[1]张君 李淼 张倩楠.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[J].卒中与神经疾病杂志,2023,30(03):235-241.[doi:10.3969/j.issn.1007-0478.2023.03.001]
　Zhang Jun*,Li Miao,Zhang Qiannan*..Lupinol inhibits OGD/R-induced neuronal autophagy by modulating MAPK/ERK/mTOR signaling pathway[J].Stroke and Nervous Diseases,2023,30(03):235-241.[doi:10.3969/j.issn.1007-0478.2023.03.001]
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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()
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《卒中与神经疾病》杂志[ISSN:1007-0478/CN:42-1402/R]

卷:
第30卷
期数:
2023年03期
页码:
235-241
栏目:
论著
出版日期:
2023-06-20

文章信息/Info

Title:
Lupinol inhibits OGD/R-induced neuronal autophagy by modulating MAPK/ERK/mTOR signaling pathway
文章编号:
1007-0478(2023)03-0235-07
作者:
张君 李淼 张倩楠
116000 辽宁省大连医科大学附属二院神经内科[张君 张倩楠(通信作者)],药学部(李淼)
Author(s):
Zhang Jun*;  Li Miao;  Zhang Qiannan*.
*Department of Neurology, the Second Hospital of Dalian Medical University, Dalian 116000
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
Keywords:
Lupinol; Neurons; MAPK/ERK/mTOR signaling pathway; OGD/R; Autophagy
分类号:
R743.3
DOI:
10.3969/j.issn.1007-0478.2023.03.001
文献标志码:
A
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、80 μmol/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+10 μmol/L羽扇豆醇)、叔丁基对苯二酚(Tert-butylhydroquinone,TBHQ)组(OGD/R+10 μmol/L羽扇豆醇+50 μmol/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0 μmol/L比较,5、10、20、40 μmol/L羽扇豆醇均能提高神经元存活率(P<0.05),但10 μmol/L羽扇豆醇时神经元存活率最高; 与CON组比较,OGD/R组神经元存活率、SOD,GSH以及p62和p-mTOR蛋白表达水平显著降低(P<0.05),LDH,MDA,ROS水平、Ca2+平均荧光强度、自噬小体个数、黄色荧光颗粒、红色荧光颗粒以及LC3-Ⅱ/Ⅰ,Beclin 1和p-ERK1/2蛋白表达水平显著上升(P<0.05); 与OGD/R组比较,羽扇豆醇组SOD,GSH以及p62和p-mTOR蛋白表达水平显著升高,LDH,MDA,ROS水平、Ca2+平均荧光强度、自噬小体个数、黄色荧光颗粒、红色荧光颗粒以及LC3-Ⅱ/Ⅰ,Beclin 1和p-ERK1/2蛋白表达水平显著降低(P<0.05); MAPK激活剂TBHQ逆转了羽扇豆醇对OGD/R海马神经元的损伤改善作用。结论 羽扇豆醇通过调节MAPK/ERK/mTOR信号通路来抑制OGD/R诱导的神经元自噬,起到保护大鼠海马神经元效果。
Abstract:
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, 80 μmol/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+10 μmol/L lupinol), and TBHQ group(OGD/R+10 μmol/L lupinol+50 μmol/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 0 μmol/L, 5, 10, 20, and 40 μmol/L lupinol improved the neuron survival rate(P<0.05). However, the neuron survival rate was the highest at 10 μmol/L lupinol. Based on the comprehensive results, the concentration of 10 μmol/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 OGD/R group, SOD, GSH, and the levels of p62 and p-mTOR in the lupinol group were greatly increased; the LDH, MDA, ROS, Ca2+ mean fluorescence intensity, the number of autophagosomes, yellow fluorescent particles, red fluorescent particles, and levels of LC3-II/I, Beclin 1 and p-ERK1/2 were greatly decreased(P<0.05). The MAPK activator TBHQ reversed the negative effects of lupinol on OGD/R hippocampal neurons.Conclusion Lupinol inhibits OGD/R-induced neuronal autophagy by regulating the MAPK/ERK/mTOR signaling pathway and protects rat hippocampal neurons.

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更新日期/Last Update: 2023-06-20