新近发现,老年性痴呆（AD）脑内Aβ与tau相互作用的毒性损伤是导致认知损害的关键，其核心作用环节与mTOR通路及PAR-1/MARK-tau轴有关。中医认为AD核心病机在于脑髓空虚，痰浊蒙窍，而"髓空"在治法上有补肾生髓与补脾胃元气生精养髓之偏重。洗心汤是补气生精，化痰开窍治呆名方。前期研究发现，该方能保护轴突内微管结构，抑制tau蛋白磷酸化及tau毒性。那么，洗心汤是否对Aβ与tau相互作用的毒性损伤有保护作用，值得深入研究。项目采用分子生物学、膜片钳等方法，通过体内外实验，从关键部位形态功能及毒性标志物的变化，探讨Aβ与tau毒性作用途径及洗心汤的干预效应；从Akt/mTOR通路、PAR-1/MARK-tau轴及神经元凋亡与线粒体变化，探讨其干预机制，从而证实该方可能抑制Aβ/tau毒性而实现神经保护的假说。研究将对"髓空痰浊"的实质及其治法理论作出新的诠释，为AD防治提供新的思路。

Recent study has shown that toxic injury from interaction of Aβ and tau in AD (Alzheimer's Disease) constitutes the main reason for cognitive impairment, with its key role bearing a relationship with mTOR pathway and PAR-1/MARK-tau axis. According to traditional Chinese Medicine, main pathogenesis of AD lies in the state of "hollowness of brain marrow and obstruction of orifices by turbid phlegm". Treatment of "hollowness of brain marrow" mainly includes nourishing kidney to generate marrow and replenishing spleen and stomach to produce essence with the aim at nourishing marrow. Xixin Decoction is a famous formula which invigorates qi to generate essence and eliminates phlegm for resuscitation. Our previous study has demonstrated that it can protect the structure and function of microtubes as well as prevent axons from injury，inhibit the phosphorylation of tau protein and its following toxicity. Thus, it is worthwhile to further study whether Xixin Decoction has the therapeutic effect of protecting the toxic injury of Aβ and tau interaction. To confirm our hypothesis, various state-of-the-art methods such as molecular biology and patch clamp will be used in vivo and in vitro experiment. The toxicity of Aβ and tau interaction and intervention effects of Xixin Decoction will be tested by observing the morphological and functional changes of synapses, axon, and dendrite and the changes of some toxicity markers. In the meantime, its effects on Akt/mTOR pathway, PAR-1/MARK-tau axis, neuronal apoptosis and mitochondria functions will be also evaluated. This study will propose a new explanation for the pathogenesis of "hollowness of brain marrow and obstruction of orifices by turbid phlegm" and its corresponding treatment theory, thus providing a new way and experimental basis for AD precaution and treatment.

本项目基于老年性痴呆（Alzheimer's disease，AD）“髓空痰浊”病机学说，提出名方洗心汤可能通过抑制AD脑内Aβ/tau毒性而实现神经保护作用的假说，并从关键部位形态功能及毒性标志物的变化，探讨Aβ与tau毒性作用途径及洗心汤的干预效应；从Akt/mTOR通路、PAR-1/MARK-tau轴及神经元凋亡与线粒体变化，探讨其干预机制。在体研究中发现：洗心汤能够改善不同时程APP/PS1双转基因小鼠的学习记忆能力；对不同时程APP/PS1双转基因小鼠海马神经元突触、轴突、树突等超微结构具有保护作用；洗心汤在不同时程，均能有效增加 APP/PS1 双转基因小鼠皮层组织及海马区域 PSD95、MBP、NMDAR2B、GAP43蛋白的表达，减少NR2B、FYN的含量，其可能通过保护MBP 、PSD-95、NMDAR2B、GAP43免受Aβ等毒性物质攻击，减少NR2B的过度表达，并干扰FYN等调节酶对PSD-95等与NR2B过度结合的介导，进而降低两者过度结合所产生病理作用；同时，洗心汤能增加小鼠脑内NEP、IDE的表达，减少PS2、BACE1的含量，恢复Aβ1-40 与Aβ1-42蛋白的正常生成和代谢，并通过调节Cdk5和PP2A的表达水平，减轻tau蛋白异常过度磷酸化。因此，通过调节Aβ/tau蛋白的生成代谢及其相互作用，从而保护AD小鼠海马神经元免于Aβ/tau的毒性损伤是洗心汤防治AD，抑制AD病理进展的重要机制。体外研究显示：以中、高浓度洗心汤含药脑脊液作用24～48h，可明显降低Aβ1-42诱导的神经细胞凋亡率，提高细胞活力，改善凋亡细胞形态，对海马神经元损伤具有明显保护作用。进一步研究发现，洗心汤含药脑脊液通过影响大鼠海马神经元PI3K/AKT/mTOR通路，增强PAR-1、MARK4表达，并通过与PAR-4的相互调节效应，影响PAR-1/MARK-tau轴，对Aβ/tau的毒性损伤起到抑制作用；洗心汤含药脑脊液同时可降低大鼠海马神经元 P13K、 Akt表达，抑制Caspase-3、8、9的生成，改善Bcl-2/Bax水平，从而阻断 Caspases 家族凋亡蛋白激活引发的凋亡级联效应，减轻Aβ毒性损伤，起到神经保护作用。本项目研究对“髓空痰浊”的内涵及其治法理论作出了新的诠释，为AD防治提供了新思路，为从脾从痰论治AD提供了科学依据。