艾灸督脉清除APP/PS1双转基因小鼠Aβ的自噬调控机制研究

Moxibustion on DU meridian points was a treating method of Alzheimer's disease (AD), which can activate Du meridian and regulate the mind . Preliminary clinical study have confirmed that it is quite effective in improving learning ability and memory.. As widely accepted, β-amyloid protein (Aβ) plays a central role in AD pathogenesis. The abnormal release and deposition of Aβ is the initiating factor to form senile plaque and the important components of its core. Autophagy, the basic intracellular mechanism for catabolic and continuous clearance of unnecessary or dysfunctional components, occupies a crucial role in AD . Amyloid⁃β protein (Aβ) can be eliminated during autophagy. Observation will be made on the effects of event-related potentials (ERP)on the AD transgenic mice behavior during water maze test. Western blot will be applied to analyze the autophagy-related protein levels of LC3-I, LC3-II and the autophagy substrate P62. To verify whether mTOR signal pathway was involved in autophagy infuenced by moxibustion,the effect on phosphorylation of the mTORCl substrate p70 will be investigated. For further determination of the autophagy levels in the brain of mice, transmission electron microscopy will also be used to observe autophagicvacuoles in cortical neurons from mouse. Moxibustion on DU meridian points　can antagonize against　β-amyloid protein induced neuronal damage　and improve the learning and memory function of AD rats through regulating the expression of PI3K/Akt/mTOR signaling pathway. At present, we are attempting to elucidate the molecule mechanism of the effect of Moxibustion on DU meridian points, e.g. action target, signal transduction pathway and related gene expression, in order to provide a more extensive theoretical basis for the clinical application of this method .

艾灸督脉具有通督益肾、生髓健脑、开窍调神功效，临床研究发现其治疗AD前期疗效确切。脑内Aβ沉积是AD发病的中心环节，细胞自噬是清除有害的Aβ的重要途径，调控细胞自噬促进Aβ清除，是治疗AD的关键靶点，据此提出“艾灸督脉通过调控PI3K/Akt/mTOR通路诱导自噬清除Aβ蛋白从而改善认知功能障碍”的科学假说。本实验通过观察艾灸督脉对AD转基因小鼠行为学评分、事件相关电位、海马Aβ、与自噬有关的蛋白(LC3-I ,LC3-II,自噬底物蛋白P62, Beclin 1，Bcl-2)以及电镜观察神经元内自噬泡的含量变化，获取艾灸督脉改善认知功能、诱导自噬清除Aβ的直接证据；通过检测PI3K、AKT、p-AKT 、mTOR、p-mTOR蛋白表达来探明该灸法是否通过调控PI3K/Akt/mTOR通路来上调自噬，促进Aβ清除，深入阐释艾灸督脉治疗AD的作用机理，旨在为该灸法临床应用提供重要的理论依据。

艾灸督脉具有通督益肾、生髓健脑、开窍调神功效，临床研究发现其改善认知功能障碍疗效确切。脑内Aβ沉积是AD发病的中心环节，细胞自噬是清除Aβ的重要途径，调控细胞自噬可以促进Aβ清除，是治疗AD的关键靶点，通过检测APP/PS1双转基因AD小鼠行为学评分、事件相关电位，观察艾灸督脉改善小鼠认知功能作用；运用免疫组化观察AD转基因小鼠皮质和海马脑区Aβ1-42沉积状态，电镜观察皮层和海马内自噬泡的含量变化以及western-blot法检测自噬相关蛋白标志物，发现艾灸督脉对自噬促Aβ清除的证据；利用western-blot法检测PI3K、AKT、p-AKT、mTOR、p-mTOR蛋白表达来发现艾灸督脉上调自噬的调控通路。研究结果显示：从神经行为学评分及事件相关电位P300测定数据发现艾灸督脉能改善小鼠认知功能能，提高AD转基因小鼠的学习记忆能力；免疫组化结果显示，与正常组相比，模型组皮质区和海马脑区Aβ1-42阳性表达增多，艾灸督脉减少APP/PS1双转基因AD小鼠脑内Aβ的聚集，减轻Aβ毒性对神经元的损害；透射电镜结果显示艾灸督脉增加海马区内自噬泡和溶酶体数量； western-blot法检测显示艾灸督脉降低LC3-I、P62、p-P70S6K蛋白表达水平与PI3K、Akt、p-Akt、mTOR、p-mTOR蛋白表达，同时升高LC3-II蛋白表达及LC3-II/LC3-I比值。课题研究结果表明艾灸督脉具有抑制PI3K/Akt/mTOR信号通路的异常激活，发挥增强或恢复细胞自噬水平，促进机体清除Aβ沉积，进而改善认知功能作用。本研究结果有助于从分子生物学角度揭示艾灸督脉治疗AD作用机制，为艾灸督脉治疗AD临床应用提供重要的理论依据。