Figure 5

Digitoflavone activates the Nrf2/ARE axis and induces cytoprotective effect via the p38 MAPK pathway. (A) Effects of p38 signaling inhibitoγ-SB202190 on digitoflavone-induced Nrf2 expression. Caco-2 cells were pretreated with various concentration of p38 signaling inhibitors-SB202190 for 2 hours then treated with 10 μM digitoflavone for an additional 8 hours. Nrf2, γ-GCSc and TR protein expression was determined using Western blot analysis. (B) Effects of p38 signaling inhibitor-SB202190 on digitoflavone-induced Nrf2 translocation. Caco-2 cells were pretreated with 10 μM SB202190 for 2 hours then treated with 10 μM digitoflavone for 0, 4, 8 hours. Nrf2 in nucleus (N), cytoplasm (C) were determined using Western blotting and appropriate specific antibodies. Lamin B and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. (C) Cell viability analysis. Caco-2 cells were preincubated with or without 10 μM SB202190 then treated with various concentrations of digitoflavone for 4 hours before espoused to 500 μM H₂O₂ for an additional 24 hours. Cell viability was determined using MTT assay. (D) Flow cytometry analysis of the intracellular ROS. Caco-2 cells were preincubated with or without 10 μM SB202190 then treated with various concentrations of digitoflavone for 4 hours before espoused to 500 μM H₂O₂ for an additional 4 hours. Intracellular ROS levels were measured using DCF fluorescence. (E) Statistical analysis of the flow cytometry date. (F) Flow cytometry analysis of the apoptotic rate. Caco-2 cells were preincubated with or without 10 μM SB202190 then treated with various concentrations of digitoflavone for 4 hours before espoused to 500 μM H₂O₂ for an additional 6 hours. cells were stained with FITC-Annexin V-PI, flow cytometry measured the apoptotic rate. (G) Statistical analysis of the apoptotic rate. The values represent the means ± SD of triplicate experiments. Significantly different: *p<0.05, **p<0.01.