Netrin-1通过ERK信号通路对原代皮层神经元OGD/R后氧化应激及突起生长的影响  

Netrin-1 Affects Oxidative Stress and Neurite Outgrowth in Primary Cortical Neurons Post OGD/R via the ERK Signaling Pathway

作　　者：陈佳静 赵梓钧 詹恒 郑志坚 陈鸿宾 CHEN Jiajing;ZHAO Zijun;ZHAN Heng;ZHENG Zhijian;CHEN Hongbin(Department of Neurology,Fujian Medical University Union Hospital,Fuzhou 350001,China;The School of Basic Medical Sciences,Fujian Medical University,Fuzhou 350122,China;Department of Neurology,Zhangzhou Affiliated Hospital of Fujian Medical University,Zhangzhou 363000,China)

机构地区：[1]福建医科大学附属协和医院神经内科,福州350001 [2]福建医科大学基础医学院,福州350122 [3]福建医科大学附属漳州市医院神经内科,漳州363000

出　　处：《福建医科大学学报》2024年第5期289-297,共9页Journal of Fujian Medical University

基　　金：福建省自然科学基金项目(2021J01765);福建医科大学附属协和医院优秀青年人才培育项目(2022XH026)。

摘　　要：目的探讨氧糖剥夺/复糖复氧(OGD/R)后,Netrin-1改善原代皮层神经元氧化应激和促进突起生长的作用机制。方法大鼠原代皮层神经元随机分为对照组(Control组)、OGD/R模型组(OGD/R组)、OGD/R+过表达Ntn1组(OGD/R+LV-Ntn1组)、OGD/R+Ntn1干扰组(OGD/R+LV-sh_Ntn1组)、OGD/R+过表达Ntn1+ERK1/2抑制剂组(OGD/R+LV-Ntn1+U0126组)。建立OGD/R模型后,采用CCK-8法检测细胞活力,采用乳酸脱氢酶(LDH)检测细胞毒性,采用丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)和还原谷胱甘肽/氧化谷胱甘肽二硫化物(GSH/GSSG)检测细胞氧化应激水平,采用免疫荧光染色检测细胞突起状态,采用qPCR检测PSD 95和GAP 43的mRNA表达水平,采用Western-blot检测ERK、PSD95和GAP43蛋白的表达水平。结果与Control组比较,OGD/R可上调ERK通路的表达水平(P<0.01),抑制原代皮层神经元的细胞活力(P<0.001),增加LDH释放(P<0.001),显著增加MDA含量(P<0.001),减低SOD活性、GPx活性和GSH/GSSG(P<0.001),显著降低最长突起长度和一级突起数量(P<0.001),降低PSD 95、GAP 43的mRNA和蛋白水平的表达(P<0.001);与OGD/R组比较,过表达Netrin-1显著上调ERK通路的表达水平(P<0.001),增强原代皮层神经元的细胞活力(P<0.001),减少LDH释放(P<0.001),显著降低MDA含量(P<0.001),加强SOD活性、GPx活性和GSH/GSSG(P<0.001),增加最长突起长度和一级突起数量(P<0.001),上调PSD 95和GAP 43 mRNA和蛋白水平的表达(P<0.001);与Netrin-1过表达组比较,U0126可以逆转Netrin-1对大鼠原代皮层神经元上述指标的影响(P<0.05)。结论Netrin-1可通过ERK信号通路改善大鼠原代皮层神经元OGD/R后氧化应激、促进突起生长。Objective To investigate the mechanism by which Netrin-1 improves oxidative stress and promotes neurite outgrowth following oxygen-glucose deprivation/reoxygenation(OGD/R)in primary cortical neurons.Methods Primary cortical neurons of rats were randomly assigned to the following groups:control group(Control group),oxygen-glucose deprivation/reoxygenation model group(OGD/R group),OGD/R+overexpression of Netrin-1 group(OGD/R+LV-Ntn1 group),OGD/R+Netrin-1 interference group(OGD/R+LV-sh_Ntn1 group),and OGD/R+overexpression of Netrin-1+ERK1/2 inhibitor group(OGD/R+LV-Ntn1+U0126 group).After establishing the OGD/R model,cell viability was assessed using the CCK-8 assay,cell toxicity was evaluated via lactate dehydrogenase(LDH)assay,oxidative stress levels were measured by malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxidase(GPx),and the ratio of reduced glutathione(GSH)to oxidized glutathione(GSSG),immunofluorescence staining was employed to examine neurite morphology,qPCR was used to measure mRNA expression levels of PSD 95 and GAP 43,Western\|blot was performed to analyze protein expression levels of ERK,PSD95,and GAP43.Results Compared to the Control group,the OGD/R model significantly upregulated the expression levels of the ERK pathway(P<0.01),inhibited the cell viability of primary cortical neurons(P<0.001),increased LDH release(P<0.001),significantly elevated MDA levels(P<0.001),reduced SOD activity,GPx activity,and the ratio of GSH/GSSG(P<0.001),markedly decreased the length of the longest neurite and the number of primary neurites(P<0.001),and decreased the expression levels of PSD 95 and GAP 43 mRNA and proteins.In comparison to the OGD/R group,overexpression of Netrin-1 significantly upregulated the expression levels of the ERK pathway(P<0.001),enhanced the cell viability of primary cortical neurons(P<0.001),decreased LDH release(P<0.001),significantly reduced MDA levels(P<0.001),increased SOD activity,GPx activity,and the ratio of GSH/GSSG(P<0.001),increased the length of the longest neurit

关 键 词：NETRIN-1 氧糖剥夺/复糖复氧 ERK信号通路 氧化应激 突起生长 

分 类 号：R743.3[医药卫生—神经病学与精神病学]