrocytic function,and protect BBB. We assume that the formula may intervene Ast response to Aβ, regulate glial - vascular signal coupling to protect the integrity of the BBB, and thus treat and prevent AD. This reaserch intends to observe the targets and mechanisms of BYHWT protecting BBB, issuing the endfeet of Ast AQP4 polarity distribution and paracrine SHH pathway as a starting point,through the rapid aging mouse SAMP8 AD model and in vitro Ast and brain microvascular endothelial cell co-culture system, using functional antagonist and molecular biology technical inspection.This study will inject new scientific connotation on Yiqitongluo formula for prevention and treatment of AD.

阿尔茨海默病（AD）发病率逐年上升，成为老年痴呆症的主要元凶。中医认为AD主要由气虚血瘀，毒损脑络所致。血脑屏障（BBB）遭破坏是毒损脑络的现代病理生理基础。星形胶质细胞(Ast)介导的胶质-血管耦合是维持BBB完整性的结构功能基础。AD中存在由Aβ介导的Ast活化，且该现象明显早于BBB破坏。然Aβ介导的Ast改变与BBB功能间的关系，尚未阐明。益气通络方药被临床广泛应用于防治AD，且疗效显著，而防治机制需深入研究。结合前期工作，假定干预Ast对Aβ的反应性，调控胶质-血管耦合，进而影响BBB完整性，是该方防治AD的主要机制。本课题拟以SAMP8小鼠AD模型、Ast与脑微血管内皮细胞接触共培养体系为研究对象，以Ast的AQP4极性分布与旁分泌SHH通路为切入点，采用功能蛋白拮抗剂方法，结合分子生物学技术阐明该方干预BBB完整性的机制。为益气通络方药防治AD注入新的科学内涵。

阿尔茨海默病（AD）发病率逐年上升，成为老年痴呆症的主要元凶。中医认为AD主要由气虚血瘀，毒损脑络所致。血脑屏障（BBB）遭破坏是毒损脑络的现代病理生理基础。星形胶质细胞(Ast)介导的胶质-血管耦合是维持BBB完整性的结构功能基础。AD中存在由Aβ介导的Ast活化，且该现象明显早于BBB破坏。然Aβ介导的Ast改变与BBB功能间的关系，尚未阐明。益气通络方药被临床广泛应用于防治AD，且疗效显著，而防治机制需深入研究。. 以SAMP8小鼠AD模型为研究对象,整体观察益气活血方药对AD 的防治作用。选用不同月龄的SAMP8鼠分为模型组、安理申组和补阳还五汤组，并设同月龄SAMR1鼠为对照组。药物干预8周，在干预期间记录SAM 鼠的基本情况用于评价老化程度；干预结束通过水迷宫实验评价行为学变化； 结果表明受试药物能够延缓衰老进程，改善脑老化及行为认知功能（P<0.05）。ELISA法检测血清Aβ40 及Aβ42 的水平，发现血清Aβ水平随SAMP8 鼠年龄增加而增高，5月与11月组间比较具有显著差异性（P<0.05）；药物能抑制Aβ40的增高（P<0.05）。免疫组织化学染色标记6E10，结果显示11月龄SAMP8 鼠海马区神经元内有Aβ沉积，药物减少了其沉积。采用免疫荧光染色及蛋白印迹法观察ZO-1、Occludin、GFAP、AQP4及SHH的表达，评价BBB完整性及AST活化状况。结果表明SAMP8 鼠与同龄SAMR1 鼠相比，两指标的表达有所下降；8月龄SAMP8 鼠较5月龄SAMP8 鼠降低更为明显。SAMP8 鼠皮质中Occludin及ZO-1的蛋白水平随增龄而降低；给予药物干预后，两蛋白水平有所增加。且随着增龄，AST 过度活化，其极性及维持血脑屏障完整性的SHH蛋白水平不断下降。. 体外以原代培养AST的细胞珠C8-D1A为研究对象，观察不同刺激作用下AST活化的异质性（A1型促炎，A2型神经保护）。通过ELISA 法及RT-PCR 检测活化AST不同亚型相关因子的表达，结果表明LPS能够诱导A1亚型，低氧及低浓度Aβ能诱导A2亚型。药物能降调促炎因子的表达，而促进A2型分泌因子的表达（P<0.01）。. 结论：益气活血方药能够延缓SAM 鼠老化进程，改善AD症状，可能与抑制AST 过度活化、调控AST活化异质性有关。