FIGURE 6.

The antioxidative activity and the inhibitory effect on the expression of apoptosis-related factors of fisetin were disappeared in the presence of ZnPP in H₂O₂-treated ARPE-19 cells. Cells exposed to 20 μM fisetin and/or 5 μM ZnPP for 1 h were further treated with 500 μM H₂O₂ for 1 h (A,B) or 24 h (C,D). (A,B) The levels of ROS generation were determined using DCF-DA dye. (A) Representative results from flow cytometry analyses are presented. (B) The graph represents the mean with SD (n = 4, ^* p ˂ 0.05 and ^*** p ˂ 0.001 compared with control group; ^### p ˂ 0.001 compared with H₂O₂ treatment group; ^$$$ p ˂ 0.001 compared with fisetin + H₂O₂ treatment group). Suppression of DCF-positive cells in the cells co-treated with fisetin and H₂O₂ was significantly restored in the presence of HO-1 inhibitor ZnPP. (C–E) The expression change of the indicated protein was confirmed using the extracted whole protein or mitochondrial and cytoplasmic proteins. The down-regulation of the expression of pro-apoptotic Bax, cleaved PARP, cytosolic cytochrome c, and p-γH2AX in the cells co-treated with fisetin and H₂O₂ was significantly restored by ZnPP.