脯氨酰羧肽酶在脑梗死后的动态表达及其对缺血再灌注损伤的影响及机制研究

Early revascularization of blocked blood vessel is the most effective treatment for acute ischemic stroke, but the following reperfusion injury keeps a problem to plague clinical treatment and prognosis. Maintaining the integrity of blood brain barrier can reduce reperfusion injury and improve stroke outcome. Prolidylcarboxy peptidase (PRCP) is an important proteolytic enzyme that can not only regulate both the kallikrein-kinin system and the renin-angiotensin system, but also participate in regulation of endothelial function, oxidative stress and angiogenesis through direct activation mechanism. However, the role of PRCP in cerebral ischemia-reperfusion has not been reported. In the present study, we will adopt a model of cerebral ischemia-reperfusion injury in mice to observe the dynamic changes of PRCP expression during reperfusion and to study the effect of PRCP on early brain injury and long-term neurological function after stroke through changing its expression and enzymatic hydrolysis products. Besides, endothelial cells are cultured in vitro to observe the role of PRCP and its possible signal pathway after oxygen-glucose deprivation/reoxygenation injury. This study provides an important theory significance for deep understanding the pathogenesis of cerebral ischemia-reperfusion injury and exploring more effective molecular targets.

急性缺血性脑卒中最有效的治疗是早期实现血管再通。但血流再通的同时带来的再灌注损伤一直困扰着临床的治疗和预后。维持血脑屏障的完整性，可降低再灌注损伤的程度，改善脑卒中转归。脯氨酰羧肽酶（prolylcarboxy peptidase, PRCP)是机体内重要的蛋白水解酶，不仅同时调控激肽释放酶—激肽系统和肾素—血管紧张素系统两大血管调节系统，还可通过自身直接激活机制参与血管内皮功能、氧化应激及血管再生过程。但PRCP在脑缺血再灌注损伤中的作用尚未有相关报道。本课题拟采用小鼠脑缺血再灌注损伤模型，观察PRCP在脑缺血再灌注中的动态变化，探究干预PRCP表达及其酶解产物对脑梗死后早期脑损伤及远期神经功能修复的影响。并体外培养内皮细胞，给予氧糖剥夺/复氧损伤，进一步观察PRCP的作用及其介导的信号通路。研究成果将为深入探究脑缺血再灌注损伤的发病机制，寻找更加有效地治疗靶点提供理论依据。

脑血管内皮细胞在缺血性损伤后继发的血管炎症是加重血脑屏障破坏、促进血管源性脑水肿，进而恶化神经功能的重要原因。脯氨酰羧肽酶（prolylcarboxy peptidase, PRCP)是机体内重要的蛋白水解酶，不仅同时调控激肽释放酶—激肽系统和肾素—血管紧张素系统两大血管调节系统，还可通过自身直接激活机制参与血管内皮功能、氧化应激及血管再生过程。但PRCP在脑缺血再灌注损伤中的作用尚未有相关报道。为探讨PRCP在脑梗死后的表达及作用，我们利用在体脑缺血再灌注模型，发现早期缺血脑组织中PRCP表达显著降低，随后逐渐增加，再灌注14天时高于假手术组，而且主要表达内皮细胞上。体外试验除了在小鼠脑微血管内皮细胞系中进行，还在人脑微血管内皮细胞中进行了验证。进一步构建PRCP干扰病毒和过表达病毒，发现与对照组相比，过表达PRCP组小鼠脑缺血再灌注后缺血脑组织中炎性因子表达降低，血脑屏障破坏减轻，梗死体积缩小，神经功能缺损明显改善；而干扰PRCP组小鼠血脑屏障破坏增加，脑梗死体积和神经功能缺损明显加重。为进一步探究PRCP在脑梗死发生发展中的作用机制，我们在干扰PRCP表达后联合Ang(1-7)预处理内皮细胞，发现干扰PRCP表达后内皮细胞的损伤和凋亡增加，但是可以Ang(1-7)逆转，提示PRCP介导的脑保护作用可能是通过分解产物Ang(1-7)产生的。利用在体脑缺血模型，我们还发现过表达PRCP组小鼠脑缺血后14天的缺血脑组织中新生血管密度增加，小鼠运动能力有所改善。该研究揭示了PRCP在脑梗死发生发展中的作用及机制，为探索脑梗死新的治疗靶点提供了重要依据。