FIGURE 4.

Fisetin ameliorated H₂O₂-induced mitochondrial dysfunction in ARPE-19 cells. Cells were treated with or without 20 μM fisetin for 1 h and then treated with 0.5 mM H₂O₂ for 24 h (A) mitochondrial membrane potential (MMP) was examined by the change in JC-1-derived fluorescence. Nuclei were counterstained with DAPI (blue). After staining, representative images of JC-1 aggregates (red, representing high potential) and monomers (green, representing low potential) of cells of each treatment group were captured using a fluorescence microscope. Pretreatment of fisetin reversed down-regulation of the ratio of JC-1 aggregates/monomers following H₂O₂-induced MMP depolarization. (B) To evaluate the expression and localization of cytochrome c, mitochondrial (M. F.) and cytoplasmic (C. F.) fractions were isolated from cells and assessed by Western blot analysis. cytochrome c was released from the mitochondria to the cytoplasm by H₂O₂, while pretreatment of fisetin was reversed H₂O₂-induced cytochrome c release. (C) Changes in the expression of Bcl-2, Bax, caspase-3, and PARP were confirmed using the extracted total proteins. (D) Caspase-3 activity was determined using a commercially available kit, and the concentration of p-nitroanilide released by activated caspase-3 was presented as a relative value compared to the control. H₂O₂ down-regulated the expression of anti-apoptotic Bcl-2 protein while up-regulating the expression of pro-apoptotic Bax, and induced the activation of caspase-3 and degradation of poly (ADP-ribose) polymerase (PARP). However, pretreatment of fisetin clearly reversed these H₂O₂-mediated alterations. All bar graphs represent mean ± SD (n = 3, ^* p ˂ 0.05, and ^*** p ˂ 0.001 compared with control group; ^### p ˂ 0.01 compared with H₂O₂ treatment group).