Tier 3 — preclinical

Intermittent Supplementation With Fisetin Improves Physical Function and Decreases Cellular Senescence in Skeletal Muscle With Aging: A Comparison to Genetic Clearance of Senescent Cells and Synthetic Senolytic Approaches

Kevin O. Murray, Sophia A. Mahoney, Katelyn R. Ludwig, Jill H. Miyamoto-Ditmon, Nicholas S. VanDongen, Nirad Banskota, Allison B. Herman, Douglas R. Seals, Robert T. Mankowski, Matthew J. Rossman, Zachary S. Clayton

Aging Cell 2025 24(8):e70114

Bibliography

DOI: 10.1111/acel.70114
PubMed: PMID 40437670
PubMed Central: PMC12341784
Funding: Supported by U.S. NIH and American Heart Association, and VIVA Physician Awards: F32HL167552 & 23POST1025630 (K.O.M.), K99HL159241 (Z.S.C.), F31HL165885 (S.A.M.), 23CDA1053582 (M.J.R.), R01AG055822 & R01AG055822-04S1 (D.R.S. & Z.S.C.), plus the Intramural Research Program of the National Institute of Aging (A.B.H. & N.B.).
Competing interests: The authors declare no conflicts of interest.

Study snapshot

Design	Preclinical randomised controlled study in aged mice with three parallel intervention arms (fisetin, genetic senolysis via p16-3MR/GCV, synthetic senolysis via ABT-263)
Model	C57BL/6N wildtype and p16-3MR mice; young 6–8 months, old males 27 months, old females (p16-3MR only) 29 months
Sample	Fisetin arm: Young Veh N=9, Young Fisetin N=11, Old Veh N=31, Old Fisetin N=39. GCV arm: Young Veh N=18, Young GCV N=17, Old Veh N=18, Old GCV N=20. ABT-263 arm: Young Veh N=18, Young ABT-263 N=11, Old Veh N=15, Old ABT-263 N=10.
Intervention	Fisetin 50 mg/kg/day via oral gavage, 1 week on – 2 weeks off – 1 week on. Vehicle: 10% EtOH, 30% PEG400, 60% Phosal 50 PG.
Duration	Fisetin and ABT-263 arms: 4-week intermittent intervention (1 week on – 2 weeks off – 1 week on), with in vivo outcome assessment 3–4 weeks after intervention completion (total ~7–8 weeks per animal). p16-3MR/GCV arm: 5 consecutive days of intraperitoneal GCV (25 mg/kg), with outcome assessment 3–4 weeks after (total ~4–5 weeks per animal).
Endpoints	31-point clinical frailty index (Whitehead 2014); Forelimb grip strength normalised to body weight; Skeletal muscle mass (soleus, gastrocnemius, tibialis anterior, quadriceps); Bulk RNA sequencing of quadriceps skeletal muscle with KEGG pathway analysis; RT-PCR of Cdkn1a (p21), Cdkn2a (p16), Pai1, Lmnb1; BayesPrism cell-type deconvolution analysis

What the study showed, in plain terms

Murray and colleagues at the University of Colorado Boulder tested whether intermittent oral fisetin — 50 mg/kg/day, one week on, two weeks off, one week on — could reduce frailty and preserve grip strength in ageing mice. The study ran three parallel intervention arms in old (27-month) mice: fisetin, a genetic senolytic (p16-3MR mice given ganciclovir to selectively clear p16-positive senescent cells), and the synthetic senolytic ABT-263.

Fisetin reduced the 31-point frailty index score by 15% and increased forelimb grip strength by 14% in old mice, with no effect in young mice. Bulk RNA sequencing of quadriceps muscle showed that fisetin attenuated broad age-related shifts in gene expression, particularly in cellular senescence and p53 signalling pathways. The senescence marker Cdkn1a (encoding p21) was normalised in aged fisetin-treated muscle by approximately 46% relative to aged vehicle.

The comparative finding is what makes this paper editorially interesting. The magnitude of fisetin's effect on frailty and grip strength was statistically indistinguishable from genetic clearance of p16-positive senescent cells — the preclinical gold-standard senolytic intervention — and from ABT-263. For a natural food-derived compound, head-to-head equivalence with a genetic intervention is a striking result.

The most important caveat is that fisetin did not reduce Cdkn2a (p16) expression, which both comparator interventions did. Physical function outcomes were equivalent across the three arms, but the underlying senescence marker profile was not identical. Whether the p21-pathway effect alone is enough to explain the functional benefit, or whether fisetin is acting through a mechanism distinct from classical senescent-cell clearance, is unresolved. The authors themselves acknowledge that senolytic activity was inferred from gene expression only, without cell-experiment or histological confirmation.

Key findings

Fisetin reduced the frailty index score by 15% (0.23 vs 0.27 AU, p = 0.0264) and increased forelimb grip strength by 14% (3.30 vs 2.90 g/g body weight, p = 0.0038) in old mice. No effect in young mice.
Fisetin normalised Cdkn1a (p21) expression in aged skeletal muscle by approximately 46%, bringing it close to young mouse levels.
Fisetin lowered Ddit4 (Redd1) expression in aged skeletal muscle — a gene that itself induces cellular senescence via upregulation of Cdkn1a.
Cellular senescence and p53 signalling KEGG pathways were enriched with ageing but no longer enriched after fisetin treatment.
Effects on frailty and grip strength with fisetin were statistically indistinguishable from genetic p16-positive senescent cell clearance and from synthetic senolytic ABT-263.
Fisetin did not reduce Cdkn2a (p16) expression, whereas both comparator interventions did — a mechanistic divergence despite equivalent functional outcomes.
BayesPrism deconvolution analysis suggested fisetin's effect on Cdkn1a and Ddit4 spans multiple cell types in muscle, including myonuclei, pericytes, fibro-adipogenic progenitors, and neural cells.

What this study can and cannot tell us

Preclinical mouse study — no human translation demonstrated. The corresponding human trials (AFFIRM, Fisetin HIGH, PROFFi) remain unreported.
Senolytic mechanism was inferred from bulk RNA sequencing gene expression only. The authors explicitly acknowledge in the discussion that senolytic effects were not confirmed with cell experiments or histological evaluation.
Fisetin did not reduce Cdkn2a (p16), which is the defining senescence marker cleared by both comparator interventions (GCV in p16-3MR mice; ABT-263). The mechanistic basis for equivalent physical function outcomes despite this divergence is not established.
Internal dose inconsistency in the published paper: methods section states fisetin was given at 50 mg/kg/day, but Figure 1A labels the dose as 100 mg/kg/day. Methods dosing appears to be authoritative since it matches the same group's prior Mahoney 2024 paper.
Female representation in the wildtype fisetin arm is limited: Old Fisetin N=39 comprised 31 males and 8 females. The p16-3MR arm was more balanced. Sex-stratified analyses were reported as showing no differences but were combined for the primary comparisons.
Vehicle contained 10% ethanol; potential vehicle effects on aged skeletal muscle beyond the vehicle-only comparator arm not directly quantified.
The intermittent dosing pattern (one week on – two weeks off – one week on) differs from the Mayo Clinic clinical trial protocol (20 mg/kg × 2 days every 28 days). Translational dose extrapolation is not straightforward.
Grip strength trials in which the mouse "forcefully jerked the bar rather than simply releasing its grasp were included" (methods section 2.6). Whether this measurement approach affects effect-size estimation is not discussed.

Tier 3

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Tier 2

Fisetin as a senotherapeutic agent — evidence and perspectives for age-related diseases

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