Hydrogen peroxide induces oxidative stress and the mitochondrial pathway of apoptosis in RAT intestinal epithelial cells (IEC-6)

In order to investigate the mechanism of apoptosis in rat intestinal epithelial cells (IEC-6) induced by hydrogen peroxide (H2O2), IEC-6 cells were subjected to 20 μmol/L H2O2 and cell proliferation activity was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide. Cell morphology was observed by microscopy and cell apoptosis was detected by acridine orange and ethidium bromide staining and the portion of apoptotic cells was measured by flow cytometry. Genes and proteins related to cell apoptosis were detected by RT-PCR and Western blotting, and the mitochondrial membrane potential was evaluated by fluorescence probes. Results: Significant morphology damage was caused by exposure to H2O2, and results showed that ROS generation significantly increased (P < 0.01). The activity of superoxide dismutase decreased significantly (P < 0.05), malondialdehyde content increased (P < 0.05), and expression of both catalase and glutathione peroxidase decreased significantly (P < 0.05) in the H2O2 treatment group. Mitochondrion membrane potential was reduced, cytochrome released into the cytoplasm and caspase-9 and caspase-3 were significantly increased (P < 0.01) after treatment with H2O2. Moreover, the ratio of Bax/Bcl-2 and apoptosis were significantly increased (P < 0.01) in the H2O2 group. In conclusion, the present study indicated that the mitochondrial pathway plays a vital role in H2O2 induced IEC-6 cell apoptosis.