芦丁对肝细胞氧化应激损伤的保护作用及其机制  

Protective effect of rutin on oxidative stress injury of HepG2 cells and its mechanism

作　　者：金芳多 张天 张钊 尹学哲[1] 全吉淑[1] JIN Fangduo;ZHANG Tian;ZHANG Zhao;YIN Xuezhe;QUAN Jishu(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Yanbian University,Yanji 133002,China)

机构地区：[1]延边大学基础医学院生物化学与分子生物学教研室,吉林延吉133002

出　　处：《吉林大学学报（医学版）》2020年第6期1117-1123,I0001,共8页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(81760659,81360651)。

摘　　要：目的:探讨芦丁对肝癌HepG2细胞氧化应激损伤的保护作用,并阐明其作用机制。方法:培养人肝癌HepG2细胞,取处于对数生长期的HepG2细胞分为对照组、叔丁基过氧化氢(TBHP)组(给予400μmol·L^-1TBHP)和0.25、0.50、1.00μmolL^-1芦丁组(给予0.25、0.50和1.00μmol·L^-1芦丁+400μmol·L^-1 TBHP)。采用四甲基偶氮唑盐(MTT)法检测各组HepG2细胞活性和各组肝癌HepG2细胞存活率,比色法检测各组肝癌HepG2细胞中丙二醛(MDA)和还原型谷胱甘肽(GSH)水平及超氧化物歧化酶(SOD)活性,采用二氯荧光素乙酰乙酸盐(DCFH-DA)荧光染色法检测各组肝癌HepG2细胞中活性氧(ROS)水平,Western blotting法检测各组肝癌HepG2细胞中磷脂酰肌醇3激酶(PI3K)、磷酸化磷脂酰肌醇3激酶(p-PI3K)、蛋白激酶B(Akt)、磷酸化AktB(p-Akt)、核转录因子κB(NF-κB)、磷酸化NF-κB(p-NF-κB)和p53蛋白表达水平。结果:MTT检测,芦丁摩尔浓度≤1μmol·L^-1时,肝癌HepG2细胞的存活率均>90%,不显示细胞毒性;当芦丁摩尔浓度>1μmol·L^-1时,肝癌HepG2细胞存活率逐渐降低,均<90%,显示细胞毒性。与对照组比较,TBHP组HepG2细胞存活率明显降低(P<0.01);与TBHP组比较,不同浓度芦丁组HepG2细胞存活率明显升高(P<0.01)。比色法检测,与对照组比较,TBHP组肝癌HepG2细胞中MDA水平明显升高(P<0.01),GSH水平和SOD活性明显降低(P<0.01);与TBHP组比较,不同浓度芦丁组肝癌HepG2细胞中MDA水平明显降低(P<0.01),GSH水平和SOD活性明显升高(P<0.01)。DCFH-DA染色,与对照组比较,TBHP组肝癌HepG2细胞中ROS水平明显升高;与TBHP组比较,不同浓度芦丁组肝癌HepG2细胞中ROS水平降低。Western blotting法检测,与对照组比较,TBHP组肝癌HepG2细胞中PI3K、p-PI3K、Akt和p-Akt蛋白表达水平降低(P<0.05),NF-κB、p-NF-κB和p53蛋白表达水平升高(P<0.05);与TBHP组比较,不同浓度芦丁组肝癌HepG2细胞中PI3K、p-PI3K、Akt和p-Akt蛋白表达水平升高(P<0.05),NF-κB、p-NObjective:To explore the protective effect of rutin on the oxidative stress injury of liver cancer HepG2 cells,and to clarify its mechanism.Methods:The liver cancer HepG2 cells in the logarithmic growth phase were divided into control group,tert-butyl hydroperoxide(TBHP)group(400μmol·L^-1 TBHP),0.25,0.50,and 1.00μmol·L^-1 rutin groups(0.25,0.50,1.00μmol·L^-1 rutin+400μmol·L^-1 TBHP).The vitalities of liver cancer HepG2 cells in various groups and the survival rates of liver cancer HepG2 cells in various groups were detected by MTT assay;the levels of malondialdehyde(MDA)and glutathione(GSH)and activities of superoxide dismutase(SOD)in the liver cancer HepG2 cells in various groups were assessed by colorimetric method;the levels of reactive oxidative species(ROS)in the liver cancer HepG2 cells in various groups were quantified with DCFH-DA immnofluorescence staining.The expression levels of PI3K,p-PI3K,Akt,p-Akt,NF-κB,p-NF-κB,and p53 proteins in the liver cancer HepG2 cells in various groups were mesured by Western blotting method.Results:The MTT results showed that when the concentrations of rutin were less than or equal to 1.00μmol·L^-1,the vitality of liver cancer HepG2 cells was higher than 90%,and the rutin had no toxic effect;when the concentration of rutin was higher than 1.00μmol·L^-1,the vitality of the liver cancer HepG2 cells was lower than 90%,and the rutin had toxic effect.Compared with control group,the survival rate of the HepG2 cells in TBHP group was significantly decreased(P<0.01);compared with TBHP group,the survival rates of the HepG2 cells in different concertrations of rutin groups were significantly increased(P<0.01).Compared with control group,the level of MDA in the HepG2 cells in TBHP group was significantly decreased(P<0.01),the level of GSH and the activity of SOD were significantly increased(P<0.01);compared with TBHP group,the levels of MDA in the HepG2 cells in different concentrations of rutin groups were significantly increased(P<0.01),the levels of GSH and the activi

关 键 词：芦丁 叔丁基过氧化氢 氧化应激 肝细胞损伤 肝细胞保护 

分 类 号：R282.7[医药卫生—中药学]