基于mTOR信号通路探讨首乌丸对D-半乳糖衰老大鼠海马神经元突触可塑性的影响  

Shouwuwan Regulates Synaptic Plasticity of Hippocampal Neurons in Rat Model of D-galactose-induced Aging via mTOR Signaling Pathway

作　　者：张超 加秀凤 黄万凌 周勇[3] 汤琪[1] 陈刚 ZHANG Chao;JIA Xiufeng;HUANG Wanling;ZHOU Yong;TANG Qi;CHEN Gang(College of Basic Medicine,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei College of Chinese Medicine,Jingzhou 434020,China;Hubei Provincial Hospital of Traditional Chinese Medicine,Wuhan 430061,China)

机构地区：[1]湖北中医药大学基础医学院,武汉430065 [2]湖北中医药高等专科学校,湖北荆州434020 [3]湖北省中医院,武汉430061

出　　处：《中国实验方剂学杂志》2023年第17期116-125,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：湖北省第二届医学领军人才工程培养对象暨湖北名医工作室负责人项目[鄂卫通(2019)47号];湖北省卫生计生委中西医结合重点科研项目[鄂卫生计生通(2017)20号];湖北省卫生健康委员会中医药科研项目青年人才项目(ZY2021Q031);湖北省卫健委面上重点项目(WJ2019H422);武汉市卫健委面上重点项目(WZ19A15)。

摘　　要：目的:探讨首乌丸基于哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对D-半乳糖衰老大鼠海马神经元突触可塑性的影响。方法:将50只SPF级SD雄性大随机分为正常组、模型组、维生素E组、首乌丸低剂量组及首乌丸高剂量组,除正常组外,其余4组均用D-半乳糖(120 mg·kg^(-1))制造衰老模型,同时给予维生素E(0.018 g·kg^(-1)),首乌丸低、高剂量(1.08、2.16 g·kg^(-1))灌胃。造模6周后Morris水迷宫检测行为学变化,取全脑、海马组织,免疫组化检测海马突触后密度蛋白-95(PSD-95)、突触素(SYN)的表达,高尔基染色观察大鼠神经元形态及功能的变化,蛋白免疫印迹法(Western blot)检测海马组织中mTOR、磷酸化(p)-mTOR、p70核糖体蛋白S6激酶(p70S6K)、磷酸化(p)-p70S6K、真核翻译起始因子4E结合蛋白2(4EBP2)、磷酸化(p)-4EBP2蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测海马mTOR、p70S6K、4EBP2 mRNA表达水平。结果:与正常组比较,模型组大鼠平均游泳速度减慢(P<0.01),游泳总路程延长(P<0.05),上平台潜伏期延长(P<0.01),穿越平台次数明显减少(P<0.01),海马CA1区PSD-95、SYN蛋白表达下降(P<0.01),染色效果最淡,区域最小,在同心圆距胞体100、140、180、200μm时,海马神经元树突与同心圆交点数量减少(P<0.01),树突棘长度及密度均下降(P<0.01),p-mTOR、p-p70S6K蛋白表达升高(P<0.01),4EBP2、p-4EBP2蛋白表达显著下降(P<0.01),mTOR、p70S6K mRNA表达上升(P<0.01),4EBP2 mRNA表达降低(P<0.01)。与模型组比较,首乌丸低、高剂量组大鼠的平均游泳速度加快(P<0.01),上平台潜伏期缩短(P<0.01),穿越平台次数增加(P<0.01),海马CA1区PSD-95、SYN蛋白表达升高(P<0.01),在同心圆距胞体100、140、180、200μm时,海马神经元树突与同心圆交点数量增加(P<0.01),首乌丸低、高剂量组大鼠的树突棘数量、长度、密度均升高(P<0.01),p-mTOR、p-p70S6K蛋白表达下降(P<0.01),4EBP2、p-4EBP2蛋白表达Objective:To investigate the effect of Shouwuwan on the synaptic plasticity of hippocampal neurons in the rat model of D-galactose-induced aging via the mammalian target of rapamycin(mTOR)signaling pathway.Method:A total of 50 male SPF-grade SD rats were randomized into normal group,model group,vitamin E(0.018 g·kg^(-1))group,and low-and high-dose(1.08,2.16 g·kg^(-1),respectively)Shouwuwan groups.Except the normal group,the other four groups were treated with D-galactose(120 mg·kg^(-1))for the modeling of aging.The rats were simultaneously administrated with corresponding agents by gavage.After six weeks of modeling,Morris water maze test was carried out to examine the behavioral changes.The whole brain and hippocampus samples were collected.The expression of postsynaptic density protein-95(PSD-95)and synaptophysin(SYN)in the hippocampus was detected by immunohistochemistry.Golgi staining was employed to observe the changes in the morphology and function of neurons.Western blot and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)were respectively employed to determine the mRNA and protein levels of mTOR,phosphorylated(p)-mTOR,p70 ribosome protein S6 kinase(p70S6K),phosphorylated(p)-p70S6K,eukaryotic translation initiation factor 4E-binding protein 2(4EBP2),and phosphorylated(p)-4EBP2 in the hippocampus.Result:Compared with the normal group,the model group showed slow swimming(P<0.01),extended total swimming distance(P<0.05),prolonged latency(P<0.01),and decreased crossing number(P<0.01).The modeling inhibited the expression of PSD-95 and SYN in the CA1 region of the hippocampus(P<0.01),with the weakest staining effect and the smallest region,decreased the intersections of hippocampal neuron dendrites with concentric circles at the concentric distance of 100,140,180,and 200μm from the cell body(P<0.01),and reduced the length and density of dendritic spine(P<0.01).In addition,the modeling up-regulated the mRNA levels of mTOR and p70S6K and the protein levels of pmTOR and p-p70S6K(P<

关 键 词：脑衰老 首乌丸 哺乳动物雷帕霉素靶蛋白(mTOR)信号通路 突触可塑性 

分 类 号：R2-0[医药卫生—中医学] R33R289R745.1