Hippo-YAP信号通路调控紧密连接蛋白参与蛛网膜下腔出血后血脑屏障损伤的作用及机制研究

Subarachnoid hemorrhage (SAH) is a deadly cerebrovascular disease with high morbidity and mortality. In recent years，research interest has been shifted to the early brain injury (first 72h) evoked by SAH. However, relative clinical trials cannot be proved effective so far, because they merely targeting injured neurons after SAH. These disappointing results might remind us that we should pay more attention into the widespread contacts among all intracranial cell types, as known as the Vascular Neural Network. The blood brain barrier is the most typical neurovascular unit in this vascular neural network, and also blood brain barrier damage is thought to be one of the main pathophysiological processes in SAH patients. In this case, repairing blood brain barrier has been considered as one of the most promising therapy strategies in rescuing and repairing brain injury after SAH. Thus, as the key factor of blood brain barrier structural proteins, the tight junction proteins become the high interested repairing target. Our previous study demonstrated thatβ-catenin nucleic translocation in cerebral endothelial cells was reduced after SAH, leading to reduced tight junction protein levels. Meanwhile, a key protein of Hippo-YAP pathway, MST1, significantly increased at 24 hours after SAH. Furthermore, recent studies indicated that Hippo-YAP pathway might involve in the blood brain barrier injury after SAH. Another study showed that YAP could inhibit the downstream transcription of β-catenin by combined withβ-catenin in cytoplasm. Therefore, based on current evidences, we purpose that Hippo-YAP pathway could inhibit tight junction protein transcription by reducing β-catenin nucleic translocation, resulting in blood brain barrier injuried, which finally leads to neurological deficits after SAH. In the current project, by using two photon microscopy, immunofluorescence, laser scanning confocal microscope, behavior evaluation, cellular and molecular biology techniques，we will document the role of Hippo-YAP pathway in blood brain barrier injury after SAH, and then, identify the mechanisms of how Hippo-YAP pathway inhibiting tight junction proteins transcription. We hope that the current project could clarify the role and mechanism of Hippo-YAP pathway involving in blood brain barrier injury after SAH, and finally provide an innovational strategy to prevent early brain injury and improve clinic outcome in SAH patients.

血脑屏障(BBB)损伤是蛛网膜下腔出血(SAH)后早期脑损伤主要病理生理改变之一，其中针对紧密连接蛋白这一关键因素的调控，可能是促进BBB修复最具前景的策略。前期研究证实：SAH后内皮细胞β-Catenin 核转位受抑制、紧密连接蛋白表达降低，而Hippo-YAP通路关键蛋白MST1显著升高。近期文献亦提示：该通路可能参与SAH后BBB中内皮细胞的损伤，其中YAP能够抑制β-Catenin 下游转录，其机制是通过YAP与β-Catenin形成复合体，减少β-Catenin 核转位。据此我们推测：Hippo-YAP通路可能通过抑制内皮细胞β-Catenin 核转位及紧密连接蛋白表达参与SAH后BBB损伤。本项目拟以SAH救治新靶点——BBB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Hippo-YAP通路的作用及机制，提供有助于SAH患者临床救治的实验依据。

本研究立足于前期研究基础，通过干预Hippo通路中的关键分子，探讨hippo通路在SAH引起的早期脑损伤中的作用，为临床转化提供实验证据。. 1. 首次证实了hippo通路在蛛网膜下腔出血后的早期脑损伤的发生发展过程中发挥着重要的调节作用。运用MST1特异性抑制剂XMU-MP-1以及MST1 shRNA后可以显著改善SAH后小鼠神经功能，减少神经元损伤、减轻脑水肿、改善血脑屏障渗透性、减少白质损伤。其机制可能与抑制NF-κB P65入核以及MMP9的表达以及紧密连接蛋白的降解有关。抑制MST1可能成为减轻SAH后早期脑损伤的重要靶点。. 2. SAH后被抑制的YAP能够显著促进炎症反应的产生，而其相关机制很有可能是通过上调STAT3实现的。在星形胶质细胞中过表达YAP能够显著减少SAH引起的神经功能损伤，减轻脑水肿，保护血脑屏障，减少白质损伤。而这些保护作用的实现都是通过抑制STAT3表达实现的。因此，针对YAP的治疗可能成为提高SAH病人预后的重要靶点，为后续的临床转化奠定基础。. 3、率先研究了SAH后小鼠白质区域的microRNA变化情况，通过microRNA 4.0芯片筛选以及后续的实验验证后，发现SAH后小鼠大脑白质区域内的miR-706的表达水平出现了明显降低，利用miR-706 agomir过表达miR-706后，能够显著减轻小鼠髄鞘以及轴突损伤，改善SAH引起的小鼠运动神经功能以及学习记忆能力损伤，同时下调SAH引起的PKCα、P-MST1、P-P65的升高，而PKCα激动剂PMA能够逆转miR-706对神经功能的保护作用，废除其对PKCα、P-MST1、P-P65的调节作用。笔者证明了SAH后降低的miR-706可能通过激活PKCα/MST1/NF-κB信号通路对小鼠的白质纤维产生损伤作用。针对miR-706的调控可能成为控制SAH后白质损伤的重要靶点