Fisetin

Twelve-week-old male C57BL/6 mice were purchased from Modelorg (Shanghai Model Organisms Company, Shanghai, China). The mice were randomly divided into three groups with each group comprising eight mice as follows: F group (fisetin + MPTP), orally administered with fisetin (100 ng/kg bodyweight) (Sigma-Aldrich, St Louis, United States) for 30 consecutive days; MPTP group (PBS + MPTP), orally administered with phosphate-buffered saline (PBS PH7.4) for 30 consecutive days; normal control group, simultaneous intragastric and intraperitoneal administration of PBS. On day 25 post-PBS/fisetin administration, the F and MPTP groups were intraperitoneally injected with MPTP (30 mg/kg bodyweight) (Sigma-Aldrich, St Louis, United States) once a day for five consecutive days. During administration of MPTP, mice of the fisetin + MPTP group were also given fisetin. All traumatic operations were performed under anesthesia after intraperitoneal injection of pentobarbital sodium (50 mg/kg bodyweight). Every effort was made to minimize the suffering of the animals. This study was approved by the Ethics Committee of Shanghai Model Organisms Company (IACUC No.2018-0005). All experiments were performed in compliance with China’s National Science and Technology Commission Laboratory Animal Regulations.

C2C12 myoblasts (CRL-1772, ATCC, USA) were cultured as previously described [16 (link)]. To analyze the protective effect of fisetin (Sigma-Aldrich Co., USA) on oxidative stress caused by H₂O₂ (Sigma-Aldrich Co.), C2C12 cells were treated with fisetin and H₂O₂ for 24 h, or cells pretreated with fisetin, N-acetyl-L-cysteine (NAC, Thermo Fisher Scientific, USA) or zinc protoporphyrin IX (ZnPP, Sigma-Aldrich Co.) for 1 h were further treated with H₂O₂ for 24 h. To evaluate the inhibitory action of fisetin on ROS accumulation by H₂O₂, cells were treated with fisetin or NAC for 1 h and then treated with H₂O₂ for 1 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Thermo Fisher Scientific) assay was used to investigate the protective potential of fisetin on the suppression of cell viability by H₂O₂ according to the previously described method [17 (link)]. Morphological changes of cells were observed using a phase-contrast microscope (Olympus, Japan). Stock solutions of fisetin and H₂O₂ were prepared by dissolving in dimethyl sulfoxide (DMSO, Sigma-Aldrich Co.), and the highest concentration of DMSO was less than 0.05%, showing no cytotoxicity.

ARPE-19 cells were grown on a 96 well plate overnight and then exposed to room-air or CSE (50%) with appropriate treatments of cysteamine (250μM, Sigma) or fisetin (40 μM, Sigma) for 24 hours. Cells were the incubated with 1.2 mM MTS (Life Technologies) reagent for 1 hour at 37°C and 5% CO₂. Absorbance was then measured at 490 nm and data analyzed by SoftMax Pro 6.0 analysis software. For the assessment of cell viability using propidium iodide (PI) staining, cells were exposed to room-air or CSE (50%) and/or parallel treatments with cysteamine (250μM, Sigma) or fisetin (40 μM, Sigma) for 12 hours. The cells were harvested, washed with PBS (2x) and incubated with PI staining solution (10 μg/ml), and data was immediately acquired using the BD FACSAria flow cytometer using the FL2 (PE) channel. Subsequently, data was analyzed using the BD FACSDiva software as we previously described [24 (link), 25 (link)].