微小RNA-155通过Notch信号通路对脑缺血-再灌注损伤的影响

The effect of microRNA-155 on cerebral ischemia-reperfusion injury through regulation of Notch signaling

目的探讨微小 RNA(miR)在小鼠大脑中动脉闭塞(MCAO)所造成缺血-再灌注(I-R)损伤中的调控作用。方法构建 C57BL/6 小鼠 MCAO 模型,采用氯化三苯四氮唑(TCC)染色和神经功能评分了解脑 I-R 损伤对小鼠神经功能的影响。实时定量聚合酶链反应(RT-qPCR)技术检测 miR-155 表达,苏木精-伊红(HE)染色观察 miR- 155 对脑组织病理学的影响,伊文氏蓝(EB)和脑组织含水量检测观察miR- 155 对血脑屏障(BBB)通透性的影响,一氧化氮(NO)含量和内皮型内皮型一氧化氮合酶(eNOS)表达检测评估 miR- 155 对血管内皮细胞功能的影响,免疫印迹法检测 Notch1、Notch1 胞内结构域(NICD)、Jagged1 和 Hes1 表达, RT-qPCR 检测 Notch1 和 Hes1 mRNA 水平,以明确 miR- 155 在 Notch 信号通路中的作用。结果 miR-155 缺失提高 Notch1、NICD、Hes1 表达,降低脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色阳性细胞百分比和半胱氨酸天冬氨酸特异性蛋白酶(caspase)- 3 水平;干扰miR-155 表达增加 NO 产生和 eNOS 表达,导致脑组织含水量和 EB 含量下调。 miR-155 过度表达使所有这些改变恢复至脑 I-R 损伤水平。 Notch1、NICD、Hes1 表达减轻脑 I-R 损伤状态。结论 miR-155 可通过Notch 信号通路阻断正常 NO 产生和 eNOS 表达,这一调控机制可能是未来缺血性脑卒中治疗潜在靶点之一。

Objective To investigate the molecular mechanism of microRNA- 155 (miR- 155) in the regulation of cerebral ischemia-reperfusion (I-R) injury caused by middle cerebral artery occlusion (MCAO) in experimental mice. Methods The MCAO model was established in C57BL/6 mice. The effect of cerebral I- R injury on the neural function of mice was evaluated by triphenyltetrazole chloride (TTC) staining and nerve function scoring. The real-time quantitative polymerase chain reaction (RT- qPCR) technique was used to test the expression of miR- 155. By using hematoxylin- eosin (HE) staining the effect of miR- 155 on brain histopathology was assessed. The effect of miR- 155 on the permeability of blood brain barrier was evaluated by measuring Evans blue (EB) and water content in brain tissue. The effect of miR - 155 on endothelial function was estimated by measuring nitric oxide (NO) content and expression of endothelial nitric oxide synthase (eNOS). Immunoblotting was employed to check the expressions of Notch1, Notch1 intracellular domain (NICD), Jagged1 and Hes1, and RT- qPCR was adopted to check Notch1 and Hes1 mRNA levels in order to clarify the role of miR- 155 in Notch signaling pathway. Results The deletion of miR- 155 enhanced the expressions of Notch1, NICD, Hes1 and decreased the percentage of TUNEL staining positive cells and the caspase- 3 levels. Interference with the expression of miR- 155 could increase NO production and eNOS expression, leading to downregulation of the brain water content and EB level. However, over- expression of miR- 155 could restore all these changes back to the cerebral I- R injury levels. The expression of Notch1, NICD and Hes1 could improve the cerebral I-R injury status. Conclusion The results of this study indicate that miR- 155 can block normal NO production and eNOS expression through the Notch signaling pathway. This regulatory mechanism may be one of the potential targets for the treatment of ischemic stroke in the future.