电针对抗缺氧性脑损伤的分子机理：DOR对微小RNA的调控

Hypoxic brain injury is a common and serious medical condition. Unfortunately, there is a lack of effective therapy up to date. It is of utmost to search for novel strategies against hypoxic brain injury. Toward the goal, we have found that electroacupuncture (EA) can reduce the death rate of the animals exposed to prolonged hypoxia and attenuate hypoxic/ischemic brain injury and neural deficits, which is dependent on the activity of the dealt-opioid receptor (DOR). The activation and up-expression of DOR can mimic the EA-induced neuroprotection. However, the molecular mechanism for the EA-DOR neuroprotection remains poorly understood. Recently, we found that hypoxia alters the expression of the microRNAs that are relevant to neural responses to hypoxia, while DOR activation modulates such microRNA reaction. Based on these new observations and our other work, we hypothesize that EA regulates microRNAs via the DOR pathway and then stabilizes the membrane ionic hemostasis and upregulates intracellular Nrf2 signaling, thus rendering the brain more tolerant to hypoxic stress. This project is proposed to test this novel hypothesis with in vivo and in vitro models using molecular, transgenic and neurophysiological approaches. We will specifically ask the following three fundamental questions: 1) how EA alters the expression of DOR and microRNAs in the brain and attenuates hypoxic brain injury? 2) If selective up- or down-regulation of EA/DOR-sensitive microRNAs affects the EA- and DOR-induced neuroprotection? and 3) if the EA- and DOR-induced neuroprotection is dependent on the stabilization of membrane ionic homeostasis and the upregulation of Nrf2?

我们发现电针可减少慢性缺氧动物的死亡率和减轻缺氧缺血性脑损伤；激活或过表达Delta-阿片受体(DOR)可模拟电针的保护效应，抑制DOR则阻碍电针作用。我们还发现，缺氧改变微小RNA (miRNA)和 Nrf2的表达、引发膜离子稳态的失衡，激活DOR可调控这些变化。因而我们设想：电针通过DOR调控微小RNA的表达，籍此上调Nrf2介导的神经保护信号、减轻缺氧性膜离子紊乱，增强脑的缺氧耐受性。本项目拟用分子生物学、转基因、神经电生理等多种技术在动物和细胞模型上验证这一新学说。重点探索三个关键问题：①电针如何改变不同脑区内DOR和miRNA的表达并减轻缺氧性损伤？②选择性调控与电针/DOR相关的miRNA是否影响电针/DOR的神经保护效应？③电针-DOR-miRNA的神经保护效应是否依赖于胞膜离子的稳定和胞内Nrf2的上调？研究结果有助于针刺对抗缺氧性脑病的深入探索和促进传统与现代医学的融合。

缺氧可影响脑内多种微小RNA的表达与功能，导致神经元功能异常，引起或恶化多种退行性神经系统疾病的病理性变化。业已表明，脑卒中、老年性痴呆和巴金森氏病等多种难治性神经系统疾病均与缺氧性脑损伤有关。然而，这些世界性医学难题目前依然缺乏卓有成效的临床举措。本课题从缺氧对脑内微小RNA的影响中寻找线索，探索delta-阿片受体（DOR）对缺血缺氧脑细胞的保护效应和在电针脑保护中的作用，并进而拓展研究范围，取得了系列新成果。我们的结果已表明，缺氧导致脑内各种微小RNA的差异性变化，而DOR的激活能选择性地良性调控微小RNA的病理性变化。通过微小RNA特异性调控靶基因的线索，我们进而发现：1）增强DOR的活动可大幅减少ROS刺激从而减轻缺氧性神经元的损伤；2）DOR的激活与表达能促进缺氧时促炎性小胶质细胞向抗炎性小胶质细胞转化，从而抑制TNF等炎症因子的产生；3）DOR的激活可减轻巴金森氏病样病理损伤，其机制与加强PINK1/Parkin-调控的线粒体自噬功能有关；4）活化DOR的信号通路能有效抑制BACE1的表达与功能，减轻阿尔茨海默氏病样损伤，而与DOR同一家族的MOR活动则导致相反的变化；5）电针能上调DOR的活动，并调控某些微小RNA的活动而抑制退行性神经系统病变，如实验性巴金森氏病。为进一步明确脑细胞反应的特异性和其它细胞反应的特殊性，我们还比较研究了DOR对缺血缺氧性敏感（如大脑和肾脏）和不敏感（如肝脏）器官内微小RNA的影响。我们发现，神经细胞与同为缺氧敏感的肾细胞相比，两者的DOR保护机制有所不同，并籍此发现了对抗缺氧性肾损伤的新途径。我们的系列结果可能为防治缺氧敏感器官的缺氧缺血性损伤及其它退行性疾病提供新线索。在项目申请书的预期目标中，我们原计划发表SCI 论文5-7 篇。实际执行课题以来，我们已发表8篇SCI论著和2部由Springer出版的有关针刺研究的大型英文专著, 并申报了一项国家发明专利。此外，我们的工作还获得了首届 Zayed国际传统医学奖金质奖章和其它奖项。