抄録
Background: The benefit of dietary n-3 polyunsaturated fatty acids (PUFAs) in the reduction of cardiovascular disease has been recognized for many years. Epoxydocosapentanoic acids (EDPs) are lipid mediators produced from the metabolism of docosahexaenoic acids (DHA) by cytochrome P450 epoxygenase. In this study, we investigated the mechanism of the effect of DHA and 19,20-EDP on H9c2 cells cultured under glycolytic and OXPHOS conditions.
Methods: H9c2 cells were cultured in DMEM containing either normal (25 mM) or low (5.5 mM) glucose to change the metabolic state. Cells were treated with or without 19,20-EDP (1 μM), DHA (100 μM), MSPPOH (50 μM) or myriocin (1 μM) for 24 h. Cellular viability, caspase-3 activity, ATP production and autophagic response were assessed by fluorometric assay. Ceramide levels were measured by LC/MS after cell fractionation. Mitochondrial function was determined by assessing respiration and enzymatic activities of complex IV and citrate synthase.
Results: H9c2 cells cultured under normal glucose conditions had lower respiration rates indicating a more glycolytic phenotype compared to cells under low glucose conditions (OXPHOS). DHA and 19,20-EDP decreased cell viability, ATP production and mitochondrial function. Inhibition of de novo synthesis of ceramide by myriocin blocked the cell injury effects. Moreover, DHA and 19,20-EDP increased caspase-3. Importantly, MSPPOH, a CYP epoxygenase inhibitor, attenuated DHA-induced effects in cells treated under normal glucose conditions, indicating the CYP-derived metabolite, 19,20-EDP, is the active lipid mediator.
Conclusion: Our data suggest that 19, 20-EDP induces a cell death associated with autophagy due to ceramide-induced mitochondrial dysfunction under normal glucose conditions. Thus reflecting a differential response where 'aerobic glycolytic' states are more susceptible to effects observed in conditions of cells under more 'oxidative phosphorylation' states. Thus, 19,20-EDP has different effects on H9c2 cell survival depending on the metabolic state.
