Centella asiatica fraction-3 suppresses Nrf-2 antioxidant pathway and enhances apoptotic cell death in HEK293 kidney cells

Abstract BackgroundThe traditional medicinal plant Centella asiatica (C. asiatica) is commonly used in Chinese and Ayuvedic medicine due to its vast range of therapeutic properties. Previously, the ethanolic C. asiatica leaf extract was subjected to silica column fractionation and the C3 fraction was obtained. We investigated the antioxidant and anti-proliferative effects of C3 in embryonic kidney (HEK293) cells.MethodsIn HEK293 cells, C3 cytotoxicity was assessed (Viability assay; 24 hours; [0.2–3 mg/ml]) and a half maximal inhibitory concentration (IC50) determined. Malondialdehyde (MDA), lactate dehydrogenase (LDH) (spectrophotometry), mitochondrial depolarization (Δψm), intracellular reactive oxygen species (flow cytometry), glutathione (GSH), oxidized glutathione (GSSG) concentrations, caspase activities, ATP levels (luminometry) and fragmentation of DNA (SCGE assay) were evaluated. Protein expressions were assessed by western blotting. Gene expressions were quantified by qPCR.ResultsCell viability in HEK293 cells was decreased in a dose dependent manner by C3. MDA, Δψm, LDH, caspase activities and DNA fragmentation (p < 0.0004) were significantly increased by C3. Nrf-2 protein expression, GSH and GSSG concentrations were increased whereas antioxidant (Nrf-2, GPx, SOD and CAT) gene expression was significantly decreased by C3 (p < 0.001). C3 decreased both Bax and Bcl-2 protein expression (p < 0.03). Gene expression of c-myc was significantly increased whereas OGG-1 was significantly reduced by C3 (p < 0.05).ConclusionCentella asiatica extract, C3, reduced antioxidant gene expression and elevated anti-proliferative effects in HEK293 cells, suggesting that C3 is potentially toxic to kidney cells at high concentrations thus rendering cause for concern from its association with human use.