Chu Heling, Yang Xiaobo, Huang Chuyi, Gao Zidan, Tang Yuping, Dong Qiang
Department of Neurology, Huashan Hospital, State Key Laboratory of Medical Neurobiology, Shanghai, China.
Cerebrovasc Dis. 2017;44(1-2):10-25. doi: 10.1159/000460261. Epub 2017 Apr 13.
Apelin-13 has been found to have protective effects on many neurological diseases, including cerebral ischemia. However, whether Apelin-13 acts on blood-brain barrier (BBB) disruption following cerebral ischemia is largely unknown. Aquaporin-4 (AQP4) has a close link with BBB due to the high concentration in astrocyte foot processes and regulation of astrocytes function. Here, we aimed to test Apelin-13's effects on ischemic BBB injury and examine whether the effects were dependent on AQP4.
We detected the expression of AQP4 induced by Apelin-13 injection at 1, 3, and 7 days after middle cerebral artery occlusion. Meanwhile, we examined the effects of Apelin-13 on neurological function, infarct volume, and BBB disruption owing to cerebral ischemia in wild type mice, and tested whether such effects were AQP4 dependent by using AQP4 knock-out mice. Furthermore, we assessed the possible signal transduction pathways activated by Apelin-13 to regulate AQP4 expression via astrocyte cultures.
It was found that Apelin-13 highly increased AQP4 expression as well as reduced neurological scores and infarct volume. Importantly, Apelin-13 played a role of BBB protection in both types of mice by reducing BBB permeability, increased vascular endothelial growth factor, upregulated endothelial nitric oxide synthase, and downregulated inducible NOS. In morphology, we demonstrated Apelin-13 suppressed tight junction opening and endothelial cell swelling via electron microscopy detection. Meanwhile, Apelin-13 also alleviated apoptosis of astrocytes and promoted angiogenesis. Interestingly, effects of AQP4 on neurological function and infarct volume varied with time course, while AQP4 elicited protective effects on BBB at all time points. Statistical analysis of 2-way analysis of variance with replication indicated that AQP4 was required for these effects. In addition, Apelin-13 upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and Akt as well as AQP4 protein in cultured astrocytes. The latter was inhibited by ERK and phosphatidylinositol 3'-kinase (PI3K) inhibitors.
Our data suggest that Apelin-13 protects BBB from disruption after cerebral ischemia both morphologically and functionally, which is highly associated with the increased levels of AQP4, possibly through the activation of ERK and PI3K/Akt pathways. This study provides double targets to protection of ischemic BBB damage, which can present new insights to drugs development.
已发现Apelin-13对包括脑缺血在内的多种神经疾病具有保护作用。然而，Apelin-13是否作用于脑缺血后的血脑屏障（BBB）破坏在很大程度上尚不清楚。水通道蛋白4（AQP4）由于在星形胶质细胞足突中的高浓度以及对星形胶质细胞功能的调节而与血脑屏障密切相关。在此，我们旨在测试Apelin-13对缺血性血脑屏障损伤的影响，并研究这些影响是否依赖于AQP4。
我们在大脑中动脉闭塞后1、3和7天检测了注射Apelin-13诱导的AQP4表达。同时，我们研究了Apelin-13对野生型小鼠脑缺血所致神经功能、梗死体积和血脑屏障破坏的影响，并通过使用AQP4基因敲除小鼠来测试这些影响是否依赖于AQP4。此外，我们通过星形胶质细胞培养评估了Apelin-13激活的可能信号转导途径以调节AQP4表达。
发现Apelin-13高度增加AQP4表达以及降低神经评分和梗死体积。重要的是，Apelin-13通过降低血脑屏障通透性、增加血管内皮生长因子、上调内皮型一氧化氮合酶和下调诱导型一氧化氮合酶，在两种类型的小鼠中均发挥血脑屏障保护作用。在形态学上，我们通过电子显微镜检测证明Apelin-13抑制紧密连接开放和内皮细胞肿胀。同时，Apelin-13还减轻了星形胶质细胞的凋亡并促进了血管生成。有趣的是，AQP4对神经功能和梗死体积的影响随时间进程而变化，而AQP4在所有时间点均对血脑屏障产生保护作用。重复测量的双向方差分析的统计分析表明这些作用需要AQP4。此外，Apelin-13上调了培养星形胶质细胞中细胞外信号调节激酶（ERK）和Akt的磷酸化以及AQP4蛋白。后者被ERK和磷脂酰肌醇3'-激酶（PI3K）抑制剂抑制。
我们的数据表明，Apelin-13在形态和功能上保护血脑屏障免受脑缺血后的破坏，这与AQP4水平的升高高度相关，可能是通过激活ERK和PI3K/Akt途径。本研究为缺血性血脑屏障损伤的保护提供了双重靶点，可为药物开发提供新的见解。
