Fan Ruiming, Enkhjargal Budbazar, Camara Richard, Yan Feng, Gong Lei, Tang Jiping, Chen Yangmei, Zhang John H
Department of Neurology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China; Department of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA 92354, United States.
Department of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA 92354, United States.
Exp Neurol. 2017 Oct;296:41-48. doi: 10.1016/j.expneurol.2017.07.003. Epub 2017 Jul 8.
Early brain injury (EBI) is reported as a primary cause of mortality in subarachnoid hemorrhage (SAH) patients. Eph receptor A4 (EphA4) has been associated with blood-brain barrier integrity and pro-apoptosis. We aimed to investigate a role of EphA4 in EBI after SAH. One hundred and seventy-nine male adult Sprague-Dawley rats were randomly divided into sham versus endovascular perforation model of SAH groups. SAH grade, neurological score, Evans blue dye extravasation, brain water content, mortality, Fluoro-Jade staining, immunofluorescence staining, and western blot experiments were performed after SAH. Small interfering RNA (siRNA) for EphA4, recombinant Ephexin-1 (rEphx-1), and Fasudil, a potent ROCK2 inhibitor, were used for intervention to study a role of EphA4 on EBI after SAH. The expression of EphA4, Ephexin-1, RhoA, and ROCK2 significantly increased after SAH. Knockdown of EphA4 using EphA4 siRNA injection intracerebroventricularly (i.c.v) reduced Evans blue extravasation, decreased brain water content, and alleviated neurobehavioral dysfunction after SAH. Additionally, the expression of Ephexin-1, RhoA, ROCK2 and cleaved caspase-3 were decreased. Tight junction proteins increased, and apoptotic neuron death decreased. The effects of EphA4 siRNA were abolished by rEphx-1. In contrast, Fasudil abolished the effects of rEphx-1. These results suggest that EphA4, a novel and promising target for treatment, exacerbates EBI through an Ephexin-1/ROCK2 pathway after SAH.
早期脑损伤（EBI）被报道为蛛网膜下腔出血（SAH）患者死亡的主要原因。Eph受体A4（EphA4）与血脑屏障完整性和促凋亡有关。我们旨在研究EphA4在SAH后EBI中的作用。179只成年雄性Sprague-Dawley大鼠被随机分为假手术组和SAH血管内穿刺模型组。SAH后进行SAH分级、神经评分、伊文思蓝染料外渗、脑含水量、死亡率、荧光金染色、免疫荧光染色和蛋白质印迹实验。使用针对EphA4的小干扰RNA（siRNA）、重组Ephexin-1（rEphx-1）和强效ROCK2抑制剂法舒地尔进行干预，以研究EphA4对SAH后EBI的作用。SAH后EphA4、Ephexin-1、RhoA和ROCK2的表达显著增加。脑室内注射EphA4 siRNA敲低EphA4可减少SAH后的伊文思蓝外渗，降低脑含水量，并减轻神经行为功能障碍。此外，Ephexin-1、RhoA、ROCK2和裂解的半胱天冬酶-3的表达降低。紧密连接蛋白增加，凋亡神经元死亡减少。rEphx-1消除了EphA4 siRNA的作用。相反，法舒地尔消除了rEphx-1的作用。这些结果表明，EphA4是一个新的有前景的治疗靶点，在SAH后通过Ephexin-1/ROCK2途径加重EBI。
