平滑肌源性ATP-P2X1/X4在蛛网膜下腔出血后的调节反应及其在脑血管痉挛中的作用机制

Cerebral vasospasm (CVS), a severe complication of subarachnoid hemorrhage (SAH), determines the prognosis of this disease mostly, however, the key pathogenic molecule, resulting in excessive depolarization of cerebrovascular smooth muscle cells (CVSMCs) after SAH, is still unidentified. Adenosine triphosphate (ATP), a crucial extracellular signaling molecule as well as a vasoactive substances, is confirmed to cause CVS, but its origin and signaling pathway is remaining unknown. Most recently, a few studies found the local ATP release via CVSMCs, also termed as myogenic ATP, was sensitive to various diseases or stresses. Additionally, the myogenic ATP and its ionotropic receptor P2X (P2X1/X4), also expressed in CVSMCs, were shown to be involved in several vascular or neurological diseases. Based on the findings above and our interest in this area, preliminary studies showed that constricted cerebral artery has been dilated significantly through the inhibition of myogenic ATP and P2X after SAH. We therefore hypothesize myogenic ATP-P2X is upregulated and contributing to CVS significantly after SAH. Accordingly, this pending project is aiming to investigate this hypothesis though well-organized experiments, including molecular biological and cytological studies, gating properties research of ion channels, vascular contractility and hemodynamics studies in vivo and in vitro. This research group, including several dedicated cerebrovascular scientists, has extensive experience and ability to complete this project. The devices, equipments and other experimental necessities for the entire project are already definitely available. Taken together, we are dedicated to obtaining more evidence to consolidate the myogenic signaling concept in CVS pathogenesis, and, additionally, make a remarkable progress and provide a novel target for CVS research and intervention through studying myogenic ATP-P2X following SAH.

脑血管痉挛(CVS)是自发性蛛网膜下腔出血(SAH)的特异并发症,与预后关系密切且具有重大治疗意义,迄今其关键病理生理环节(脑动脉平滑肌细胞过度去极化)的分子机制尚未阐明。ATP是重要的胞外信号分子和血管活性物质,也是致CVS因子之一,但其来源和作用机制不明。受平滑肌释放ATP现象及其受体P2X的研究进展启发,本课题组开展前期研究并发现分别抑制脑动脉平滑肌ATP和P2X均可使CVS显著缓解,故提出科学假设:脑动脉平滑肌ATP-P2X的整体功能在SAH后呈现出上调反应,从而以细胞过度去极化作为始发效应并导致CVS发生。项目将通过分子生物学、微透析并色谱分析、膜电位及离子通道门控、血管机能和脑血流动力学等从微观到宏观的研究方法,以明确平滑肌ATP-P2X在SAH后的活性变化及其在CVS中的作用机制。最终从肌源性信号角度为CVS的基础研究提供崭新视野和实质性创新,也为CVS的治疗提供实践指导。

蛛网膜下腔出血（SAH）是一类世界范围内公认的具有高致死率和致残率的重症脑血管疾病。微循环障碍是SAH后发生的主要病理变化,也是影响SAH患者预后最主要的危险因素,会导致微血栓的大量形成，进一步造成局部脑组织的缺血，SAH后微循环障碍的概念提出距今已有30多年的历史，近年来由于针对主要动脉痉挛的治疗没有带来可靠的临床预后效果，因此为显著改善SAH的预后,若能够发现微循环障碍发病机制中某一关键的分子或靶点便尤为重要。在前期研究所提供科学依据的基础上,本课题拟将Pannexin-1-ATP进一步引入SAH和微血管的研究领域,为微循环障碍的防治提供新兴的科学线索和理论依据。本人于2018年1月—2021年12月以负责人身份主持国家自然科学基金地区基金“平滑肌源性ATP-P2X1/X4在蛛网膜下腔出血后的调节反应及其在脑血管痉挛中的作用机制(项目编号：H0906/8176050228)”的研究工作,其主要研究内容为：(1)证实了蛛网膜下腔出血对Pannexin-1通道蛋白的表达具有上调作用；还证实Pannexin-1通道蛋白在脑血管中的细胞定位分布，从而阐明了蛛网膜下腔出血后血管平滑肌细胞上的Pannexin-1通道蛋白表达的分子机制。(2)发现Pannexin-1通道蛋白阻滞剂(CBX)对蛛网膜下腔出血后微血管管径明显扩张,从而在宏观层面肯定了Pannexin-1在微循环障碍中的作用。(3)通过膜片钳实验来证实蛛网膜下腔出血后Pannexin-1通道电流的存在,为Pannexin-1通道蛋白致微循环障碍提供了分子层面的依据。(4)证实了ATP的特异性受体P2X1/P2X4在蛛网膜下腔出血后表达上调，为Pannexin-1-ATP-P2X1/P2X4这一通路致微循环障碍提供了依据。综上所述,该课题较为完整的阐明了平滑肌源性ATP-P2X1/X4在蛛网膜下腔出血后微循环障碍中的作用机理。在完成所资助研究内容的前提下,本课题组还开展了一些与脑血管病相邻范畴基础研究的探索,上述内容均从不同角度与所资助研究内容相关或为延续和深入关系。在该研究周期内至美国梅奥医学中心进行研修数月,发表课题相关SCI论文、北大核心期刊论文。获得数项省市级人才项目及荣誉称号并晋升正高职称,培养硕士研究生3名，在读博士1名。