激活嗅觉通路的“嗅三针”调控阿尔茨海默病转基因模型小鼠Aβ-MAPK-MG的机制研究

Alzheimer's disease(AD) is a kind of senile disease which arouse severe harm to the health. We applied olfactory three needle therapy and have got good curative effect in clinic,but the mechanism is still not clear. The newest studies show that AD has close relationship with two aspects, firstly,β-amyloid protein(Aβ)deposition results in the activation of microglia(MG),then activated MG mediates inflammatory reaction,secondly,(mitogen-activated protein kinase)MAPK signal pathway mediates inflammatory reaction,and Inflammation can directly lead to neuronal damage, accelerates the development of AD .In preliminary study,we have observed that olfactory three needle therapy can reduced the level of inflammatory factor and Aβ-MAPK expression in hippocampus of AD mice under the integrity of olfactory pathway.This might be one of the effective pathways for olfactory three needle therapy. Consequently,we intend to establish the relationship among Aβ-MG-MAPK. Based on the same inflammatory response link, we will take AD transgenic mice as research object,with its’ olfactory pathway getting cut and kept, and apply molecular biology as test technique.We will undergo a systematic study on the chain effect of “Aβdeposition- the activation of MAPK pathway-MG activation- inflammatory factor release”.We initially reveal the mechanism of olfactory three needle therapy ,that is to say this therapy maybe benignly adjust the Aβ-MAPK-MG mutual regulation through activating olfactory pathway. At last,we systematically elucidate the molecular mechanism of olfactory three needle therapy in the treatment of AD through olfactory pathway，meanwhile,we expect to provide a theoretical and experimental basis for clinical treatment of AD with this therapy.

阿尔茨海默病（AD）是严重危害患者健康的老年性疾病，临床上我们应用嗅三针对其有较好疗效，但作用机制尚不明确。最新研究显示，本病与由β-淀粉样蛋白（Aβ）沉积促小胶质细胞(MG)活化启动和丝裂原活化蛋白激酶(MAPK)通路介导的炎症反应密切相关，而炎症反应能够直接导致神经元损伤，加速AD的发展。预实验中，我们观察到嗅三针能在嗅觉通路完整的情况下降低AD小鼠海马炎症因子水平和Aβ-MAPK表达，这可能是嗅三针的效应途径之一。因此，本研究拟将Aβ-MAPK-MG相联系，基于炎症反应这一相通环节，以切断和保留嗅觉通路的转基因AD小鼠为实验对象，采用分子生物学等技术，对“Aβ沉积-MAPK通路激活-MG活化-炎症因子释放”的链条效应进行系统研究，揭示嗅三针通过激活嗅觉通路发挥对Aβ-MAPK-MG的良性调控的作用机制，系统阐明嗅三针通过嗅觉通路治疗AD的分子机制，对本病的临床治疗具有理论和实际意义

本项目按照计划书要求，如期进行了“激活嗅觉通路的“嗅三针”调控阿尔茨海默病转基因模型小鼠Aβ-MAPK-MG的机制研究”。项目将Aβ-MAPK-MG相联系，基于炎症反应这一相通环节，以切断和保留嗅觉通路的转基因AD小鼠为实验对象，采用分子生物学等技术，对“Aβ沉积-MAPK通路激活-MG活化-炎症因子释放”的链条效应进行系统研究。第一，采用Morris水迷宫，开展了嗅三针对AD小鼠认知能力的干预效应研究，研究结果证实嗅三针能够显著提高AD小鼠学习能力和空间记忆能力；第二，采用RT-PCR和琼脂糖凝胶电泳法，开展了嗅三针对AD小鼠海马MG炎性因子TNFα、IL-6、IL-1β表达干预效应研究，研究结果证实嗅三针能够抑制AD小鼠海马IL-6、TNF-a、IL-1β等细胞因子的释放，减轻炎症反应的泛化和持续，降低MG激活引发的炎症反应；第三，采用免疫荧光化学法和流式细胞术，开展了嗅三针对AD小鼠MG活化的干预效应研究，通过观察嗅三针对MG的特异性标志物CD68表达的影响，研究结果证实嗅三针可降低AD小鼠海马MG活化数量并降低海马活化的MG比例，进而抑制小鼠海马MG的活化；第四，采用Western Blot和免疫组化法，开展了嗅三针对AD小鼠APP/Aβ及MAPK信号通路活化的干预效应研究，研究结果证实嗅三针能够减轻小鼠海马区p-p38MAPK、p-JNK、p-ERK1/2和 p-tau Thr181的表达，以上四部分干预效应的产生均有赖于嗅觉通路的激活。初步阐明了嗅三针通过激活嗅觉通路改善AD小鼠认知障碍，提高学习能力和空间记忆能力，减轻Aβ淀粉样沉积，继而抑制MG产生炎性细胞因子，减轻脑内的炎症反应, 减少神经元损伤，并与抑制海马MAPK信号通路、下调tau蛋白过度磷酸化具有一定相关性。通过本项目研究，解决了嗅三针影响AD的作用途径和干预机制这一关键科学问题，为针刺治疗AD的临床应用提供了理论和实验依据。