ABSTRACT

Chronic alcohol consumption leads to hippocampal neuronal impairment, which related to neuronal death, oxidative stress, and inflammatory response. Rho-associated protein kinase 2 (ROCK2) is a major regulator in the central nervous system injury. However, the effects of ROCK2 in ethanol-induced brain injury have not been explored. In this work, we investigated the neuroprotective effects and the mechanism of ROCK2 inhibition in vivo. Wistar rats were exposed to 37% ethanol for 8 weeks to establish brain injury models. Morris water maze test was performed to evaluate cognitive function, and we found that the down-regulation of ROCK2 reduced the escape latency and increased the passing times and percentage of time spent in the target quadrant of rats. The results of H&E staining and Nissl staining showed that ROCK2 inhibition alleviated the pathological injury induced by ethanol. PI staining and Western blot confirmed that inhibiting ROCK2 attenuated the neuronal death and apoptosis as reflected by the reduced PI-positive neurons and the decreased expression of cleaved-caspase-3 and cleaved-caspase-9. Furthermore, the down-regulation of ROCK2 ameliorated the oxidative stress and inflammatory response induced by ethanol in rats as reflected by the up-regulation of IL-10, SOD, and GSH and reduction of TNF-α, IL-6, and MDA respectively. Additionally, Western blot and EMSA analysis revealed that the down-regulation of ROCK2 suppressed the nuclear transfer of NF-κB p65. In conclusion, our data suggested that ROCK2 inhibition ameliorated ethanol-mediated hippocampal neuronal impairment by anti-apoptotic, anti-inflammatory, anti-oxidative effects at least partially through the suppression of the NF-κB pathway.

摘要

长期饮酒会导致海马神经元损伤，这与神经元死亡、氧化应激和炎症反应有关。Rho 相关蛋白激酶 2 (ROCK2) 是中枢神经系统损伤的主要调节因子。然而，尚未探索 ROCK2 在乙醇诱导的脑损伤中的作用。在这项工作中，我们研究了体内ROCK2 抑制的神经保护作用和机制. Wistar大鼠暴露于37%乙醇中8周以建立脑损伤模型。进行Morris水迷宫试验以评估认知功能，我们发现ROCK2的下调减少了逃逸潜伏期并增加了通过时间和在目标象限中花费的时间百分比。H&E 染色和 Nissl 染色结果表明 ROCK2 抑制减轻了乙醇诱导的病理损伤。PI 染色和蛋白质印迹证实抑制 ROCK2 减弱了神经元死亡和细胞凋亡，这表现为 PI 阳性神经元减少以及 cleaved-caspase-3 和 cleaved-caspase-9 表达降低。此外，如 IL-10 的上调所反映的，ROCK2 的下调改善了乙醇诱导的大鼠氧化应激和炎症反应，SOD 和 GSH 以及 TNF-α、IL-6 和 MDA 的减少。此外，蛋白质印迹和 EMSA 分析显示 ROCK2 的下调抑制了 NF-κB p65 的核转移。总之，我们的数据表明 ROCK2 抑制通过抗凋亡、抗炎、抗氧化作用至少部分通过抑制 NF-κB 通路改善了乙醇介导的海马神经元损伤。