基于调控Nedd4-2/TREM2/DAP12信号通路研究解毒化瘀法治疗阿尔茨海默病的作用机制

Studies have shown that Trem2 genetic mutations increase risk in Alzheimer disease (AD), besides, TREM2/DAP12 mediate the microglial Aβ phagocytosis and suppress neuroinflammation in the pathological process of AD. Our previous results have shown that, ubiquitin ligase E3 Nedd4-2 mediate the ubiquitination and degradation of TREM2 in AD. In traditional Chinese medicine, “brain collateral damage by internal toxin” is considered as the main pathogenesis of AD. Based on the therapeutic methods of heat-clearing and detoxifying, and removing blood stasis and dissipating phlegm, a decoction named Jie-Du-Hua-Yu-Tang (JDHYT) was created by us and the previous researches showed that, JDHYT can inhibit the overactive microglia and the release of inflammatory cytokines, in addition, it can also down-regulate Nedd4-2 protein expression and up-regulate TREM2/DAP12 protein expression. These results indicate that JDHYT can regulate the homeostasis between Aβ phagocytosis and neuroinflammation via modifying the ubiquitination of TREM2/DAP12 in AD. In this project, 5×FAD and Trem2-/- mouse models will be utilized to clarify whether JDHYT regulate the homeostasis between Aβ phagocytosis and neuroinflammation via Nedd4-2/TREM2/DAP12 signaling in AD. The above researches may clarify the mechanisms of JDHYT in treatment of AD with a view to providing experimental evidence for clinical application of the detoxifying and removing blood stasis methods in AD treatment and for further potential drug discovery.

研究表明Trem2基因突变增加患阿尔茨海默病（AD）的风险，并且TREM2/DAP12参与调控小胶质细胞对Aβ的吞噬和抑制脑内炎症反应，从而影响AD病理进程，我们新发现AD病理过程中泛素连接酶E3 Nedd4-2介导TREM2泛素化降解。中医学认为“毒损脑络”是AD的主要病机，以清热解毒、祛瘀豁痰为法则创立的解毒化瘀汤可以抑制小胶质细胞激活和炎性因子释放，并且可以下调Nedd4-2表达，上调TREM2/DAP12表达，提示AD中解毒化瘀汤可能会通过泛素化修饰TREM2/DAP12调控Aβ吞噬与神经炎症反应之间稳态，从而发挥神经保护作用。本项目拟在前期基础上采用5×FAD和Trem2-/-小鼠模型，阐明解毒化瘀汤是否通过调节Nedd4-2/TREM2/DAP12通路影响Aβ吞噬与神经炎症反应之间的稳态，揭示解毒化瘀法治疗AD的具体机制，为该法的临床应用及进一步药物研发提供研究基础。

本项目研究了以清热解毒、祛瘀豁痰为法则创立的黄连解毒汤加味方（解毒化瘀汤）对阿尔茨海默病模型小鼠学习、记忆障碍的影响及调控机制。通过研究发现，解毒化瘀汤显著改善了阿尔茨海默病（AD）模型小鼠的学习、记忆障碍。超高效液相色谱质谱联用（UPLC-Q-TOF/MS）结果显示，和原方黄连解毒汤相比，在解毒化瘀汤中，谷氨酸和天冬氨酸等氨基酸含量更丰富。分子生物学结果提示解毒化瘀汤上调了AD模型小鼠海马NMDA受体相关亚基的表达。基于飞行时间质谱联用技术（GC-TOF/MS）的代谢组学检测结果表明，相较黄连解毒汤，解毒化瘀汤显著提高了AD模型小鼠海马的腺苷水平。解毒化瘀汤同时也上调了腺苷下游的ATPase/AMPK信号通路。16S rDNA肠道菌群检测结果表明，解毒化瘀汤调控了AD模型小鼠肠道Firmicutes, Bacteroidetes, Proteobacteria等菌属的多样性。通过对代谢组学和肠道菌群进行的相关性分析，我们最终鉴定出Dorea是响应解毒化瘀汤上调AD模型小鼠海马腺苷水平的肠道菌属。我们的研究提示，解毒化瘀汤可能是治疗AD的新型有效药物。基于该项目，我们研究了中药复方相关单体——人参的主要有效单体成分—人参皂苷Rb1在神经退行性疾病中发挥神经保护作用的机制。此外，依托本项目我们也揭示了包括CNTNAP4、GLUD2和GLT-1在内的基因/蛋白和帕金森病（PD）发病的关系，我们首次提出多巴胺神经元表达的CNTNAP4基因缺陷小鼠是PD的一个新的、有效模型。