黄芩素对HT22细胞痴呆模型PI3K/Akt信号通路的影响  

Effect of Baicalein on PI3K/Akt Signaling Pathway in HT22 Cell Dementia Model

作　　者：叶佳希 朱敏 陶彦谷[1] 黄启辉[1] YE Jiaxi;ZHU Min;TAO Yangu;HUANG Qihui(Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University,Guangzhou Guangdong 510120,China)

机构地区：[1]中山大学孙逸仙纪念医院,广东广州510120

出　　处：《新中医》2025年第1期209-216,共8页New Chinese Medicine

基　　金：广东省自然科学基金-面上项目(2019A1515011414,2023A1515011136);广东省中医药局科研项目(20211072)。

摘　　要：目的:观察黄芩素对Aβ_(25-35)诱导的HT22细胞PI3K/Akt信号通路相关因子表达的影响。方法:Aβ_(25-35)40μmol·L^(-1)干预HT22细胞24 h,建立痴呆细胞模型。采用4个不同浓度(5、10、20、40μmol·L^(-1))黄芩素干预痴呆细胞模型24 h,CCK-8法检测细胞活力,选取细胞活力最高的黄芩素浓度用于后续实验。实验分为对照组、Aβ_(25-35)组和黄芩素组。流式细胞仪和Hoechest 33342染色检测细胞凋亡情况;JC-10法检测线粒体膜电位情况;DCFH-DA法检测活性氧水平;免疫荧光检测微管相关蛋白轻链3(LC3)蛋白表达情况;Western blot法检测选择性自噬接头蛋白(p62)、自噬关键分子酵母Atg6同系物1(Beclin-1)、磷脂酰肌醇3-激酶(PI3K)、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、丝/苏氨酸蛋白激酶(Akt)、磷酸化丝/苏氨酸蛋白激酶(p-Akt)和哺乳动物雷帕霉素靶蛋白(mTOR)表达。并与PI3K特异性抑制剂LY294002进行比较。结果:最终选择黄芩素浓度10μmol·L^(-1)作为后续实验。与对照组比较,Aβ_(25-35)组细胞凋亡率、ROS水平、p62蛋白表达、mTOR蛋白表达升高(P<0.05),MMP水平、LC3表达、Beclin-1蛋白表达、p-PI3K/PI3K比值、p-Akt/Akt比值降低(P<0.05);与Aβ_(25-35)组比较,黄芩素组细胞凋亡率、ROS水平、p62蛋白表达、mTOR蛋白表达减少(P<0.05),MMP水平、LC3表达、Beclin-1蛋白表达、p-PI3K/PI3K比值、p-Akt/Akt比值升高(P<0.05)。与黄芩素组比较,加入PI3K抑制剂LY294002后,p-PI3K/PI3K、p-Akt/Akt的比值降低,mTOR蛋白表达升高(P<0.05)。结论:黄芩素能够明显改善Aβ_(25-35)诱导的HT22细胞的线粒体功能,减少细胞凋亡和氧化应激,其抗痴呆作用与激活PI3K/AKT信号通路、提高细胞自噬水平有关。Objective:To observe effect of baicalein on the expression of PI3K/Akt signaling pathway related factors in HT22 cells induced by Aβ_(25-35).Methods:The Aβ_(25-35)at 40μmol·L^(-1)was used to intervened in HT22 cells for 24 hours to establish a dementia cell model.Four different concentrations(5,10,20,40μmol·L^(-1))of baicalein were used to intervene in dementia cell models for 24 hours.The cell viability was detected by CCK-8 method,and the baicalein concentration with the highest cell viability was selected for subsequent trials.The trial models were divided into the control group,the Aβ_(25-35)group,and the baicalein group.Flow cytometry and Hoechest 33342 staining were used to detect cell apoptosis;JC-10 method was used to detect mitochondrial membrane potential;DCFH-DA method was used to detect levels of reactive oxygen species;Immunofluorescence was used to detect microtubule associated protein light chain 3(LC3)protein expression;Western blot was used to detect the expression of selective autophagy adaptor protein(p62),autophagy key molecule yeast Atg6 homolog 1(Beclin-1),phosphatidylinositol 3-kinase(PI3K),phosphorylated phosphatidylinositol 3-kinase(p-PI3K),serine/threonine protein kinase(Akt),phosphorylated serine/threonine protein kinase(p-Akt),and mammalian target of rapamycin(mTOR),and compared with the PI3K specific inhibitor LY294002.Results:Finally,a concentration of 10μmol·L^(-1)of baicalein was chosen for the subsequent trial.Compared with those in the control group,the apoptosis rate,ROS level,p62 protein expression,and mTOR protein expression were increased in the Aβ_(25-35)group(P<0.05),while MMP level,LC3 expression,Beclin-1 protein expression,p-PI3K/PI3K ratio,and p-Akt/Akt ratio were decreased(P<0.05);Compared with those in the Aβ_(25-35)group,the cell apoptosis rate,ROS level,p62 protein expression,and mTOR protein expression in the baicalein group were decreased(P<0.05),while MMP levels,LC3 expression,Beclin-1 protein expression,p-PI3K/PI3K ratio,and p-Akt/Akt ratio were raised

关 键 词：阿尔茨海默病 黄芩素 自噬 线粒体膜电位 氧化应激 细胞凋亡 

分 类 号：R749.16[医药卫生—神经病学与精神病学]