FIGURE 5.

Fisetin activated Nrf2-mediated activation of HO-1 in H₂O₂-treated ARPE-19 cells. Cells were pretreated for 1 h with or without 20 μM fisetin or 5 μM ZnPP, and then treated with 500 μM H₂O₂ for another 24 h. (A) Total protein was isolated and followed by Western blotting was performed using antibodies against Nrf2, p-Nrf2, and HO-1. The expression of Nrf2 and HO1, as well as the phosphorylation of Nrf2 were enhanced by pretreatment of fisetin. (B) After treatment, cells were double-stained with p-Nrf2 (red) and DAPI (blue), and representative immunofluorescence images observed are shown. Phosphorylated form of Nrf2 was over-expressed in the nucleus following pretreatment of fisetin. (C) The activity of HO-1 was measured by the HO-1 activity assay kit. The increased activity of HO-1 in cells co-treated with fisetin and H₂O₂ was significantly restored in the presence of ZnPP, a specific inhibitor for HO-1. Results are presented as mean ± SD (n = 4, ^* p ˂ 0.05 and ^*** p ˂ 0.001, compared with the control group; ^### p ˂ 0.001 compared with H₂O₂ treatment group. ^$$$ p ˂ 0.001 compared with fisetin + H₂O₂ treatment group.