TRPV4调控应力纤维生成在脑出血后血肿机械性应力损伤中的作用与机制研究

Intracerebral hemorrhage (ICH) is the most lethal and disabling cerebrovascular disease with no satisfactory therapy. Though the mechanical stress produced by hematoma is the key to the primary injury after ICH, there are few related studies. One of the important reasons is the lack of standardized, effective and reproducible animal model. Based on the predilection sites and clinical features of ICH, our previous studies successfully established a simple and repeatable "balloon-induced stress damage” model and found transient receptor potential cation channel subfamily V4 (TRPV4) may mediate the hematoma-induced mechanical stress damage. Previous studies have confirmed that TRPV4 activation can induce a large number of calcium influxs, leading to PKCα activation which further activate RhoA, and exert biological effects. Recent studies have demonstrated that RhoA activation could regulate the actin remodeling in the vascular endothelial cells after ICH, resulting in stress fiber formation, which leads to the degradation of tight junction proteins and destruction of the blood-brain barrier (BBB). Our previous work also found that inhibition of RhoA activation could increase the expression of E-cadherin and tight junction proteins and preserve BBB after ICH. Therefore, based on current evidences, we purpose that TRPV4 could regulate stress fiber formation and disrupt BBB through PKCα/RhoA/p-MLC2 signaling pathway, and participate in hematoma-induced mechanical stress damage after ICH. In the current project, by establishing in.vivo and in vitro models, combined with MRI diffusion tensor imaging, two photon microscopy, immunofluorescence, laser scanning confocal microscope, behavior evaluation, cellular and molecular biology techniques, we will document the role and mechanisms of TRPV4 in hematoma-induced mechanical stress damage after ICH. We hope that the current project could help us to better understand the occurrence and development of ICH and provide an innovational strategy to improve clinical diagnosis and treatment.

脑出血（ICH）是致死和致残率最高的脑血管病。血肿对周围脑组织产生的机械性应力是造成ICH原发性损伤的关键，但目前相关研究很少。基于ICH的好发部位及临床特点，课题组成功建立了一种大鼠球囊应力损伤模型并发现瞬时受体电位阳离子通道亚家族V4（TRPV4）激活可能是介导血肿应力损伤的重要环节，但其具体机制尚不清楚。既往研究证实：TRPV4激活后可引起大量钙离子内流，导致PKCα活化并进一步激活RhoA，发挥生物学效应。新近研究提示：ICH后RhoA活化可促使血管内皮细胞应力纤维生成，导致紧密连接蛋白降解并最终破坏血脑屏障。据此我们推测：TRPV4激活可通过PKCα及RhoA信号促使应力纤维生成，破坏血脑屏障，参与ICH后血肿应力损伤。本项目拟以血肿应力损伤为切入点，建立在体和离体模型，结合分子生物学、双光子显微成像等技术，探讨TRPV4的作用及机制，为ICH临床诊治提供新思路。

血肿对周围脑组织产生的机械性应力是造成脑出血原发性损伤的关键，本研究立足于前期研究基础，通过干预应力损伤中的关键分子TRPV4以及Piezo1，探讨其在脑出血后应力损伤中的作用及机制，为临床转化提供实验证据。.1、血肿机械性应力损伤对血脑屏障有直接破坏作用，本研究首次发现脑出血后血肿周围的应力相关敏感分子TRPV4表达增高并被激活，引起大量钙离子内流，导致PKCα活化并进一步激活RhoA信号促使应力纤维生成，破坏血脑屏障，参与ICH后血肿应力损伤。抑制TRPV4可能成为减轻脑出血后应力损伤的重要靶点。.2、脑出血后白质损伤是血肿机械应力的直接破坏靶点，本研究首次发现脑出血后血肿周围的应力相关敏感分子Piezo1表达明显增高，而piezo1中等剂量抑制剂可以减轻脑出血后的白质损伤，减少少突胶质胶质细胞的凋亡，其机制与减轻内质网应激时动未折叠蛋白反应（UPR）有关。 抑制Piezo1可能成为减轻脑出血后应力损伤的重要靶点。.3、本研究率先建立了模拟神经细胞机械性应力损伤的体外模型，为进一步阐明脑出血后血肿应力损伤的机制提供研究基础。