DHF对过氧化氢诱导H9c2心肌细胞损伤保护作用(1)
[摘要] 目的 探討7，8-二羟基黄酮(DHF)对过氧化氢(H2O2)诱导的H9c2心肌细胞氧化应激损伤的保护作用及机制。
方法首先观察DHF对H2O2诱导的H9c2细胞损伤的保护作用(实验1)，H9c2细胞分为对照组(无药物处理)、H2O2组(700 μmol/L H2O2孵育24 h)、DHF+H2O2组(1、5、10、20 μmol/L DHF预处理1 h，700 μmol/L H2O2孵育24 h)、DHF组(20 μmol/L DHF孵育24 h)。为观察各组Akt 蛋白水平的改变(实验2)，H9c2细胞分为对照组、H2O2组(700 μmol/L H2O2孵育24 h)、DHF+H2O2组(10 μmol/L DHF预处理1 h，余同H2O2组)、LY294002+DHF+H2O2组(先加入10 μmol/L LY294002孵育30 min，其余处理同DHF+H2O2组)、DHF组(10 μmol/L DHF孵育24 h)。为观察PI3K阻断剂LY294002对DHF保护作用的影响(实验3)，实验分5组：前4组分组及处理方法同实验2，第5组为LY294002组(加入10 μmol/L LY294002孵育24 h)。采用MTT法测定各组细胞存活率，Western blot法检测p-Akt蛋白表达。
结果实验1结果表明，H2O2处理后细胞存活率降低至(54.1±5.8)%，而5～20 μmol/L DHF发挥明显的细胞保护作用(n=6，F=16.50，q=1.95～4.76，P<0.05)。实验2结果显示，H2O2能显著减少p-Akt蛋白表达，而DHF预处理上调了p-Akt蛋白水平，该作用可被PI3K抑制剂LY294002所阻断，单独使用DHF对Akt的磷酸化无明显影响(n=3，F=18.67，q=6.17～10.47，P<0.01)。实验3结果表明，与对照组比较，H2O2组细胞存活率明显降低，该作用可被10 μmol/L DHF所拮抗，而DHF的保护作用可部分被LY294002所阻断(n=6，F=44.75，q=8.52～17.16，P<0.01)。
结论DHF对H2O2诱导H9c2心肌细胞损伤具有明显的保护作用，该作用可能与PI3K/Akt 信号通路激活密切相关。
[关键词] 7，8-二羟基黄酮;过氧化氢;氧化性应激;肌细胞，心脏
[中图分类号] R541
[文献标志码] A
[文章编号] 2096-5532(2019)01-0035-05
PROTECTIVE EFFECT OF 7，8-DIHYDROXYFLAVONE AGAINST H2O2-INDUCED INJURY IN H9c2 CARDIOMYOCYTES
LU Chang， SONG Yu， ZHU Wenzhen， FENG Manman， LI Jiawei， HAN Xiaohua
(Department of Physiology， School of Basic Medicine， Qingdao University， Qingdao 266071， China)
[ABSTRACT]ObjectiveTo investigate the protective effect of 7，8-dihydroxyflavone (DHF) against H2O2-induced oxidative stress injury in H9c2 cardiomyocytes and related mechanism.
MethodsIn order to observe the protective effect of DHF against H2O2-induced injury in H9c2 cardiomyocytes (experiment 1)， H9c2 cardiomyocytes were divided into control group (without drug treatment)， H2O2 group (treated with 700 μmol/L H2O2 for 24 h)， DHF+H2O2 group (pretreated with 1，5，10， and 20 μmol/L DHF for 1 h， followed by the treatment with 700 μmol/L H2O2 for 24 h)， and DHF group (treated with 20 μmol/L DHF for 24 h). In order to investigate the change in the level of Akt protein (experiment 2)， H9c2 cardiomyocytes were divided into control group， H2O2 group (treated with 700 μmol/L H2O2 for 24 h)， DHF+H2O2 group (pretreated with 10 μmol/L DHF for 1 h， followed by the same treatment as the H2O2 group)， LY294002+DHF+H2O2 group (firstly treated with 10 μmol/L LY294002 for 30 min， followed by the same treatment as the DHF+H2O2 group)， and DHF group (treated with 10 μmol/L DHF for 24 h). In order to investigate the effect of LY294002， a PI3K antagonist， on the protective effect of DHF (experiment 3)， H9c2 cardiomyocytes were divided into five groups; the former four groups and their treatment were the same as experiment 2 and the fifth group was LY294002 group (treated with 10 μmol/L LY294002 for 24 h). MTT assay was used to measure cell viability， and Western blot was used to measure the protein expression of Akt., http://www.100md.com(卢畅 宋妤 朱文珍 冯慢慢 李佳玮 韩晓华)
方法首先观察DHF对H2O2诱导的H9c2细胞损伤的保护作用(实验1)，H9c2细胞分为对照组(无药物处理)、H2O2组(700 μmol/L H2O2孵育24 h)、DHF+H2O2组(1、5、10、20 μmol/L DHF预处理1 h，700 μmol/L H2O2孵育24 h)、DHF组(20 μmol/L DHF孵育24 h)。为观察各组Akt 蛋白水平的改变(实验2)，H9c2细胞分为对照组、H2O2组(700 μmol/L H2O2孵育24 h)、DHF+H2O2组(10 μmol/L DHF预处理1 h，余同H2O2组)、LY294002+DHF+H2O2组(先加入10 μmol/L LY294002孵育30 min，其余处理同DHF+H2O2组)、DHF组(10 μmol/L DHF孵育24 h)。为观察PI3K阻断剂LY294002对DHF保护作用的影响(实验3)，实验分5组：前4组分组及处理方法同实验2，第5组为LY294002组(加入10 μmol/L LY294002孵育24 h)。采用MTT法测定各组细胞存活率，Western blot法检测p-Akt蛋白表达。
结果实验1结果表明，H2O2处理后细胞存活率降低至(54.1±5.8)%，而5～20 μmol/L DHF发挥明显的细胞保护作用(n=6，F=16.50，q=1.95～4.76，P<0.05)。实验2结果显示，H2O2能显著减少p-Akt蛋白表达，而DHF预处理上调了p-Akt蛋白水平，该作用可被PI3K抑制剂LY294002所阻断，单独使用DHF对Akt的磷酸化无明显影响(n=3，F=18.67，q=6.17～10.47，P<0.01)。实验3结果表明，与对照组比较，H2O2组细胞存活率明显降低，该作用可被10 μmol/L DHF所拮抗，而DHF的保护作用可部分被LY294002所阻断(n=6，F=44.75，q=8.52～17.16，P<0.01)。
结论DHF对H2O2诱导H9c2心肌细胞损伤具有明显的保护作用，该作用可能与PI3K/Akt 信号通路激活密切相关。
[关键词] 7，8-二羟基黄酮;过氧化氢;氧化性应激;肌细胞，心脏
[中图分类号] R541
[文献标志码] A
[文章编号] 2096-5532(2019)01-0035-05
PROTECTIVE EFFECT OF 7，8-DIHYDROXYFLAVONE AGAINST H2O2-INDUCED INJURY IN H9c2 CARDIOMYOCYTES
LU Chang， SONG Yu， ZHU Wenzhen， FENG Manman， LI Jiawei， HAN Xiaohua
(Department of Physiology， School of Basic Medicine， Qingdao University， Qingdao 266071， China)
[ABSTRACT]ObjectiveTo investigate the protective effect of 7，8-dihydroxyflavone (DHF) against H2O2-induced oxidative stress injury in H9c2 cardiomyocytes and related mechanism.
MethodsIn order to observe the protective effect of DHF against H2O2-induced injury in H9c2 cardiomyocytes (experiment 1)， H9c2 cardiomyocytes were divided into control group (without drug treatment)， H2O2 group (treated with 700 μmol/L H2O2 for 24 h)， DHF+H2O2 group (pretreated with 1，5，10， and 20 μmol/L DHF for 1 h， followed by the treatment with 700 μmol/L H2O2 for 24 h)， and DHF group (treated with 20 μmol/L DHF for 24 h). In order to investigate the change in the level of Akt protein (experiment 2)， H9c2 cardiomyocytes were divided into control group， H2O2 group (treated with 700 μmol/L H2O2 for 24 h)， DHF+H2O2 group (pretreated with 10 μmol/L DHF for 1 h， followed by the same treatment as the H2O2 group)， LY294002+DHF+H2O2 group (firstly treated with 10 μmol/L LY294002 for 30 min， followed by the same treatment as the DHF+H2O2 group)， and DHF group (treated with 10 μmol/L DHF for 24 h). In order to investigate the effect of LY294002， a PI3K antagonist， on the protective effect of DHF (experiment 3)， H9c2 cardiomyocytes were divided into five groups; the former four groups and their treatment were the same as experiment 2 and the fifth group was LY294002 group (treated with 10 μmol/L LY294002 for 24 h). MTT assay was used to measure cell viability， and Western blot was used to measure the protein expression of Akt., http://www.100md.com(卢畅 宋妤 朱文珍 冯慢慢 李佳玮 韩晓华)