Fisetin Supplementation Attenuates Premature Vascular Aging Induced by Doxorubicin via Suppression of Cellular Senescence and Mitochondrial Oxidative Stress

Figures

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  FIGURE 1. Treatment with fisetin reduces Doxorubicin (Doxo)‐induced cellular senescence in endothelial cells. Cultured human aortic endothelial cells (HAECs) were treated with Doxo (200 nM) or control for 24 h, treated with fisetin (0, 0.25, 0.5, or 1.0 μM) or control for 48 h, then recovered for 4 days (A). Senescence‐associated β‐galactosidase (SA‐β‐Gal) signal was measured in HAECs following treatments, representative images below (B). Gene expressions of cellular senescence biomarkers were measured in the HAECs following Doxo and 1.0 μM fisetin treatment (C). Data are mean ± SEM; n = 3–4/group; *p < 0.05 versus Control; ‡ p < 0.05 versus Doxo.

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  FIGURE 2. Oral intermittent fisetin supplementation reduces aortic expression of cellular senescence and senescence‐associated secretory phenotype (SASP) markers in Doxorubicin (Doxo)‐treated mice. Young adult (4 months) male and female p16‐3MR mice received a single intraperitoneal injection of sham (saline) or Doxo (10 mg/kg in saline). Four weeks later, animals were treated with vehicle (Veh; 10% ethanol, 30% PEG400, 60% Phosal 50 PG) or fisetin (Fis; 100 mg/kg/day in the vehicle solution) via oral gavage using an intermittent dosing paradigm (A). Aortic gene expression of cellular senescence (B) and SASP (C) markers in Sham‐Veh, Doxo‐Veh, and Doxo‐Fis treated groups. Data are mean ± SEM; n = 9–12/group; *p < 0.05 versus Sham‐Veh, ‡ p < 0.05 versus Doxo‐Veh.

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  FIGURE 3. Oral intermittent fisetin supplementation improves endothelial dysfunction caused by Doxorubicin (Doxo) administration by suppressing excess cellular senescence. Endothelium‐dependent dilation (EDD) to increasing doses of acetylcholine (ACh) (A). Peak EDD following ex vivo ganciclovir (GCV, 5 μM) incubation compared with incubation with ACh alone (B). Endothelium‐independent dilation (EID) to the exogenous nitric oxide donor, sodium nitroprusside (SNP) following GCV incubation (C). Data are mean ± SEM; n = 5–7/group; *p < 0.05 versus Sham‐Vehicle, ‡ p < 0.05 versus Doxo‐Vehicle, ^p < 0.05 versus Doxo‐Vehicle ACh alone.

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  FIGURE 4. Oral intermittent fisetin supplementation improves endothelial dysfunction resulting from Doxorubicin (Doxo) administration by increasing nitric oxide (NO) bioavailability. NO–mediated, endothelium‐dependent dilation was calculated after ex vivo incubation with the NO synthase inhibitor L‐NAME as follows: Maximal dilation to acetylcholine (ACh) minus maximal dilation to ACh in the presence of L‐NAME (A). Endothelium‐independent dilation (EID) to the exogenous NO donor, sodium nitroprusside (SNP) (B). Data are mean ± SEM; n = 5–12/group; *p < 0.05 versus Sham‐Vehicle; ^p < 0.05 versus – L‐NAME; ‡ p < 0.05 versus Doxo‐Vehicle.

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  FIGURE 5. Oral intermittent fisetin supplementation restores the Doxorubicin (Doxo)‐induced decrease in endothelial function by lowering mitochondrial oxidative stress. Aortic mitochondrial reactive oxygen species (ROS) levels of the Sham‐Vehicle (Veh), Sham‐Fisetin, Doxo‐Veh, and Doxo‐Fisetin treated groups (A). Peak endothelium‐dependent dilation (EDD) with acetylcholine (ACh) alone versus ACh with MitoQ, a mitochondrial‐targeted antioxidant (B). Data are mean ± SEM; n = 5–7/group; *p < 0.05 versus Sham‐Vehicle, ‡ p < 0.05 versus Doxo‐Vehicle, ^p < 0.05 versus Doxo‐Vehicle ACh alone.

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  FIGURE 6. Oral intermittent fisetin supplementation reverses Doxorubicin (Doxo)‐induced aortic stiffness by suppressing excess cellular senescence and modulating the circulating milieu. In vivo aortic pulse wave velocity (PWV) of treatment groups following sham/Doxo administration (solid) and intermittent vehicle/fisetin supplementation (hatched) (A). Aortic elastic modulus of isolated aorta rings from Sham‐Vehicle, Sham‐Fisetin, Doxo‐Vehicle, and Doxo‐Fisetin treated groups following 48 h incubation in control media alone (solid) or with ganciclovir (GCV, 5 μM; hatched), a genetic senescent cell‐clearing compound in the p16‐3MR mouse model (B). Paired aortic elastic modulus of young, intervention‐naïve mice following plasma exposure from the Sham‐Vehicle, Doxo‐Vehicle, and Doxo‐Fisetin treated mice (C). Data are mean ± SEM; n = 5–13/group; *p < 0.05 versus Sham‐Vehicle, ‡ p < 0.05 versus Doxo‐Vehicle, ^p < 0.05 versus Post‐Doxo.