Effect of asiaticoside on generation of reactive oxygen species by induction of 4-hydroxy-2-nonenal in ECV-304 cells

Abstract

4-hydroxy-2-nonenal (4-HNE), the most abundant aldehyde product of lipid peroxidation, was shown to exert several effects on pathological processes. Its effects were shown to be tightly associated with an ability to induce intracellular reactive oxygen species (ROS) generation. The purposes of this study were to elucidate the possible cytotoxic role of 4-HNE and underlying mechanisms on ECV-304 cells. Moreover, this study investigated the protective effect of asiaticoside, the major active component of Centella asiatica (Linn.), against 4-HNE mediated cytotoxicity. Exposure with 4-HNE decreased cell viability in a concentration dependent manner over a wide range concentrations (0-40 μM) analyzed by MTT assay. In addition, Hoechst 33342 and propidium iodide staining assay showed a significant increase (P < 0.05) in apoptotic and necrotic cells at 12 hours. Addition of antioxidant, N-acetyl-L-cysteine (NAC) or glutathione (GSH), significantly inhibited (P < 0.05) 4-HNE induced cytotoxicity measured by MTT assay, apoptotic cell death analyzed by Hoechst 33342 staining assay, and necrotic cell death determined by propidium iodide staining assay. Flow cytometric analysis of ROS-specific 2’, 7’ –dichlorodihydrofluorecein diacetate fluorescent dye indicated that 4-HNE treatment strongly significant up-regulated (P < 0.05) intracellular ROS, suggesting the involvement of ROS on 4-HNE induced cytotoxicity. However, asiaticoside which has been reported to be an antioxidant showed antioxidant effect only at high concentrations (200 µM) in this study. These findings provided evidence that antioxidant effect of asiaticoside might not be involved in the protection of ECV-304 cells against 4-HNE induced toxicity. Nevertheless, the precise mechanisms of asiaticoside in protection of 4-HNE induced cytotoxicity remain to be defined.