Ghrelin protects H9c2 cells from hydrogen peroxide-induced apoptosis through NF-?B and mitochondria-mediated signaling
Abstract
Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. Herein we investigate the protective effects of ghrelin in H(2)O(2)-induced apoptosis of H9c2 cells, as well as the possible molecular mechanisms involved. To study apoptosis, the cells were assessed by morphologic examination, MTS assay, Annexin V-propidium iodide dual staining and TUNEL analysis. Intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential were also measured. To investigate the underlying molecular mechanisms, the expression of Bcl-2, Bax, active caspase-9 and NF-?B were assessed by Western blotting, and caspase-3 activity was determined by a colorimetric activity assay kit. After stimulation with H(2)O(2) for 18h, H9c2 cells viability decreased significantly; a large fraction of cells underwent apoptosis. We observed a dose-dependent rescue of H9c2 cells from H(2)O(2)-induced apoptosis in the presence of different ghrelin concentrations. Preincubation with ghrelin also restored the ROS and mitochondrial membrane potential levels that had been altered by H(2)O(2) treatment. Moreover, ghrelin decreased H(2)O(2)-induced Bax production and caspase-9 activation, and increased Bcl-2 levels. NF-?B phosphorylation was also significantly inhibited by ghrelin in H(2)O(2)-treated cells. Caspase-3 activation was suppressed by ghrelin in H(2)O(2)-treated H9c2 cells in a dose-dependent manner. In summary, ghrelin protects H9c2 cells from oxidative stress-induced apoptosis through downregulation of Bax expression, caspase-9 activation and NF-?B phosphorylation, and upregulation of Bcl-2 expression. Caspase-3 activation was also reduced in a dose-dependent manner. These data suggest that ghrelin might protect against cardiovascular disease by protecting the mitochondria.
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