君迁子叶杨梅苷诱导HepG2细胞凋亡及其作用机制

Myricitrin from Diospyros lotus L.Leaves Induced Apoptosis in Hepatocellular Carcinoma HepG2 Cells and Its Underlying Mechanism

目的:探究君迁子叶杨梅苷诱导人肝癌细胞HepG2细胞凋亡及其作用机制。方法:噻唑蓝法测定杨梅苷对细胞存活率的影响;激光共聚焦显微镜结合Hoechst 33342染色观察杨梅苷对细胞形态的影响;流式细胞分析仪检测杨梅苷对细胞凋亡、周期阻滞、胞内活性氧(reactive oxygen species,ROS)水平和单丹璜酰戊二胺(monodansylcadaverine,MDC)荧光强度的影响;Western blot检测杨梅苷对细胞凋亡和自噬相关蛋白表达量的影响。结果:浓度为10~200μmol/L的杨梅苷对人正常肝细胞L-02细胞无显著影响(P>0.05),但可显著降低HepG2细胞的存活率(P<0.05),促进其凋亡,提升ROS水平,增加MDC和细胞核荧光强度,将细胞阻滞在G2/M期;此外,可显著上调HepG2细胞中Bax、细胞色素c、Apaf-1、Caspase-9、Caspase-3、Beclin 1、Atg5和LC3-II的相对表达量(P<0.05),下调Bcl-2和LC3-I的相对表达量(P<0.05)。结论:杨梅苷具有抗肝癌作用,其机制与激活线粒体介导的凋亡通路、阻滞细胞周期、提升ROS水平和促进细胞自噬有关。实验可为杨梅苷作为天然抗肝癌药物的研究及应用提供参考。

Objective:To investigate the effect of myricitrin from Diospyros lotus L.leaves on inducing apoptosis in human hepatocellular carcinoma HepG2 cells and its underlying mechanism.Methods:Cell viability was determined by the methyl thiazolyl tetrazolium assay.Laser confocal microscopy with Hoechst 33342 staining was used to observe cell morphology.Meanwhile,apoptosis,cell cycle arrest,intracellular reactive oxygen species(ROS)levels and monodansylcadaverine(MDC)mean fluorescence intensity in HepG2 cells were detected using a flow cytometer.Finally,Western blot was used to measure the expression levels of proteins associated with apoptosis and autophagy in HepG2 cells.Results:Myricitrin at concentrations of 10–200μmol/L had no significant effect on human normal liver L-02 cells(P>0.05),but could significantly reduce the survival rate of HepG2 cells(P<0.05).Myricitrin could dramatically increase the apoptosis rate,ROS level,MDC mean fluorescence intensity and nuclear fluorescence intensity,and induce cell cycle arrest in the G2/M phase.In addition,myricitrin could up-regulate the expression levels of Bax,cytochrome c,Apaf-1,Caspase-9,Caspase-3,Beclin 1,Atg5 and LC3-II proteins significantly(P<0.05),and down-regulate the expression levels of Bcl-2 and LC3-I proteins significantly(P<0.05).Conclusion:Myricitrin has an anti-liver cancer effect,and its underlying mechanism is related to the activated mitochondrial-mediated apoptosis pathway,cell cycle arrest,increased intracellular ROS levels and promoted autophagy.These results lay a foundation for the development and application of myricitrin as a natural anti-hepatoma drug.