Nrf2携带酸敏感离子通道（ASICs）出核在脑缺血星形胶质细胞损伤中的作用及分子机制研究

The injury of astrocytes is a critical event in cerebral ischemia. Investigation of the mechanism of astrocyte injury is important for searching new therapeutic strategies for brain ischemia. Acid sensitive ion channels (ASICs) are a group of ion channels with non-selective permeability to various cations such as Na+ and Ca2+. They distribute mainly in the nucleus in astrocytes. However, the pathological significance of this distribution remains unclear. Our recent work showed that stimulation of astrocytes with oxygen and glucose deprivation/reperfusion (OGD/R) induced a significant export of nuclear ASICs. We also showed that ASIC1 lacks the nuclear export signals and the nuclear localization sequences, suggesting that the export of nuclear ASICs needs the assistance of other molecules. Disturbing the nuclear export signals in an anti-oxidant protein NF-E2-related factor 2 (Nrf2) markedly suppressed the export of nuclear ASICs. Given the fact that ASICs are permeable to Ca2+ and Ca2+ overloading is critical to cell injury, we will investigate the sub-cellular locations and functional changes of ASICs in astrocytes, as well as their pathological significances using ischemia-reperfusion injury models. We will also investigate how Nrf2 responds to cerebral ischemia and assists the export of nuclear ASICs in astrocytes. The ultimate results will clarify the role of ASICs in ischemia-induced injuries of astrocytes, and provide a new basis for the exploration of novel protective mechanisms and drugs for astrocyte injury.

星形胶质细胞损伤是脑缺血发展的关键环节，深入研究其机制对寻找脑缺血治疗方案具有重要意义。酸敏感离子通道（acid sensing ion channels, ASICs）是一组对阳离子具有非选择通透性的离子通道，在星形胶质细胞主要分布于细胞核内，但ASICs此种分布的病理学意义尚不清楚。我们发现：OGD/R能使星形胶质细胞ASICs转出细胞核；ASIC1缺乏出核和核定位信号，提示ASICs出核需要其它分子的携带；干扰抗氧化蛋白Nrf2出核信号显著抑制ASIC1出核。鉴于ASICs能通透Ca2+，而钙超载又是诱发细胞损伤的关键，本研究将借助缺血/再灌损伤模型，分析ASICs在星形胶质细胞的亚细胞定位与功能变化及病理学意义，并阐明Nrf2响应脑缺血、携带ASICs出核的分子机制。研究结果将明确ASICs在星形胶质细胞缺血损伤中的作用，为寻找星形胶质细胞损伤的保护机制及药物研发提供新的思路。

酸敏感离子通道(acid sensing ion channels, ASICs)是一组对阳离子具有非选择通透性的离子通道，其在星形胶质细胞中的功能尚不清楚，本项目针对该问题展开深入研究。发现ASIC1、ASIC2和ASIC3表达于大鼠原代培养星形胶质细胞，且主要分布于细胞核中；OGD/R刺激能诱导ASIC1、ASIC2和ASIC3从细胞核转位到细胞浆中，并使ASICs表现出离子通透功能；ASIC1而非ASIC2或ASIC3能够介导与OGD/R相关的星形胶质细胞内Ca2+增加及星形胶质细胞凋亡；MCAO/R刺激能使大鼠前额皮层星形胶质细胞ASIC1从细胞核转位到细胞浆中。我们还发现OGD/R能显著上调与ASIC1直接结合的Nrf2在星形胶质细胞细胞浆中的表达水平，与此同时显著下调Nrf2在星形胶质细胞细胞核中的表达水平；当遭受OGD/R刺激时，Nrf2会从星形胶质细胞细胞核转位到细胞浆中，这种转位效应的意义在于促进ASIC1发生细胞浆转位。功能学研究显示，Nrf2基因沉默能抑制由OGD/R引起的原代培养大鼠星形胶质细胞细胞浆ASIC1蛋白增加及由OGD/R引起的细胞核ASIC1蛋白减少；此外，Nrf2基因沉默还能抑制由OGD/R引起的大鼠原代培养星形胶质细胞ASIC样电流幅值增加、细胞内Ca2+浓度升高、星形胶质细胞凋亡、及由MCAO/R引起的大鼠前额皮层星形胶质细胞凋亡。以往研究认为主要是神经元中的ASICs介导脑缺血等神经系统疾病的发生、发展，我们对ASICs在神经系统其它细胞中的功能了解得还很少，本研究以星形胶质细胞ASICs为中心，从创新性角度探索ASICs在神经系统的新功能，阐明Nrf2在介导星形胶质细胞ASIC1细胞浆转位中的关键作用，这对我们深入理解缺血再灌注诱导星形胶质细胞ASIC1细胞浆转位的分子机制具有重要意义。