Decorin对急性缺血性卒中后血脑屏障中ZO-1蛋白的作用及机制研究

Stroke is the leading cause of death and disability of adults in China, and the majority is ischemic. The blood-brain barrier disruption after acute ischemic stroke is the key factor for the subsequent brain damage. Decorin is a kind of proteoglycans existing profoundly in extracellular matrix of the central nervous system vasculature. In a previous study , in animal model, DCN highly expressed in the vascular struture of acute infarction. The serum decorin level was enhanced after being operated with middle cerebral artery occlusion in rats during 3 days after onset, and decreased from 3 days to 14 days. DCN can reduce the expression of MMP - 9 in the cerebral microvascular endothelial cell during the hypoxia/reoxygenation environment, meanwhile, it can reduce the degradation of ZO-1 due to the hypoxia damage.Latest research shows that MMP-9 was related to the tight junction proteins disruption after acute ischemic stroke. We speculated that DCN may play a direct or indirect role in regulating the expression of MMP-9 and protecting the degradation of ZO - 1.On this basis, to examine the function of decorin in ischemic stroke process, we planed to explore the changes and mechanism of DCN after stroke. We wanted to explore the effect of DCN to the ZO-1 and its related signal transduction pathways. Also we wanted to explore its correlation with the prognosis of stroke by regulating its expression. These researches will be helpful to provide new experimental basis and new targets for stroke therapy.

急性缺血性卒中后血脑屏障的破坏是引起后续脑损伤的关键因素。蛋白聚糖Decorin（DCN）广泛存在于神经血管单元外基质中，但DCN在脑卒中过程中的作用机制还不清楚。前期动物实验中发现DCN在急性缺血大鼠血管组织中高表达；在急性缺血后0-3天内小鼠血清DCN表达升高，3-7天内血清DCN表达降低。脑微血管内皮细胞缺氧/复氧诱导实验提示DCN预处理能降低MMP-9的表达，减少缺氧损伤造成的紧密连接蛋白ZO-1的降解。已有研究证实MMP-9与急性缺血性卒中后紧密连接蛋白的破坏有关。我们推测DCN可能通过直接或者间接作用调控MMP-9及相关因子的表达对急性缺血性卒中后紧密连接蛋白ZO-1的破坏起到保护作用。本研究拟在已有的基础上，通过动物实验和细胞水平研究，研究脑卒中后DCN变化机制，及其对ZO-1影响及其相关信号通路，并通过调控DCN表达研究其与卒中预后的相关性，为缺血性脑卒中防止提供新的思路。

缺血性卒中是临床上最常见的脑血管疾病，其发病率、死亡率和致残率一直居高不下，严重威胁到人类的健康和生命。缺血性卒中的病理生理学过程是一个多因素、多机制、多环节的恶性级联过程，细胞毒性反应、氧化应激反应、炎症级联反应、血脑屏障破坏、细胞外机制重建是造成缺血性脑损伤的中心环节。然而对其分子机制的研究目前为止仍然远远不足。在本研究中，我们在探索最优条件后建立了小鼠微血管内皮细胞（bEND.3细胞）的氧糖剥离（OGD）/复氧模型，通过Transwell实验检验了核心蛋白聚糖（DCN）对bEND.3细胞的血管通透性的抑制作用，然后我们选取氧糖剥离/复氧的细胞，对照组不经处理，实验组DCN处理，复氧后，通过第二代高通量测序检测相关基因的表达，根据fold change和P value筛选10个高表达和10个低表达的基因，通过qPCR对细胞中差异基因的表达进行验证，发现Claudin5和Claudin11在OGD组中的表达较正常细胞中下降，而DCN能够恢复Claudin5和Claudin11的表达。进一步对测序分析中的差异基因进行KEGG聚类分析发现Tight Junction 信号通路在OGD+DCN组中激活，意味着DCN能够一定程度上恢复紧密连接信号通路。紧接着我们构建了小鼠脑缺血再灌注模型（MCAO模型），将DCN由尾静脉注射，TTC染色发现MCAO组的梗死面积显著高于假手术组，而DCN能显著减少梗死面积。进一步通过qPCR和Western blot检测各组小鼠脑组织中ZO-1，Claudin5和Claudin11的表达时我们发现MCAO组中这些基因和蛋白的表达水平显著下降，而在DCN组中有所回复，验证了DCN能够通过影响脑微血管内皮细胞中紧密连接蛋白的表达，保护血脑屏障。这些研究将有助于阐明缺血性脑卒中的发病机理，为缺血性脑卒中的诊断、治疗提供新的实验依据和新的靶点。本项目的研究成果具有潜在的临床应用价值和应用前景。

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