基于Parkin活化探讨“通督启神”法电针调控线粒体自噬干预AD小鼠的脑保护机制

It was previously found that electroacupuncture could decrease the level of Aβ and improve the learning and memory ability in AD mice (ECAM,2016). Further more, autophagy vacuoles and mitochondria were observed in the AD mice (Journal of Neuroanatomy, 2015). New research shows that mitophagy inhibits Aβ reverses cognitive deficits in AD. In this study, APP/PS1 mice were selected as the research object. The Parkin pathway closely related to mitophagy was studied. Micro-PET, laser confocal and immunohistochemical, Western blot techniques were used. The intervention effects of electroacupuncture on AD mice Aβ and mitophagy were observed from three levels of "in vivo-tissue-molecule". The key targets of mitophagy and Parkin activation in different brain regions were detected and analyzed. The key role of Parkin activation in mitochondrial autophagy was elucidated. In order to verify the hypothesis that ‘electroacupuncture can improve the ability of learning and memory of AD model by activating Parkin to increase mitophagy to protect brain neurons and reduce Aβ precipitation’, we try to explain the mechanism of electro-acupuncture intervention in AD from the point of molecular biology. Therefore to develop a new clinical treatment method for AD and provide its experimental basis.

申请人前期发现电针可以降低Aβ水平改善AD小鼠的学习记忆能力（ECAM，2016），并观察到AD小鼠中的自噬空泡及线粒体等囊性细胞器（神经解剖学杂志，2015）。最新研究显示:线粒体自噬可抑制Aβ沉淀逆转AD的认知缺陷。本课题选用转基因APP/PS1小鼠作为研究对象，从与线粒体自噬密切相关的Parkin活化相关靶点入手，应用Micro-PET、激光共聚焦、免疫组化、Western blot多种技术，从“在体-组织-分子”三个层次进行研究，观察电针对AD小鼠Aβ表达和线粒体自噬的干预效应，分析不同脑区线粒体自噬及Parkin活化关键靶点，明确Parkin活化在电针调控线粒体自噬中的关键作用。以验证“电针通过激活Parkin增加线粒体自噬保护脑神经元，减少Aβ沉淀，从而改善AD小鼠的学习记忆能力”的假说，力求从分子生物学角度阐释电针干预AD的作用机制，为临床治疗AD开辟新的途径并提供科学依据。

阿尔茨海默病患病率高，病患群体基数大，研究针刺干预阿尔茨海默病的作用机制具有必要性，申请人前期发现电针可以降低Aβ水平改善AD小鼠的学习记忆能力，并观察到AD小鼠中的自噬空泡及线粒体等囊性细胞器。最新研究显示:线粒体自噬可抑制Aβ沉淀逆转AD的认知缺陷。本课题选用转基因APP/PS1小鼠作为研究对象，从与线粒体自噬密切相关的Parkin活化相关靶点入手，应用Micro-PET、透射电镜、尼氏染色、激光共聚焦、免疫组化、ATP测定、Westernblot多种技术，从“在体-组织-分子”三个层次进行研究。.Morris水迷宫结果显示EA组的低于M组，具有统计学意义（P < 0.05），EA+3MA组与EA组比较差异明显说明“通督启神”针法能够改善6月龄AD模型小鼠的学习与记忆能力。Micro-PET结果EA组较M组18F-AV45摄取率更低，EA＋3MA组的摄取率较高，具有统计学意义（P < 0.05）。说明“通督启神”针法对小鼠Aβ沉积具有一定的缓解作用。小鼠海马区尼氏染色的结果显示与M组相比较，EA组尼氏体数量均有所增加，说明“通督启神”法电针可改善AD模型小鼠脑内海马区神经元细胞的丢失现象。透射电子显微镜成像组间比较显示“通督启神”针法可以维护线粒体结构完整性。免疫荧光结果显示PINK1、PARKIN与TOM20存在共定位。LC3与Aβ存在共表达。Western blot检测结果表明神经元线粒体自噬相关蛋白PINK1、PARKIN、LC3、Aβ、HIF -1α、BNIP3、AMPK等蛋白相对表达量,并通过组间比较分析电针的调控效应。组间比较具有差异性且具有统计学意义（P < 0.05），说明“通督启神”法电针能够降低Aβ、Tua，上调自噬相关蛋白PINK1、PARKIN、LC3、 HIF-1α、BNIP3、Beclin1、AMPK、SIRT1、胞核FOXO3A以及下调P62、SIRT3、mTOR、胞质FOXO3A表达水平以激活AD模型小鼠神经元线粒体自噬。.综上所述，以上实验证明了“电针可以通过激活小鼠脑神经元线粒体自噬减少Aβ沉淀，从而改善AD小鼠的学习记忆能力”的假说，从神经元线粒体自噬角度阐释电针干预AD的作用机制。