依达拉奉通过Nrf2信号分子调节氧化应激减轻脑缺血再灌注诱导的神经损伤  

Edaravone Regulates Oxidative Stress to Alleviate Neuronal Damage Induced by Cerebral Ischemia Reperfusion via Nrf2

作　　者：王力波 贝赟 秦东旭 艾立瑶 宋英 WANG Li-Bo;BEI Yun;QIN Dong-Xu;AI Li-Yao;SONG Ying(College of Pharmacy,Zhejiang University of Technology,Hangzhou 310018,China;The First Affiliated Hospital of Huzhou University,Huzhou 313000,Zhejiang,China)

机构地区：[1]浙江工业大学药学院药理研究所,杭州310018 [2]湖州市第一人民医院药剂科,浙江湖州313000

出　　处：《中国生物化学与分子生物学报》2020年第3期328-336,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：浙江省自然科学基金(No.LY17H090018);湖州市科技局自然科学基金(No.2019YZ13)资助。

摘　　要：本研究旨在探讨依达拉奉(edaravone,ED)在脑缺血再灌注损伤中发挥神经元保护作用与Nrf2信号分子间的关系。体内实验利用脑内脑中动脉闭塞(middle cerebral artery occlusion model,MCAO)建立SD大鼠脑缺血再灌注损伤模型,体外实验采用过氧化氢(H2O2)损伤PC12细胞建立氧化应激模型。通过TTC染色、HE染色、Nissl染色来检测大脑的病理状态。测定活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活性,来反映氧化应激水平。此外,通过Hoechst 33342染色和线粒体膜电位(mitochondrial membrane potential,MTP)测定,探究细胞水平的损伤。采用免疫组织化学和蛋白质印记测定Nrf2的表达。构建Nrf2敲除的PC12细胞系,证实Nrf2信号分子抑制氧化应激损伤的作用。结果提示,经依达拉奉给药后,在动物体内水平,TTC染色证实,脑缺血再灌注损伤(cerebral ischemia reperfusion injury,CIRI)大鼠的脑组织梗死体积减小(P<0.001),ROS和MDA水平下降(P<0.01),SOD活性上升(P<0.01);在细胞水平,凋亡细胞减少(P<0.05),MTP上升(P<0.01),ROS和MDA水平下降,SOD活性上升(P<0.01);在分子水平,免疫组化和Western印迹结果均提示,Nrf2蛋白质含量较正常组增加。H2O2诱导Nrf2基因敲除的PC12细胞损伤加重,且依达拉奉的治疗效果明显削弱。综上所述,Nrf2在依达拉奉减轻脑缺血再灌注诱导的氧化应激损伤中发挥关键作用。The purpose of this study was to investigate the relationship between the edaravone protective effects on neurons and Nrf2 signaling molecules in cerebral ischemia-reperfusion injury.In vivo experiments were performed by establishing a middle cerebral artery occlusion model in SD rats,and in vitro experiments were performed using hydrogen peroxide(H2O2)to damage PC12 cells to establish an oxidative stress model.The pathological status of the brain was detected by TTC,HE and Nissl staining.Reactive oxygen species(ROS),malondialdehyde(MDA)contents,and superoxide dismutase(SOD)activity were measured to reflect the level of oxidative stress.In addition,Hoechst 33342 staining and measurement of mitochondrial membrane potential(MTP)were used to investigate the cell-level damage.Nrf2 expression was determined using immunohistochemistry and Western blotting.Nrf2 knockout PC12 cells were constructed to confirm the role of Nrf2 signaling molecules in inhibiting oxidative stress-induced injury.The results suggest that after edaravone administration,both animal-level and TTC staining confirmed that cerebral ischemia-reperfusion injury(CIRI)rats had reduced cerebral infarcted areas(P<0.001),ROS and MDA levels decreased(P<0.01),SOD activity increased(P<0.01);at the cell level,apoptotic cells decreased(P<0.05),MTP increased(P<0.01),ROS and MDA levels decreased,and the SOD activity increased(P<0.01);at the molecular level,immunohistochemistry and Western blotting showed that the Nrf2 protein level was increased compared with the control group.H2O2 induced damage in Nrf2-knockout PC12 cell was aggravated and the therapeutic effect of edaravone was significantly weakened.In summary,Nrf2 plays a key role in edaravone in reducing cerebral ischemia-reperfusion-induced oxidative stress injury.

关 键 词：脑缺血再灌注损伤 氧化应激 Nrf2信号分子 线粒体 

分 类 号：R96[医药卫生—药理学] Q255[医药卫生—药学]