miR-497-5p靶向AKT3对缺血/再灌注损伤诱导的神经细胞氧化应激和炎症反应的影响  

Effects of miR-497-5p targeting AKT3 on oxidative stress and inflammation induced by ischemia/reperfusion injury in neurons

作　　者：闫海清[1] 岳学静[1] 贵永堃[1] 任瑞芳[1] 王昊亮 赵君 张平[1] YAN Haiqing;YUE Xuejing;GUI Yongkun;REN Ruifang;WANG Haoliang;ZHAO Jun;ZHANG Ping(Department of Neurology,First Affiliated Hospital of Xinxiang Medical University,Xinxiang 453100,China)

机构地区：[1]新乡医学院第一附属医院神经内科

出　　处：《免疫学杂志》2019年第11期927-933,共7页Immunological Journal

基　　金：河南省科技攻关计划项目(182102310529)

摘　　要：目的探究miR-497-5p对缺血再灌注诱导的神经细胞氧化应激和炎症反应的影响以及潜在的作用机制。方法采用小鼠海马神经元细胞构建氧糖剥夺/复氧(OGD/R)损伤模型;氧糖剥夺后复氧6 h、12 h、24 h,检测氧化应激指标的含量,酶联免疫吸附实验检测炎症因子的水平,实时荧光定量PCR检测miR-497-5p的表达水平。检测miR-497-5p mimic和inhibitor的转染效率。双荧光素酶报告实验分析miR-497-5p与AKT3的靶向关系;蛋白质印迹检测miR-497-5p对AKT3表达水平的影响。miR-497-5p inhibitor与AKT3 siRNA单独或共转后OGD/R,检测氧化应激和炎症反应的变化;蛋白质印迹检测叉头转录因子O3(FOXO3)、核因子κB(NF-κB)的表达水平的变化。结果神经细胞经氧糖剥夺复氧后,细胞中ROS和MDA的含量显著升高,SOD的活性显著降低;白介素1β(IL-1β)、白介素6(IL-6)和肿瘤坏死因子α(TNF-α)的含量显著升高,miR-497-5p的含量显著升高。miR-497-5p与AKT3存在靶向抑制关系。miR-497-5p inhibitor转染神经细胞后OGD/R,细胞中氧化应激和炎症反应被抑制,细胞凋亡率显著减低;FOXO3、NF-κB的表达水平显著降低。AKT3 siRNA缓解miR-497-5p inhibitor对OGD/R模型氧化应激、炎症反应、细胞凋亡和FOXO3、NF-κB表达水平的影响。结论 miR-497-5p靶向抑制AKT3的表达,促进OGD/R模型氧化应激和炎症反应。The aim of this experiment is to investigate the effects of miR-497-5p on oxidative stress and inflammation induced by ischemia-reperfusion and its potential mechanism. Hippocampal neurons in mice was used to construct the oxygen-glucose deprivation/reoxygenation(OGD/R) injury model. Neurons was reoxygenated for 6,12, 24 hours after oxygen-glucose deprivation. Then the content of oxidative stress index was detected;the level of inflammatory factors was detected by enzyme-linked immunosorbent assay;and real-time fluorescence quantitative PCR was used to detect the expression level of miR-497-5p. The transfection efficiency of miR-497-5p mimic and inhibitor were detected. Dual luciferase reporter assay was used to analyze the targeting relationship between miR-497-5p and AKT3. Western blotting was used to detect the effect of miR-497-5p on the expression of AKT3.Furthermore, miR-497-5p inhibitor and/or AKT3 siRNA alone or co-transfected to neurons before OGD/R, and oxidative stress and inflammation changes were detected;Western blot was used to detect the changes of FOXO3 and NF-kappa B expression levels. Data showed that the contents of ROS, MDA, IL-1 beta, IL-6 and TNF-alpha,and the contents of miR-497-5p in the neurons were significantly increased after OGD. In miR-497-5p mimic and AKT3 wild-type reporter vectors co-transfected neurons, luciferase activity decreased significantly. MiR-497-5p mimic inhibited the expression of AKT3, while MiR-497-5p inhibitor inhibited the oxidative, inflammation reaction and the expression of FOXO3 and NF-κB in neurons. AKT3 siRNA alleviates the effects of miR-497-5p inhibitor on oxidative stress, inflammation and FOXO3,NF-κB expression in OGD/R model. In conclusion, miR-497-5p inhibits AKT3 expression and promotes oxidative stress and inflammatory response in OGD/R model.

关 键 词：缺血再灌注脑损伤 微小RNA 蛋白激酶B 氧化应激 炎症反应 

分 类 号：R743.31[医药卫生—神经病学与精神病学]