血红蛋白激活内皮细胞Rho/ROCK通路在脑出血后脑水肿中的作用及其机制

Hemoglobin (Hb) is an important cause for brain edema after intracerebral hemorrhage (ICH). During the natural course of intracerebral hematoma after ICH, OxyHb in the hematoma can be changed into DeoxyHb and MetHb, etc. and there are many forms of Hb. However, it is unknown which Hb is the main cause of brain edema and its molecular mechanism. Recently, it is well known Rho/ROCK pathway is a key pathway to regulate vascular permeability. In our preliminary research, we found that Hb could activate Rho/ROCK pathway and cause disruption of tight junctions of cerebral endothelial cells in blood-brain barrier (BBB). Based upon these researches, we firstly proposed that Hb could interact with adhesion molecules on cerebral endothelial cells directly or indirectly after ICH. Through the outside-in transmembrane signal transduction process, adhesion molecules can activate the Rho/ROCK pathway in endothelial cells and cause disruption of BBB. As the result of disruption of BBB caused by Hb, brain edema may form and develop after ICH. In this project, with the combination of in vitro BBB research and in vivo research, we will make use of advanced experimental techniques (siRNA and confocal microscopy, etc.) to get the evidence of activation of Rho/ROCK pathway through interaction between adhesion molecules and Hb in cerebral endothelial cells after ICH. We will clarify the molecular basis and mechanism of brain edema caused by Hb after ICH. With the results of this project, it will help us to develop new stratagem for treatment of ICH and improve the prognosis of ICH.

血红蛋白（Hb）是导致脑出血后脑水肿的重要病理介质。然而，脑出血发生后，随着血肿演变，Hb可以有OxyHb、MetHb、DeoxyHb等多种存在形式，究竟是哪一种Hb类物质在脑水肿发生过程中起主要作用、其作用分子机制尚不清楚。近年研究表明，Rho／ROCK信号通路是调控血管通透性的关键之一。我们的预试验结果也发现，Hb可以激活血脑屏障内皮细胞Rho／ROCK通路并导致血脑屏障紧密连接的破坏。由此，我们首次提出，脑出血发生后，Hb类物质可能通过作用于内皮细胞表面粘附分子并激活胞内Rho／ROCK通路进而诱导血脑屏障结构和功能的障碍并导致脑水肿的发生。本项目拟通过体外血脑屏障模型与整体动物模型的综合研究，采用siRNA、激光共聚焦等方法，旨在获得Hb通过内皮细胞粘附分子激活Rho／ROCK通路的可靠证据，本项目将阐明Hb诱导脑水肿的分子基础及其作用机制，为改善脑出血预后提供新的治疗策略和途径。

脑出血是一类发病率、致死率、致残率都很高的脑血管疾病。脑出血后脑水肿的发生、发展是导致脑出血患者神经功能严重缺损、脑疝甚至死亡的主要原因之一。血脑屏障的结构和功能障碍是导致脑出血后血管源性脑水肿发生、发展的主要原因之一。红细胞是血液中含量最多的血细胞，也是脑出血后脑内血肿的主要成分。研究表明脑出血后红细胞崩解释放的血红蛋白可能是导致血脑屏障损伤的重要病理介质之一。.本研究通过体外内皮细胞培养等研究，应用免疫组化、免疫荧光、RT-PCR，WESTERN杂交等方法，系统研究了血红蛋白对体外内皮细胞通透性、磷酸化肌球蛋白轻链、肌动蛋白F-actin及紧密连接蛋白ZO-1表达变化。同时，建立了大鼠脑内注入血红蛋白脑出血动物模型，采用伊文思蓝浓度检测评估各组大鼠血脑屏障的通透性，采用干湿法测定各组大鼠血肿周围脑组织的水含量，采用免疫组化染色(DAB染色)检测RhoA和ROCK2在各组大鼠脑中的表达分布情况及磷酸化肌球蛋白轻链(phosphorylated myosin light chain，p-MLC)和紧密连接蛋白claudin-5等的表达变化。采用荧光定量PCR测定各组大鼠脑中RhoA和ROCK2的表达情况。研究表明：①血红蛋白可引起内皮细胞间紧密连接破坏及通透性增加，进而导致内皮“屏障”功能障碍；②血红蛋白可引起内皮细胞p-MLC表达降低，进而导致细胞内F-actin大量应力纤维形成、并诱导细胞间紧密连接蛋白ZO-1表达下降，这一变化可能是脑出血后内皮细胞间紧密连接破坏的重要病理生理机制之一；③血红蛋白可使大鼠脑组织血肿周边Rho激酶Ⅱ表达增加，一方面可引起p-MLC表达增强、紧密连接破坏致使血脑屏障的通透性增加；另一方面可以直接对紧密连接蛋白claudin-5磷酸化修饰而致其表达降低、血脑屏障破坏；④Hb可引起脑实质内iNOS、eNOS表达显著增加，进而合成和释放过量的NO。脑出血后，Hb可能通过激活RhoA/ Rho激酶信号通路及诱导iNOS、eNOS过量表达、NO释放增加引起血脑屏障通透性增加，进而参与脑水肿的发生发展。.课题总体基本按照申请书研究工作计划，已基本完成预定研究目标内容。.目前，已发表SCI论文2篇，中文论文2篇。部分研究仍在进行中。部分研究论文将在后续时间内陆续发表。