高渗盐水通过抑制血管内皮因子生长因子表达保护血脑屏障通透性减轻脑水肿的分子机制研究

The up-regulated permeability of blood-brain barrier (BBB) is closely related to the formation of cerebral edema. We have confirmed that hypertonic saline (HS) could alleviate cerebral edema via reducing the permeability of BBB, furthermore, HS could inhibit the expression of vascular endothelial growth factor (VEGF). However, its mechanism is still unclear. It had shown that brain vascular endothelial cell and tight junction protein between it are key components of BBB. They together with astrocytes around brain microvascular regulate the permeability of BBB. And VEGF could down-regulate tight junction (TJ) protein expression leading to the disruption of BBB via PLCγ/PKC/eNOS signaling pathway. So we speculate that HS could protect the function of BBB through reversing down-regulation of TJ protein, which mediated by attenuation of astrocytes and endothelial-derived VEGF stimulation on PLCγ/PKC/eNOS signaling pathway, resulting in alleviation of cerebral edema. This study aimed to explore the possible molecular mechanisms underlying HS protects the permeability of BBB using western blot, RT-PCR and immunofluorescence after establishment of ischemia-reperfusion model and astrocytes/endothelial cells co-culture model. It may provide more theoretical basis for HS widely used in clinical and targeted therapy of cerebral edema.

血脑屏障(BBB)通透性增加是脑水肿形成的重要原因。前期研究发现高渗盐水(HS) 可抑制星形胶质细胞上血管内皮生长因子（VEGF）的表达，并可降低BBB的通透性，但机制不明。脑微血管内皮细胞及其间的紧密连接（TJ）蛋白是BBB的核心组成部分，与包绕脑微血管的星形胶质细胞共同调节BBB通透性。而VEGF可通过激活PLCγ/PKC/eNOS信号通路来调节TJ蛋白表达，进而影响BBB通透性。故我们推测HS通过抑制星形胶质细胞及血管内皮细胞上VEGF的表达，进而抑制血管内皮细胞中PLCγ/PKC/eNOS信号通路的激活，逆转VEGF对TJ蛋白的下调，降低BBB通透性。本研究拟制备大鼠脑缺血再灌注模型及星形胶质细胞/血管内皮细胞共培养体系，利用siRNA干扰、免疫荧光、免疫印迹及RT-PCR 等技术，探讨HS通过VEGF降低BBB通透性的具体机制，为进一步临床推广HS和脑水肿靶向治疗

缺血缺氧后脑水肿的重要成因是血脑屏障通透性增加，前期研究发现高渗盐水可以降低缺血缺氧条件下炎症因子的表达，保护血脑屏障的通透性，并且下调星形胶质细胞上血管内皮生长因子的表达，但具体机制不明，炎症因子和血管内皮生长因子与血脑屏障之间的关系亦不清楚。脑微血管内皮细胞是血脑屏障的最重要成分之一，其中脑血管内皮细胞间的紧密连接蛋白又是其通透性重要决定因素。有研究发现缺氧时小胶质细胞释放的炎症因子如IL-1β可影响血管内皮生长因子的表达，而血管内皮生长因子可通过其受体VEGFR2影响下游PLCγ/eNOS通路调控紧密连接蛋白的表达。为明确高渗盐水、IL-1β、血管内皮生长因子及其下游信号通路VEGFR2/PLCγ/eNOS之间的关系，本研究通过线栓法构建大鼠大脑中动脉局灶脑缺血再灌注模型、体外细胞低氧复氧模型模拟在体缺血再灌注损伤，采用免疫蛋白印迹、免疫荧光、信号通路相关实验技术探讨上述各因素间的关系。研究发现高渗盐水的确具有保护缺血缺氧后血脑屏障结构与功能的作用，即降低血脑屏障通透性，防止紧密连接蛋白表达下调。其机制为高渗盐水通过抑制小胶质细胞上炎症体NLRP3及配体表达，减少炎症因子IL1β的表达，以相对减少通过IL1β/IL1R1/NF-кB通路而下调星形胶质细胞及脑微血管内皮细胞上血管内皮生长因子的表达，达到抑制VEFFR2/PLCγ/PKC/eNOS信号通路的激活，防止紧密连接蛋白表达的减少，从而保护血脑屏障结构与功能。本研究明确了高渗盐水下调血管内皮生长因子的机制，为高渗盐水的临床推广和脑水肿治疗提供更多的理论依据。