磷酯酰肌醇3-激酶/蛋白激酶B信号通路介导葛根素改善缺氧复氧损伤的作用及机制  

Puerarin Reduces Apoptosis of EA.hy926 After Hypoxia-reoxygenation via Regulation of PI3K/Akt Signaling Pathway

作　　者：杨学攀 朱明明 余曦 贺稳 陈晗 施洪飞[2] YANG Xuepan;ZHU Mingming;YU Xi;HE Wen;CHEN Han;SHI Hongfei(Hanlin College,Nanjing University of Chinese Medicine,Taizhou,225300,Jiangsu,China)

机构地区：[1]南京中医药大学翰林学院,江苏省泰州市225300 [2]南京中医药大学第二临床医学院营养学教研室

出　　处：《中国循环杂志》2021年第8期814-821,共8页Chinese Circulation Journal

基　　金：国家自然科学基金(81574044)。

摘　　要：目的:探讨磷酯酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路介导葛根素改善缺氧复氧损伤的作用及机制。方法:应用噻唑蓝法筛选出合适的葛根素以及PI3K抑制剂LY294002干预人脐静脉内皮细胞EA.hy926的浓度。分为对照组、缺氧复氧组、缺氧复氧+葛根素组(葛根素组),缺氧复氧+葛根素+LY294002处理组(葛根素+抑制剂组),每组均设3个复孔。应用蛋白免疫印迹法(Western blot)检测葛根素预处理细胞后以及葛根素预处理细胞的同时加入LY294002后对B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、活化天冬氨酸蛋白水解酶-3(cleaved caspase-3)、活化天冬氨酸蛋白水解酶-9(cleaved caspase-9)、磷酸化的蛋白激酶B(p-Akt)、Akt蛋白表达的影响。应用实时荧光定量PCR检测葛根素预处理对EA.hy926细胞缺氧复氧后天冬氨酸蛋白水解酶-3(caspase-3)和Bcl-2信使核糖核酸(mRNA)表达的影响。应用Hoechest染色检测葛根素预处理对EA.hy926细胞凋亡的影响。结果:葛根素(10^(-7)mol/L)明显升高缺氧复氧后EA.hy926细胞活力(P<0.001),并显著上调Bcl-2蛋白水平的表达,下调Bax、cleaved caspase-9、cleaved caspase-3蛋白水平的表达(P均<0.01)。与缺氧复氧组相比,葛根素预处理后,p-Akt蛋白表达水平明显升高(P<0.01)。加入LY294002(10μmol/L)后p-Akt蛋白表达水平下降,且抗凋亡蛋白Bcl-2的表达水平下降,促凋亡蛋白cleaved caspase-3、cleaved caspase-9、Bax的表达水平升高(P均<0.01)。结论:葛根素对缺氧复氧诱导的EA.hy926细胞凋亡具有抑制作用,其抑制作用可能与其对PI3K/Akt信号通路的调控有关。Objectives:This study explored the effect and related mechanism of puerarin in reducing the injury induced by hypoxia and reoxygenation in human umbilical vein endothelial cells.Methods:MTT method was used to determine the appropriate concentration of puerarin and LY294002 to interfere with human umbilical vein endothelial cells EA.hy926.Cells were divided to four groups:control group,hypoxia and reoxygenation group,hypoxia and reoxygenation+puerarin group and hypoxia and reoxygenation+puerarin+LY294002 group.Western blot was used to detect the effects of puerarin pretreated cells and puerarin pretreated cells with LY294002 on the expression of Bcl-2,Bax,cleaved caspase-3,cleaved caspase-9,p-Akt and Akt.Real time PCR was used to detect the mRNA expression of caspase-3 and Bcl-2.Hoechest test was used to explore the effect of puerarin pretreatment on cell apoptosis.Results:Puerarin(10^(-7) mol/L)significantly improved cell viability after hypoxia and reoxygenation(P<0.001),and significantly up-regulated the protein expression of Bcl-2,and down-regulated the protein expression of Bax,cleaved caspase-9,and cleaved caspase-3(all P<0.01).Compared with the hypoxia-reoxygenation group,the protein expression of p-Akt was significantly increased after puerarin pretreatment(P<0.01).LY294002(10μmol/L)pretreatment reversed the effect of puerarin,as shown by the reduced expression level of p-Akt and Bcl-2,and upregulated expression levels of cleaved caspase-3,cleaved caspase-9,and Bax(all P<0.01).Conclusions:Puerarin possesses an inhibitory effect on EA.hy926 cell apoptosis induced by hypoxia and reoxygenation,and the inhibitory effect may be related to its regulation on PI3K/Akt signaling pathway.

关 键 词：细胞凋亡 内皮细胞 葛根素 心肌缺血再灌注损伤 磷酯酰肌醇3-激酶/蛋白激酶B信号通路 

分 类 号：R54[医药卫生—心血管疾病]