ASICs核膜外再分布在颅脑创伤血管源性脑水肿酸中毒微环境中的代谢机制研究

Intracellular calcium overloading is one of key causitive factors for damage of blood-brain barrier (BBB) under acidic conditions of traumatic vasogenic brain edema induced by craniocerebral trauma. Acid-sensing ion channels (ASICs), a family of ligand-gated cation channels activated by protons in acidosis, are known to be one of the most crucial ion channel family relevant to the acid metabolism and calcium overloading in brain. It was well accepted that the expression of ASICs is mainy on the cell surface of neurons, but recent studies suggested that ASICs existed in neurogliocyte. However, the functions of ASICs in non-neuronal cells in the brain are largely unknown. Our prelimilary data suggests that ASICs are highly expressed on astrocytes nuclear membrane. Interestingly, we found the re-distribution of ASICs from nuclear membrane to cytoplasm and the migration to cell surface in the cultured astrocytes under the condition of pH6.0.More importantly, we recorded increased calcium influx in these cultured astrocyte under the acidic condition. In this proposal, based on our prelimilary data, we hypothesize that activation of ASICs in BBB end-foot cells and vascular endothelial cells is critical for BBB damage caused by acidic microenvironment in traumatic vasogenic brain edema. Our experiment design in this proposal will focus on the mechanisms involved in re-distribution of ASICs and its role on the damage of BBB at molecular, cellular and entire animal model levels. Therefore, the outcome of this proposal will deepen our understanding on the cause of microvascular spasm in vasogenic cerebral edema,and will provide the experimental basis for development of ASICs as a metabolism drug-target in the treatment of traumatic cerebral edema.

细胞内钙超载是导致创伤性血管源性脑水肿与颅脑损伤后酸中毒环境下的血脑屏障破坏的主要原因之一。酸敏感性离子通道是一类在低pH环境下被激活的配体门控阳离子通道，是已知颅内与酸代谢和钙超载有关的重要通道。以往研究发现ASICs主要存在于神经元的胞膜上，近年来有报道称ASICs亦存在于神经胶质细胞，但其在神经胶质细胞上的功能未知。本课题组预实验发现ASICs在星形胶质细胞核膜上高表达，在pH为6.0的酸性环境下逐渐由核膜向胞膜上迁移再分布，同时记录到内向钙离子流明显增加。本项目在预实验的基础上，提出星形胶质细胞上的ASICs激活是酸中毒微环境下血脑屏障损伤的重要原因的假说，并将在分子、细胞和动物模型三个水平进一步验证血脑屏障终足细胞上的ASICs在酸中毒微环境下再分布对血脑屏障损伤的作用机制，揭示酸中毒环境下血管源性脑水肿微血管痉挛的原因，为ASICs作为创伤性脑水肿代谢治疗靶点提供理论依据。

细胞内钙超载是导致创伤性血管源性脑水肿与颅脑损伤后酸中毒环境下的血脑屏障破坏的主要原因之一。酸敏感性离子通道是一类在低pH环境下被激活的配体门控阳离子通道，是已知颅内与酸代谢和钙超载有关的重要通道。以往研究发现ASICs主要存在于神经元的胞膜上，近年来有报道称ASICs亦存在于神经胶质细胞，但其在神经胶质细胞上的功能未知。本课题组利用逆转录-聚合酶链反应、免疫荧光和western blot蛋白印迹等方法，证实了ASICs在星形胶质细胞细胞核膜上高表达和酸性环境下的核膜外再分布现象，亦在酸性环境下通过膜片钳技术监测到持续的微电流，与在神经元细胞上监测到的ASICs电流特征相似，此持续电流是ASICs激活导致的Ca2+内流引起的，而Ca2+内流引起的钙超载可以引起星形胶质细胞的功能障碍甚至死亡。此电流可引起血管终足细胞的死亡，进而破坏血脑屏障的结构，引起血脑屏障的损伤。星形胶质细胞上的ASICs激活是酸中毒微环境下血脑屏障损伤的重要原因，H+浓度作为此过程的重要趋化因子之一介导了ASICs的核膜外再分布，从而进一步引起胞内钙超载，引起BBB损伤，这为ASICs作为创伤性脑水肿代谢治疗靶点提供理论依据。

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