Chlorogenic acid analogues from Gynura nepalensis protect H9c2 cardiomyoblasts against HO-induced apoptosis.

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 HO-induced H9c2 cardiomyoblast damage and explored the underlying mechanisms.

Methods: H9c2 cardiomyoblasts were exposed to HO (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 DCFDA. The expression levels of relevant proteins were detected using Western blot analysis.

Results: Exposure to HO 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 HO-induced cell death. Pretreatment with compound 6 (1.56-100 μmol/L) dose-dependently alleviated all the HO-induced detrimental effects. Moreover, exposure to HO 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 HO also 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 HO-induced increase in the level of cleaved caspase-9 but not of cleaved caspase-8. It also dose-dependently suppressed the HO-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 HO-induced apoptosis, possibly by inhibiting intrinsic apoptosis and the ERK/JNK pathway.