Rutaecarpine prevents hypoxia-reoxygenation-induced myocardial cell apoptosis via inhibition of NADPH oxidases

It is proposed that myocardial cell apoptosis causes ventricular remodeling and heart failure. The aim of the present study was to determine the effects of rutaecarpine (Rut) on hypoxia-reoxygenation (H-R)-induced apoptosis in myocardial cell line H9c2, as well as the underlying mechanisms. Cultured H9c2 cells were exposed to hypoxia for 24 h, followed by 12 h reoxygenation. Rut (in concentrations of 0.1, 1, and 10 μmol/L) was added 1 h prior to H-R. Cell viability and lactate dehydrogenase were measured to evaluate the cell injuries. Apoptosis was evaluated by Hoechst 33258 staining and flow cytometry. NADPH oxidase activity was measured by assay kit; intracellular reactive oxygen species (ROS) generation was detected by 2',7'-dichlorofluorescein diacetate; and Nox2, Nox4, and p47(phox) mRNA and protein expression were analyzed by real-time PCR and Western blotting, respectively. The results showed that H-R significantly decreased cell viability and increased the lactate dehydrogenase release, as well as the apoptotic rate, concomitantly with enhanced NADPH oxidase activity. H-R also upregulated mRNA and protein expressions of Nox2, Nox4, and p47(phox) and increased ROS production. Treatment with Rut markedly reversed these effects introduced by H-R. These results suggest that the protective effects of Rut against H-R-induced myocardial cell injury and apoptosis might, at least partly, be due to the inhibition of the NADPH oxidase - ROS pathway.