血脑屏障损伤与炎症反应在缺血性脑卒中的病理过程中起重要作用，但其形成机制及治疗措施尚需深入探讨。研究表明NEDD8类泛素化修饰的激活促进肿瘤血管生长和炎症反应，但其在脑缺血中的作用未有报道。我们前期研究发现脑缺血引起整个类泛素化修饰通路的过度激活，而使用NEDD8活化酶抑制剂MLN4924阻断类泛素化修饰的激活，能有效减轻脑内中性粒细胞浸润和脑缺血损伤。本项目拟通过动物脑缺血模型和体外脑微血管通透性实验，结合运用药物干预、基因敲除、多光子显微镜活体成像、分子生物学技术和手段，研究MLN4924是否通过抑制中性粒细胞弹性蛋白酶介导的炎症反应，调控下游RhoA-Rac1-pMLC和p-IκB-α-NF-κB-MMP-3信号通路的关键分子，从而降低血脑屏障破坏，并减轻脑缺血损伤和神经功能障碍。本研究将有助于了解缺血性脑卒中后血管损伤和炎症偶联的重要环节，并为靶向血管保护药物的开发提供理论基础。

Blood-brain barrier damage and inflammatory responses play important roles in the pathophysiology of acute ischemic stroke. However, the mechanisms leading to postischemic blood-brain barrier damage and inflammatory responses have not been established, and no therapies have been identified. Recently, the activation of neddylation pathway was shown to increase tumor angiogenesis and exacerbate inflammatory responses. Our preliminary data show that the entire neddylation pathway was overactivated after cerebral ischemia. Moreover, inhibition of neddylation by the NEDD8-activating enzyme inhibitor MLN4924 significantly reduced neutrophil infiltration and ischemic cerebral injury. In this project, we will further investigate the mechanism of MLN4924 in reducing brain injury following stroke. We will employ mouse stroke model and in vitro brain microvascular permeability model, using NEDD8-activating enzyme inhibitor MLN4924, neutrophil elastase knockout mice, neutrophil elastase inhibitor, neutrophil elastase, multi-photon microscopy and molecular biological approaches to determine whether the inhibition of neutrophil elastase-mediated inflammation by MLN4924 treatment is responsible for blood-brain barrier changes, and whether these effects contribute to the reduction in brain damage and improvement of neurological functions. We will also determine whether RhoA-Rac1-pMLC and p-IκB-α-NF-κB- MMP-3 signaling pathways are involved in the effects of MLN4924 on inflammation and blood-brain barrier dysfunction following ischemic stroke. These proposed studies will not only provide novel insights into the relationships of inflammatory responses and vascular components, but should also help the development of new therapeutic strategies for limiting brain vascular damage after ischemic stroke.