Chlorogenic acid analogues from Gynura nepalensis protect H9c2 cardiomyoblasts against H2O2-induced apoptosis
Chlorogenic acid analogues from Gynura nepalensis protect H9c2 cardiomyoblasts against H2O2-induced apoptosis
复制标题
尼泊尔白花菜中的绿原酸类似物保护 H9c2 心肌细胞免受 H2O2 诱导的细胞凋亡
DOI：
10.1038/aps.2016.79
复制
发表时间：
2016-11-01
影响因子：
8.2
通讯作者：
Wang, He-yao
中科院分区：
文献类型：
--
作者：
Yu, Bang-wei;Li, Jin-long;Wang, He-yao
目的：绿原酸对心肌细胞氧化应激损伤具有保护作用。在此，我们评估了从尼泊尔Gynura nepalensis叶片中分离的9种咖啡酰奎宁酸类似物（1-9）对h2o2诱导的H9c2心肌细胞损伤的保护作用，并探讨了其潜在机制。方法：将H9c2成心肌细胞暴露于H2O2 （0.3 mmol/L）中3 h，采用MTT法检测细胞活力。Hoechst 33342染色检测细胞凋亡情况。使用JC-1检测试剂盒检测线粒体膜电位（MMPs），使用CM-H(2)DCFDA检测活性氧（ROS）生成。Western blot检测相关蛋白的表达水平。结果：H2O2显著降低H9c2细胞活力和过氧化氢酶活性，增加LDH释放和细胞内ROS生成；伴有MMP的丢失和凋亡率的增加。在9种绿原酸类似物及阳性对照药表没食子儿茶素没食子酸酯（EGCG）中，化合物6（3,5-二咖啡酰奎宁酸乙酯）对h2o2诱导的H9c2细胞死亡的保护效果最好。以化合物6 （1.56 ~ 100 μ mol/L）剂量依赖性处理可减轻h2o2诱导的所有不良反应。此外，暴露于H2O2显著增加Bax、p53、cleaved caspase-8、cleaved caspase-9水平，降低Bcl-2水平，导致细胞凋亡。H2O2暴露也显著增加了H9c2细胞中p38、JNK和ERK的磷酸化。化合物6（12.5和25 μ mol/L）预处理对h2o2诱导的裂解型caspase-9水平的升高具有剂量依赖性，但对裂解型caspase-8水平的升高没有抑制作用。它还能剂量依赖性地抑制h2o2诱导的JNK和ERK的磷酸化，但不抑制p38的磷酸化。结论：从尼泊尔山竹叶中分离得到的化合物6对h2o2诱导的H9c2心肌细胞凋亡具有保护作用，其机制可能与抑制内源性凋亡和ERK/JNK通路有关。
Aim: Chlorogenic acid has shown protective effect on cardiomyocytes against oxidative stress-induced damage. Herein, we evaluated nine caffeoylquinic acid analogues (1-9) isolated from the leaves of Gynura nepalensis for their protective effect against H2O2-induced H9c2 cardiomyoblast damage and explored the underlying mechanisms.Methods: H9c2 cardiomyoblasts were exposed to H2O2 (0.3 mmol/L) for 3 h, and cell viability was detected with MTT assay. Hoechst 33342 staining was performed to evaluate cell apoptosis. MMPs (mitochondrial membrane potentials) were measured using a JC-1 assay kit, and ROS (reactive oxygen species) generation was measured using CM-H(2)DCFDA. The expression levels of relevant proteins were detected using Western blot analysis.Results: Exposure to H2O2 markedly decreased the viability of H9c2 cells and catalase activity, and increased LDH release and intracellular ROS production; accompanied by a loss of MMP and increased apoptotic rate. Among the 9 chlorogenic acid analogues as well as the positive control drug epigallocatechin gallate (EGCG) tested, compound 6 (3,5-dicaffeoylquinic acid ethyl ester) was the most effective in protecting H9c2 cells from H2O2-induced cell death. Pretreatment with compound 6 (1.56-100 mu mol/L) dose-dependently alleviated all the H2O2-induced detrimental effects. Moreover, exposure to H2O2 significantly increased the levels of Bax, p53, cleaved caspase-8, and cleaved caspase-9, and decreased the level of Bcl-2, resulting in cell apoptosis. Exposure to H2O2 also significantly increased the phosphorylation of p38, JNK and ERK in the H9c2 cells. Pretreatment with compound 6 (12.5 and 25 mu mol/L) dose-dependently inhibited the H2O2-induced increase in the level of cleaved caspase-9 but not of cleaved caspase-8. It also dose-dependently suppressed the H2O2-induced phosphorylation of JNK and ERK but not that of p38.Conclusion: Compound 6 isolated from the leaves of Gynura nepalensis potently protects H9c2 cardiomyoblasts against H2O2-induced apoptosis, possibly by inhibiting intrinsic apoptosis and the ERK/JNK pathway.