基于“毒损脑络”病机探讨黄连解毒汤对阿尔兹海默病 Aβ毒性损伤的保护机制

It has been considered as an important factor for the Aβ-cascade reaction in pathogenesis of AD that the excessive accumulation of β-amyloid peptide can activate the microglia which then induce the inflammatory reaction . At normal state , microglia keep the balance of Aβ production and elimination in AD brains. However, excessive activation of microglia which produce many kinds of inflammatory factors can lead severe damage to the neurons, and therefore to inhibit the microglia activation and reduce the inflammation is a main target in AD therapy research. Experiments of others and ours prove that Huanglianjiedu Decoction(HLJDT) can inhibit releaseing inflammatory factors of microglia inflammation , abate inflammatory reaction and protect the hippocampal cholinergic neurons. It is unknown that whether it is by regulating the activity of microglia to abate inflammatory reaction. According to the traditional Chinese Medicine (TCM) hypothesis that “damage of brain was collateral by toxin”,we will use different methods such as detecting microglial cell surface markers of AD mice and the signal transduction pathway of toll receptor etc to investigate the effect and mechanism of HLJDT on regulation of microglia activation,to disclose the molecular mechanism of detoxicating on AD.The result has important significance in theory and practice to applicate with the detoxicating therapy on other degenerative diseases of nervous system guided by the principle of treating different diseases with the same method.

Abeta过度沉积激活小胶质细胞引起的炎症反应被认为是AD的核心病理机制；抑制小胶质细胞过度激活导致的炎症损伤是AD研究的主要靶向之一，这一特点与中医对痴呆“毒损脑络”病机及清解毒邪治法的认识不谋而合。黄连解毒汤作为清热解毒法的代表方剂之一，不仅能够清解热毒，还能够清解内生邪毒，在神经系统免疫相关疾病中应用前景广阔。我们前期研究表明，黄连解毒汤对小胶质细胞的活化及其产生的炎性因子具有一定调控作用，而这种作用有可能通过调控小胶质细胞表面的离子通道而发挥作用，但其调控的具体途径及靶点并不明确。我们拟通过在体及离体实验，采用免疫荧光、蛋白质印迹法和激光共聚焦显微镜等技术，观察黄连解毒汤对小胶质细胞表面相关受体、离子通道等指标的影响，探讨黄连解毒汤对AD小鼠脑内小胶质细胞激活与抑制失衡状态的调控作用途径及靶点，为进一步明确“毒损脑络”生物学基础及清热解毒法防治神经系统免疫相关疾病提供依据。

选用BV2小胶质细胞为研究对象，采用脂多糖/Aβ1-42诱导其激活，采用黄连解毒汤含药血清干预，并以盐酸多奈哌齐作为对照, 通过观察BV2小胶质细胞形态、细胞活性的变化，观察TLR4/NF-κB通路相关基因、蛋白等指标的变化，观察相关炎症因子含量的变化，系统整理和分析黄连解毒汤对BV2小胶质细胞激活及吞噬功能的调控作用机制；并以APP/PS1转基因AD小鼠为研究模型，采用黄连解毒汤干预，并以盐酸多奈哌齐作为对照，运用免疫组化染色、实时荧光定量PCR及蛋白质印迹等技术，观察AD小鼠海马CA1区小胶质细胞形态变化及TLR4/NF-κB通路相关基因、蛋白等指标的变化，探讨黄连解毒汤对APP/PS1转基因AD小鼠海马CA1区小胶质细胞活化的调控作用机制，以期为中医解毒法治疗神经系统变性疾病提供理论支撑和科学依据。结果表明：黄连解毒汤通过TLR4/NF-κB信号通路可有效调控体外BV2小胶质细胞及APP/PS1转基因AD小鼠海马CA1区小胶质细胞的激活，抑制其分泌释放相关炎症因子，影响BV2小胶质细胞的吞噬功能从而起到对神经炎症损伤的保护作用。本项目运用中医清热解毒治法（黄连解毒汤）反证AD“毒损脑络”的病机，进而揭示了AD中“毒”的现代科学内涵，阐明了“毒损脑络”在AD发生发展中的重要地位和具体生物学基础，从而拓宽了中医药防治AD的科学思路，也为在中医“异病同治”治则指导下运用解毒法治疗其他神经系统变性疾病提供理论支撑和实验依据。