羽扇豆醇调节MAPK/ERK/mTOR信号通路对OGD/R诱导的神经元自噬的影响  

Lupinol inhibits OGD/R-induced neuronal autophagy by modulating MAPK/ERK/mTOR signaling pathway

作　　者：张君 李淼[2] 张倩楠 Zhang Jun;Li Miao;Zhang Qiannan(Department of Neurology,the Second Hospital of Dalian Medical University,Dalian 116000)

机构地区：[1]辽宁省大连医科大学附属二院神经内科,116000 [2]辽宁省大连医科大学附属二院药学部,116000

出　　处：《卒中与神经疾病》2023年第3期235-241,共7页Stroke and Nervous Diseases

摘　　要：目的探讨羽扇豆醇调节有丝分裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)/细胞外信号调节激酶(Extracellular signal-regulated kinase,ERK)/哺乳动物雷帕霉素靶蛋白(Mammalian target of rapamycin,mTOR)信号通路对氧糖剥夺/复氧(Oxygen-glucose deprivation/Reoxygenation,OGD/R)诱导的神经元自噬的影响。方法原代培养海马神经元,建立OGD/R损伤模型;3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]法检测不同水平羽扇豆醇(0、1、5、10、20、40、80μmol/L)干预的OGD/R海马神经元存活率;将大鼠海马神经元随机分为对照(CON)组(常规培养)、OGD/R组(氧糖剥夺90 min后复氧复糖24 h)、羽扇豆醇组(OGD/R+10μmol/L羽扇豆醇)、叔丁基对苯二酚(Tert-butylhydroquinone,TBHQ)组(OGD/R+10μmol/L羽扇豆醇+50μmol/L MAPK激活剂TBHQ);试剂盒检测神经元乳酸脱氢酶(Lactate de-hydrogenase,LDH)、丙二醛(Malondialdehyde,MDA)、超氧化物歧化酶(Superoxide dismutase,SOD)及还原型谷胱甘肽(Glutathione,GSH)水平;2,7-二氯二氢荧光素二乙酸酯(2',7'-Dichlorohydrofluorescein diacetate,DCFH-DA)探针法检测神经元活性氧(Reactive oxide species,ROS)水平;钙荧光探针(Fluo-3 acetoxymethyl ester,Fluo-3/AM)法检测神经元Ca2+水平;流式细胞术检测神经元凋亡情况;透射电镜观察神经元自噬情况;自噬双标记腺病毒(Red fluorescent protein-gteen fluorescent protein-microtubule-associated protein 1 light chain 3,mRFP-GFP-LC3B)检测神经元自噬流;Western blot检测神经元微管相关蛋白1轻链3Ⅱ/Ⅰ(Microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ,LC3Ⅱ/Ⅰ)、p62,Beclin 1,ERK1/2、磷酸化细胞外信号调节激酶1/2(Phosphorylated extracellular signal-regulated kinases 1/2,p-ERK1/2)、mTOR和磷酸化哺乳动物雷帕霉素靶蛋白(Phosphorylated mammalian target of rapamycin,p-mTOR)蛋白表达水平。结果与0μmol/L比较,5、10、20、40μmol/L羽扇豆醇�ObjectiveTo investigate the influence of lupinol on oxygen-glucose deprivation/reoxygenation(OGD/R)-induced neuronal autophagy by regulating the mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase(ERK)/mammalian target of rapamycin(mTOR)signaling pathway.Methods A model of OGD/R injury was established by primary culture of hippocampal neurons;MTT method was applied to detect the survival rate of OGD/R hippocampal neurons in different concentrations of lupinol(0,1,5,10,20,40,80μmol/L).The rat hippocampal neurons were randomly separated into the CON group(conventional culture),OGD/R group(24 h reoxygenation and glucose recovery after oxygen-glucose deprivation for 90 min),lupinol group(OGD/R+10μmol/L lupinol),and TBHQ group(OGD/R+10μmol/L lupinol+50μmol/L MAPK activator TBHQ).The kits were applied to detect neuronal LDH,MDA,SOD and GSH contents.The DCFH-DA probe method was applied to detect neuronal ROS content.The Fluo-3/AM probe method was applied to detect neuronal Ca2+concentration.Flow cytometry was used to detect neuronal apoptosis;transmission electron microscopy was applied to observe neuronal autophagy.Western blots of mRFP-GFP-LC3B were applied to detect neuronal autophagic flux.Western blotting was applied to detect the levels of neuronal LC3-II/I,p62,Beclin 1,ERK1/2,p-ERK1/2,mTOR and p-mTOR.Results Compared with 0μmol/L,5,10,20,and 40μmol/L lupinol improved the neuron survival rate(P<0.05).However,the neuron survival rate was the highest at 10μmol/L lupinol.Based on the comprehensive results,the concentration of 10μmol/L lupinol was selected for subsequent experiments.Compared with the CON group,the neuronal survival rate,SOD,GSH,and the protein expression of p62 and p-mTOR in the OGD/R group were greatly decreased(P<0.05).The contents of LDH,MDA,ROS,and Ca2+mean fluorescence intensity,the number of autophagosomes,yellow fluorescent particles,and red fluorescent particles,and the levels of LC3-II/I,Beclin 1,and p-ERK1/2 were greatly increased(P<0.05).Compared with the OG

关 键 词：羽扇豆醇 神经元 有丝分裂原活化蛋白激酶/细胞外信号调节激酶/哺乳动物雷帕霉素靶蛋白信号通路 氧糖剥夺/复氧 自噬 

分 类 号：R743.3[医药卫生—神经病学与精神病学]