摘要
为了探讨木犀草素能否通过再灌注损伤挽救激酶(Reperfusion injury salvage kinase,RISK)细胞信号通路发挥抗心肌氧化应激损伤保护作用,本实验分别用1、50、100、150μmol/L的木犀草素预处理大鼠心肌来源的H9c2细胞,再使用650μmol/L的H_2O_2制作氧化应激损伤模型。利用MTT法(四甲基偶氮唑盐比色法)检测细胞存活率,然后用最适浓度的木犀草素预处理H9c2细胞并利用激光共聚焦显微镜技术检测线粒体膜电位,Western blot检测P-ERK1/2、P-Akt、P-GSK-3β以及凋亡相关蛋白细胞色素C。最后我们发现不同浓度的木犀草素预处理均能提高细胞存活率,其中在100μmol/L时达到最佳效应。与H_2O_2组相比,100μmol/L的木犀草素预处理可以使TMRE(四甲基罗丹明乙酯)强度降低程度明显减轻,对抗H_2O_2引起的细胞氧化损伤。同时木犀草素预处理可以降低细胞色素C表达,使P-GSK-3β、P-Akt、P-ERK1/2表达升高,渥曼青霉素(PI3K抑制剂)和PD98059(ERK1/2抑制剂)可以阻断这种作用。因此我们认为木犀草素预处理可以减轻H_2O_2引起的氧化应激损伤,这一作用可能是通过RISK信号通路增加P-GSK-3β表达,抑制m PTP开放实现的。
In order to investigate whether luteolin can protect heart against myocardial oxidative stress injury via reperfusion injury salvage kinase(RISK)signaling pathway we use myocardial H9c2 cells were pretreated with luteolin at 1,50,100 and 150μmol/L respectively,while cell oxidative stress injury was induced by adding 650μmol/L H 2O 2.Cell viability was detected by MTT assay(MTT assay).Then,H9c2 cells were pretreated with luteolin at the optimal concentration and mitochondrial membrane potential was detected by laser confocal microscopy.Western blot was used to detect the level of P-ERK1/2,P-Akt,P-GSK-3βand apoptosis-related protein Cytochrome c.Finally we found that compared with the control group,cells pretreated with different concentrations of luteolin showed an increase in cell survival rate,which reached the pink at 100μmol/L.Compared with H 2O 2 group,pretreatment with 100μmol/L luteolin could significantly reduce the intensity of TMRE(tetramethylrhodamine ethyl ester)and prevent oxidative damage of cells induced by H 2O 2.At the same time,luteolin pretreatment could decrease the expression of Cytochrome C,but increase the expression of P-GSK-3β,P-Akt and P-ERK1/2,which were inhibited by wortmannin(PI3K inhibitor)and PD98059(ERK1/2 inhibitor).So we think that Luteolin preconditioning reduces oxidative stress injury by decreasing GSK-3βactivity via RISK signaling pathway and then inhibiting mPTP opening.
作者
肖童
钟烨
孙雅涵
韩学超
徐森
何玛莉
徐菁蔓
田炜
XIAO Tong;ZHONG Ye;SUN Ya-han;HAN Xue-chao;XU Sen;HE Ma-li;XU Jing-man;TIAN Wei(Medical Research Center,North China University of Science and Technology;Affiliated Hospital of North China University of Science and Technolog;International Scientiec and Technology Cooperation Base of Geriatric Medicine;Tangshan Key Laboratory of Geriatric Medicine,Tangshan 063000,China)
出处
《天然产物研究与开发》
CAS
CSCD
北大核心
2018年第9期1494-1501,共8页
Natural Product Research and Development
基金
国家自然科学基金(81700324)
华北理工大学大学生创新项目(X2017365)