Effects of asiatic acid on isoproterenol induced myocardial hypertrophy

Abstract

Objective To investigate the effects of asiatic acid (AA) on isoproterenol (ISO) induced myocardial hypertrophy. Methods In-vitro cultured rat myocardial cells H9c2 were divided into the control group, ISO group and ISO+AA group. The H9c2 cells in the ISO group were treated with 10 mmol/L ISO to induce cardiomyocyte hypertrophy, those in ISO+AA group with different concentrations of AA and 10 mmol/L ISO, and those in the control group with 0.5% dimethyl sulfoxide (DMSO) as a vehicle control. Real-time quantitative PCR was performed to detect the expressions of atrial natriuretic peptide (ANP) , β-myosin heavy chain (β-MHC) , glutathione peroxidase (GSH-Px) , superoxide dismutase (SOD) and hemoglobin oxygenase 1 (HO-1) genes. Immunofluorescence staining was used to measure cross-sectional area of the cardiomyocytes. Western blot was used to detect the changes in signaling pathway related to myocardial hypertrophy. DCFH-DA fluorescent probe was used to detect the intracellular levels of reactive oxygen species (ROS). Results As shown by real-time quantitative PCR, mRNA expression of cardiac hypertrophy markers ANP and β-MHC in H9c2 cells was up-regulated after 48 h treatment with ISO compared with the control group (both P<0.05) , while 48 h intervention with 5, 10 or 20 μmol/L AA significantly reduced the ISO-induced up-regulation of ANP and β-MHC mRNA expression (all P<0.05) in a concentration-dependent manner; and treatment with 20 μmol/L AA for 12, 24 or 48 h was shown to inhibit ISO-induced ANP and β-MHC mRNA up-regulation in H9c2 cells (all P<0.05). Cross-sectional area of cardiomyocytes as measured by immunofluorescence staining was (1 946±49) μm2 in the control group, (2 952 ± 111) μm2 in the ISO group and (2 548 ± 59) μm2 in the ISO (10 mmol/L)+ AA (20 μmol/L) group. Compared with the control group, cross-sectional area of cardiomyocytes increased in ISO group, while 20 μmol/L AA could significantly reduce the ISO-induced myocardial hypertrophy (both P<0.05). Western blotting indicated that ISO could lead to activation of AKT/GSK3β and ERK/JNK related to cardiac hypertrophy, while 20 μmol/L AA could inhibit the activation of these signaling pathways. DCFH-DA fluorescent probe assay showed that 20 μmol/L AA could significantly inhibit the ISO induced increase in intracellular ROS levels. Real-time quantitative PCR showed that 20 μmol/L AA could inhibit ISO-induced up-regulation of GSH-Px, SOD and HO-1 mRNA expression. Conclusion AA may attenuate oxidative stress by blocking the AKT/GSK3β and ERK/JNK signaling pathways, thereby inhibiting the ISO-induced myocardial hypertrophy. Key words: Asiatic acid; Cardiomyopathy, hypertrophic; Isoprenaline; Oxidative stress