Chlorogenic acid analogues from Gynura nepalensis protect H9c2 cardiomyoblasts against H2O2-induced apoptosis.
OBJECTIVE
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-H2DCFDA. The expression levels of relevant proteins were detected using Western blot analysis.


RESULTS
Exposure to H2O2markedly 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 μmol/L) dose-dependently alleviated all the H2O2-induced detrimental effects. Moreover, exposure to H2O2significantly 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 H2O2also significantly increased the phosphorylation of p38, JNK and ERK in the H9c2 cells. Pretreatment with compound 6 (12.5 and 25 μmol/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.


CONCLUSIONS
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.
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|College of Pharmacy, Nanchang University, Nanchang 330038, China.
Bang-Wei
BW
Yu
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
Jin-Long
JL
Li
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
Bin-Bin
BB
Guo
Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, China.
Hui-Min
HM
Fan
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Wei-Min
WM
Zhao
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
He-Yao
HY
Wang
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D019659:Asteraceae / Q000737:chemistry*; D002726:Chlorogenic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D032386:Myoblasts, Cardiac / Q000166:cytology / Q000187:drug effects*; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation; D051381:Rats; D015398:Signal Transduction
D000975:Antioxidants; D002726:Chlorogenic Acid; D006861:Hydrogen Peroxide; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases