Tier 3 — preclinical

Effects of Fisetin Treatment on Cellular Senescence of Various Tissues and Organs of Old Sheep

Huard CA, Gao X, Dey Hazra ME, Dey Hazra RO, Lebsock K, Easley JT, Millett PJ, Huard J

Antioxidants 2023 Volume 12, issue 8, article 1646

Bibliography

DOI: 10.3390/antiox12081646
PubMed: PMID 37627641
PubMed Central: PMC10451965
Funding: Philanthropic gift from the Borgen Family and the Linda and Mitch Hart Family.
Competing interests: One author (J. Huard) received royalties from Cook Myocyte. All other authors declared no conflicts of interest.

Study snapshot

Design	Controlled large-animal study assessing cellular senescence markers across brain regions and peripheral organs after intravenous fisetin treatment, using histology, immunofluorescence and RT-qPCR.
Model	Female sheep, 6-7 years old (an aged large-animal model), treated with fisetin or vehicle.
Sample	N=6 female sheep.
Intervention	Intravenous fisetin 100 mg/kg on two consecutive days per week, for 8 weeks, versus vehicle control.
Duration	8 weeks of treatment, with tissue harvest at the time of sacrifice.
Endpoints	Senescent-associated β-galactosidase (SA-β-Gal) and GL13 (lipofuscin) staining in brain cortex, cerebellum and hippocampus; P16 and GLB1 colocalisation with neuronal (NEUN), astrocyte (GFAP) and microglial (IBA1) markers; Plasma S100B (neural injury marker); mRNA expression of antioxidant genes (SOD1, CAT), senescence genes (GLB1, P21, P53), SASP genes (IL10, IL8) and inflammasome genes (NLRP3, TREM2) across liver, spleen, lung, heart and bone marrow

What the study showed, in plain terms

Most fisetin senescence research uses mice, which are convenient but quite different from humans in size, lifespan and physiology. This study instead used aged sheep — a much larger, longer-lived animal that is considered a more translational bridge toward human biology — to ask whether fisetin, given intravenously twice a week for eight weeks, reduces senescent ('zombie') cells in the brain and other organs.

The researchers found that senescent cells were common in the grey and white matter of the cerebral cortex and cerebellum of these older sheep, and in part of the hippocampus, mostly appearing as large, aged neurons. Fisetin treatment reduced markers of senescence in brain neurons, astrocytes and microglia in several of these regions, and lowered plasma S100B, a marker of neural ageing and injury.

Beyond the brain, fisetin also shifted expression of senescence-related genes in the liver (most consistently), with smaller or non-significant trends in the lung, spleen, heart and bone marrow — suggesting the intravenous 'hit-and-run' senolytic dosing pattern used here (two days on, five days off) has its clearest effect on organs that see fisetin at highest concentration after IV dosing.

This is a genuine step up in translational relevance from mouse studies, but it used intravenous dosing at 100 mg/kg — a route and dose that bears no resemblance to an oral capsule — in a small group of six animals, and did not measure any functional or clinical outcome (frailty, cognition, mobility). It should be read as tissue-level mechanistic evidence in a large-animal model, not as evidence of a clinical benefit.

Key findings

Senescent cells (SA-β-Gal+ and GL13+) were widely present in the grey and white matter of the cerebral cortex and cerebellum, and in the non-CA area of the hippocampus, of old sheep — mostly large neurons, with grey matter showing more senescent cells than white matter.
Fisetin treatment significantly decreased SA-β-Gal+ cells in brain cortex white matter (p=0.03) and GL13+ cells in the non-CA hippocampus (p=0.002), with a decreasing trend for SA-β-Gal+ cells in cortical and cerebellar grey matter.
Fisetin significantly decreased the percentage of P16+ and GLB1+ senescent neurons, astrocytes and microglia in both grey and white matter of the cerebral cortex (multiple comparisons p<0.05), and significantly decreased senescent microglia and astrocytes in the non-CA hippocampus.
Fisetin treatment significantly decreased plasma S100B, a neural ageing and injury marker (p=0.047), while plasma MDA (lipid peroxidation) was unchanged.
At the mRNA level, fisetin significantly downregulated SOD1 in liver and spleen, downregulated GLB1 and P21 in the liver, downregulated P21 in the lung, downregulated NLRP3 in the liver and TREM2 in the lung, and upregulated CAT in the spleen — with only decreasing trends (not statistically significant) for most of these genes in lung, heart and spleen.
Fisetin did not eliminate all senescent cells in any brain region studied, and had no significant effect on senescent neurons in the CA1-4 hippocampal area, where very few neurons were senescent to begin with.

What this study can and cannot tell us

Small sample size (n=6 sheep) and a single dosing regimen (100 mg/kg IV, two consecutive days per week for 8 weeks) that cannot be compared to any oral human fisetin dose. There is no functional, cognitive or clinical outcome measured — only tissue-level senescence and gene-expression markers.

Effects were inconsistent across organs: statistically significant changes were concentrated in liver and lung, with only non-significant downward trends in spleen, heart and bone marrow, which the authors attribute to differential tissue exposure after intravenous ('hit-and-run') dosing rather than daily oral administration.

As a large-animal model, sheep sit closer to humans than rodents in some respects, but this remains preclinical animal evidence; extrapolation of dose, route, or degree of senescent-cell reduction to human ageing or supplementation is not supported by this study alone.

Tier 2

Alleviation by Fisetin of Frailty, Inflammation, and Related Measures in Older Women (AFFIRM)

AFFIRM Trial et al. · ClinicalTrials.gov (trial registry record — not yet published in a peer-reviewed journal) · 2018