五脏温阳化瘀汤对Akt-mTOR-GSK-3β-Tau蛋白降解介导动脉粥样硬化型VD的效应机制

The Tau protein builduping neurons in hippocampal CA1 area is leading to the pathological basis of vascular dementia .According to new research, Akt-mTOR-GSK-3 beta is the common link of autophagy-lysosome and ubiquitin-proteasome degradating Tau protein.Tau protein builduping is consistent with the “endogenous turbid pathogenic” of traditional Chinese medicine.Previous clininal and experimental studies have confirmed that Wuzangwenyanghuayutang can block the VD process. Therefore, our hypothesis, Wuzangwenyanghuayutang may promote the Tau lysosome and protease degradation an block VD process.This project intends to copy the AS VD model rats, electron microscope autophagy body such AS ultrastructure of hippocampal neurons, immune confocal microscope detecting protein Tau hippocampal CA1 area location , RT-PCR,immunohistochemistry and Western Blot detecting Tau protein, Akt-mTOR-GSK-3 beta pathway downstream autophagy Beclin 1 specific protein, Becl-2,LC3,P62 and ubiquitin E3, S26, pGSK-3 beta, pTau, immune coprecipitation detecting interactions between proteins.In order to clarify Akt-mTOR- GSK-3 beta pathway in autophagy-lysosome and ubiquitin-proteasome degradating Tau protein and provide the objective basis for Wuzangwenyanghuayutang preventing VD.

Tau蛋白集结神经元海马CA1区是导致血管性痴呆(VD)的病理基础。最新研究表明，Akt-mTOR-GSK-3β是自噬溶酶体与泛素蛋白酶体介导Tau蛋白降解的共同环节。Tau蛋白集结与中医“内生浊邪”相一致，前期临床与实验研究均证实了五脏温阳化瘀汤能阻断VD进程。因此，我们假说，五脏温阳化瘀汤可能是促进Tau溶酶体与蛋白酶体降解而起到阻断VD。本项目拟复制AS型VD大鼠模型，电镜观察海马神经元自噬体等超微结构，共聚焦显微镜检测Tau海马CA1区蛋白定位，RT-PCR、免疫组化、Western Blot检测Tau蛋白，Akt-mTOR-GSK-3β下游Beclin1，Bcl-2，LC3，P62及泛素，E3、S26，pGSK-3β、pTau表达，免疫共沉淀检测蛋白相互作用，阐明Akt-mTOR-GSK-3β在自噬溶酶体与泛素蛋白酶体介导Tau蛋白降解机制，为五脏温阳化瘀汤防治VD提供客观依据。

Tau蛋白过度磷酸化引起的神经纤维缠结（NFTs）是导致血管性痴呆（VD）的关键病理基础，而Akt-mTOR-GSK-3β细胞信号转导途径是自噬溶酶体介导磷酸化Tau蛋白降解的重要机制。临床实践证实五脏温阳化瘀汤对VD具有较好疗效，推测其机制或与促进磷酸化Tau蛋白降解有关。本项目采用高糖高脂饮食饲喂SAMP8快速衰老小鼠，成功构建动脉粥样硬化型血管性痴呆小鼠动物模型。以SAMR1小鼠为正常对照，以多奈哌齐组为阳性药物对照。连续给药8周。给药结束，以Morris水迷宫测试小鼠定位航行与空间探索能力；电镜观察NFTs、脑神经纤维微管超微结构；qRT-PCR检测CDK-5mRNA表达水平；WB检测蛋白磷酸酶2A甲基化、蛋白激酶A（PKA）催化亚基、Tau蛋白磷酸化及CDK-5蛋白表达水平。测试结果显示，接受五脏温阳化瘀汤治疗的小鼠，其定位航行和空间探索能力明显改善，NFTs减少，微管超微结构相对完整，蛋白磷酸酶2A甲基化、CDK-5、PKA催化亚基与Tau蛋白磷酸化表达水平显著降低。本研究部分揭示了五脏温阳化瘀汤改善AS型VD小鼠认知能力的分子机制，即多靶点调节Akt-mTOR-GSK-3β细胞信号转导途径介导的自噬清除磷酸化Tau蛋白，减少NFTs。本研究为五脏温阳化瘀汤临床防治VD的提供了新的理论依据，有助于推动中医扶阳理论的现代化进程。