Huang Lin-Qiang, Zhu Gao-Feng, Deng Yi-Yu, Jiang Wen-Qiang, Fang Ming, Chen Chun-Bo, Cao Wei, Wen Miao-Yun, Han Yong-Li, Zeng Hong-Ke
Department of Emergency & Critical Care Medicine, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, PR China.
J Neuroinflammation. 2014 Jun 11;11:102. doi: 10.1186/1742-2094-11-102.
Hypertonic saline (HS) has been successfully used clinically for treatment of various forms of cerebral edema. Up-regulated expression of Na-K-Cl Cotransporter 1 (NKCC1) and inflammatory mediators such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) has been demonstrated to be closely associated with the pathogenesis of cerebral edema resulting from a variety of brain injuries. This study aimed to explore if alleviation of cerebral edema by 10% HS might be effected through down-regulation of inflammatory mediator expression in the microglia, and thus result in decreased NKCC1 expression in astrocytes in the cerebral cortex bordering the ischemic core.
The Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of NKCC1, TNF-α and IL-1β expression using Western blotting, double immunofluorescence and real time RT-PCR, and the model also was used for evaluation of brain water content (BWC) and infarct size. SB203580 and SP600125, specific inhibitors of the p38 and JNK signaling pathways, were used to treat primary microglia cultures to determine whether the two signaling pathways were required for the inhibition of HS on microglia expressing and secreting TNF-α and IL-1β using Western blotting, double immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The effect of TNF-α and IL-1β on NKCC1 expression in primary astrocyte cultures was determined. In addition, the direct inhibitory effect of HS on NKCC1 expression in primary astrocytes was also investigated by Western blotting, double immunofluorescence and real time RT-PCR.
BWC and infarct size decreased significantly after 10% HS treatment. TNF-α and IL-1β immunoexpression in microglia was noticeably decreased. Concomitantly, NKCC1 expression in astrocytes was down-regulated. TNF-α and IL-1β released from the primary microglia subjected to hypoxic exposure and treatment with 100 mM HS were decreased. NKCC1 expression in primary astrocytes was concurrently and progressively down-regulated with decreasing concentration of exogenous TNF-α and IL-1β. Additionally, 100 mM HS directly inhibited NKCC1 up-regulation in astrocytes under hypoxic condition.
The results suggest that 10% HS alleviates cerebral edema through inhibition of the NKCC1 Cotransporter, which is mediated by attenuation of TNF-α and IL-1β stimulation on NKCC1.
高渗盐水（HS）已在临床上成功用于治疗各种形式的脑水肿。钠钾氯协同转运蛋白1（NKCC1）以及炎症介质如肿瘤坏死因子α（TNF-α）和白细胞介素-1β（IL-1β）的表达上调已被证明与多种脑损伤所致脑水肿的发病机制密切相关。本研究旨在探讨10% HS减轻脑水肿是否可能通过下调小胶质细胞中炎症介质的表达，从而导致毗邻缺血核心的大脑皮质星形胶质细胞中NKCC1表达降低来实现。
采用右侧大脑中动脉闭塞（MCAO）的Sprague-Dawley（SD）大鼠，运用蛋白质免疫印迹法、双重免疫荧光法和实时逆转录聚合酶链反应（RT-PCR）评估NKCC1、TNF-α和IL-1β的表达，该模型还用于评估脑含水量（BWC）和梗死面积。使用p38和JNK信号通路的特异性抑制剂SB203580和SP600125处理原代小胶质细胞培养物，通过蛋白质免疫印迹法、双重免疫荧光法和酶联免疫吸附测定（ELISA）来确定这两条信号通路是否是HS抑制小胶质细胞表达和分泌TNF-α和IL-1β所必需的。测定TNF-α和IL-1β对原代星形胶质细胞培养物中NKCC1表达的影响。此外，还通过蛋白质免疫印迹法、双重免疫荧光法和实时RT-PCR研究HS对原代星形胶质细胞中NKCC1表达的直接抑制作用。
10% HS治疗后BWC和梗死面积显著降低。小胶质细胞中TNF-α和IL-1β的免疫表达明显降低。与此同时，星形胶质细胞中NKCC1的表达下调。缺氧暴露并用100 mM HS处理的原代小胶质细胞释放的TNF-α和IL-1β减少。随着外源性TNF-α和IL-1β浓度降低，原代星形胶质细胞中NKCC1的表达同时且逐渐下调。此外，100 mM HS直接抑制缺氧条件下星形胶质细胞中NKCC1的上调。
结果表明，10% HS通过抑制NKCC1协同转运蛋白减轻脑水肿，这是由TNF-α和IL-1β对NKCC1的刺激减弱介导的。
