Shikonin attenuates H(2)O(2)-induced oxidative injury in HT29 cells via antioxidant activities and the inhibition of mitochondrial pathway-mediated apoptosis

Shikonin, a natural naphthoquinone extracted from the roots of Lithospermumery throrhizon, possesses multiple pharmacological properties, including antioxidant, anti-inflammatory and antitumor effects. It has been hypothesized that the properties of shikonin are associated with its oxygen free radical scavenging abilities. However, the mechanism underlying the antioxidant activity of shikonin is not completely understood. The aim of the present study was to investigate the effect of shikonin against H(2)O(2)-induced oxidative injury in HT29 cells and to explore the underlying molecular mechanism. The concentration and duration of H(2)O(2) treatment to cause maximal damage, and the effects of shikonin (2.5, 5 or 10 µg/ml) on the activity of H(2)O(2)-induced HT29 cells were determined by MTT assay. The apoptotic rate in HT29 cells was determined by annexin V/propidium iodide staining. HT29 cell cycle alteration was also analyzed by propidium iodide staining. Reactive oxygen species (ROS) production was assessed by monitoring 2',7'-dichlorofluorescin in diacetate fluorescence. Mitochondrial membrane potentials were determined by JC-1 staining. The activities of malondialdehyde, superoxide dismutase, caspase-9 and caspase-3 were measured using spectrophotometric assays. The expression levels of Bcl-2, Bax and cytochrome c were determined by western blotting. The results suggested that shikonin increased cell viability, reduced cell apoptosis and increased the proliferation index in H(2)O(2)-treated HT29 cells. Shikonin also significantly inhibited increases in intracellular reactive oxygen species (ROS), restored the mitochondrial membrane potential, prevented the release of lactic dehydrogenase and decreased the levels of superoxide dismutase and malondialdehyde in H(2)O(2)-induced HT29 cells. Furthermore, shikonin significantly decreased caspase-9 and caspase-3 activities, increased Bcl-2 expression and decreased Bax and cytochrome c expression levels in H(2)O(2)-induced HT29 cells. The results indicated that shikonin protected against H(2)O(2)-induced oxidative injury by removing ROS, ameliorating mitochondrial dysfunction, attenuating DNA oxidative damage and inhibiting mitochondrial pathway-mediated apoptosis.