高渗盐水减轻脑水肿的非渗透性分子机制研究

We have reported previously that hypertonic saline (HS) could alleviate cerebral edema more effectively than an equal volume of 20% mannitol. Interestingly, the urine output of patients did not increase substantially. It was therefore speculated that HS might reduce the cerebral edema through non-osmotic molecular mechanism. Our previous data have shown that hypertonic saline ameliorates the cerebral edema through inhibition of Na-K-Cl co-transporter (NKCC1) expression in the astrocytes. However, its underlying mechanism has remained dubious. Cerebral edema results mainly from expression and phosphorylation of NKCC1 in the astrocytes in the neighboring areas of the ischemic sites (pernumbral region) induced by some microglia-derived inflammatory mediator activated enzymes. TLR2/NF-κB signal pathway is known to be involved in microglia-derived inflammatory mediator release. Experimental studies in animals have shown that HS treatment could alleviate microglia-derived inflammatory mediator release adjacent to the ischemic sites. Therefore, it is hypothesized that HS might reduce microglia-derived inflammatory mediator release through inhibition of TLR2/NF-κB signal pathway, which would then downregulate the expression of NKCC1 in the astrocytes resulting in alleviation of cerebral edema. The information would help unravel the mechanism by which 10% HS can alleviate the cerebral edema and whether it is through inhibition of NKCC1 expression by using confocal microscopy, western blot, RT-PCR and primary cell culture. The study will shed lights on the non-osmotic molecular mechanism on how HS could reduce cerebral edema clinically.

临床研究发现高渗盐水(HS)减轻脑水肿、降低颅内压比20%甘露醇起效更快、作用更持久，但病人尿量无明显增加，仅用渗透性脱水机制难以解释，由此推测HS可能还通过非渗透性机制减轻脑水肿。前期证实HS可以通过抑制钠-钾-氯同向转运蛋白(NKCC1)在星形胶质细胞表达减轻脑水肿，但其机制不明。缺血灶周围的小胶质细胞释放的炎症介质激活星形胶质细胞内一些激酶，诱导NKCC1的表达和磷酸化是脑水肿的主要原因。TLR2/NF-κB是参与激活小胶质细胞释放炎症介质的重要信号通路。进一步动物实验显示HS可以抑制脑缺血灶周围小胶质细胞释放炎症介质。由此，我们首次提出HS通过抑制缺血缺氧诱导TLR2/NF-κB信号通路，减轻激活的小胶质细胞释放炎症介质，从而下调星形胶质细胞NKCC1的表达，最终减轻脑水肿。本项目拟进一步采用激光共聚焦、免疫印记、RT-PCR等技术，在分子水平探索HS治疗脑水肿的非渗透性分子机制。

临床研究发现高渗盐水(HS)减轻脑水肿的作用较20%甘露醇起效更快、更持久，但尿量无明显增加，用渗透性分子机制不能完全解释，推测HS 可能还通过非渗透性机制减轻脑水肿。因此本课题拟通过动物实验探讨高渗盐水对脑水肿的治疗效果及其作用靶点；同时通过体外细胞实验探讨HS减轻脑水肿的非渗透性分子机制。实验表明，HS可以减轻脑水肿，并且可减少小胶质细胞上炎症因子的表达，保护血脑屏障的通透性，还可以抑制与脑水肿密切的相关蛋白NKCC1的表达。进一步的体外细胞实验表明，HS可能通过抑制小胶质细胞上P38及JNK信号通路，减少其炎症因子的释放，并间接降低星形胶质细胞上NKCC1的表达。因此本课题通过体内外实验表明了HS盐水可能是通过减少炎症因子的释放间接抑制了NKCC1的表达，从而减轻脑水肿。本研究创新性的提出了HS减轻脑水肿的非渗透性分子机制，为HS的临床推广提供了理论依据。