类泛素蛋白NEDD8活化酶抑制剂MLN4924对缺血性脑卒中的保护作用机制研究

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.

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。

血脑屏障破坏和血管炎症在脑缺血损伤的病理生理进程中扮演关键的作用。类泛素化修饰通过神经元前体细胞表达的发育下调蛋白8（NEDD8）与底物蛋白结合调控多种细胞功能。然而，类泛素化修饰对脑缺血的作用尚属未知。我们研究发现在脑缺血急性期，NEDD8在缺血脑内表达增加，并且主要与血管内和实质内的中性粒细胞存在共定位，部分与内皮细胞共标。Neddylation特异性抑制剂MLN4924降低脑缺血后脑内NEDD8底物cullin-1的neddylation水平。并且，MLN4924减少缺血脑组织内中性粒细胞浸润，小胶质细胞激活以及活性氧释放。进一步研究发现，MLN4924降低脑缺血后血脑屏障通透性，减少TUNEL阳性内皮细胞数量，减小脑梗塞，并且改善神经功能。我们还观察到，MLN4924处理的缺血小鼠脑内CRL底物NF1表达增加，而病毒沉默NF1能够逆转MLN4924抑制中性粒细胞浸润的作用。P-selectin或ICAM-1的中和抗体可消除沉默NF1对MLN4924调控中性粒细胞浸润的作用，提示沉默NF1通过P-selectin和ICAM-1发挥作用。此外，我们发现沉默NF1可通过激活脑微血管PKCδ、pMARCKS和pMLC，逆转MLN4924对血脑屏障的保护以及神经保护作用，并且PKCδ抑制剂rottlerin可抵消NF1沉默的作用。以上结果表明，类泛素化修饰的激活促进中性粒细胞浸润，血脑屏障和脑损伤，而抑制类泛素化修饰可减轻缺血性脑损伤。