Recent work shows that senolytics such as dasatinib plus quercetin (D+Q) can selectively eliminate senescent cells and reduce the proinflammatory SASP that accumulates with age [4][3]. Concurrently, partial epigenetic reprogramming with transient expression of Yamanaka factors (OSK) reverses epigenetic age markers without causing pluripotency [5][6]. However, the inflammatory milieu created by senescent cells may impede reprogramming efficiency and increase the risk of aberrant transcription. We hypothesize that pre‑treating aged tissues with a senolytic regimen creates a permissive environment for OSK‑mediated rejuvenation by lowering SASP‑driven NF‑κB activity, thereby enhancing DNA demethylation and reducing the likelihood of oncogenic transformation.

Specifically, we predict that in mice aged 20–24 months, a single intraperitoneal dose of D+Q (5 mg/kg dasatinib + 50 mg/kg quercetin) administered 48 h before induction of a doxycycline‑inducible OSK system will produce: (1) a greater reduction in epigenetic age as measured by the Horvath mouse clock in liver, hippocampus, and skeletal muscle compared with OSK induction alone; (2) improved functional readouts such as increased hippocampal neurogenesis, enhanced grip strength, and better glucose tolerance; (3) a lower incidence of ectopic proliferation or tumor formation, assessed by Ki‑67 staining and histopathological examination over a 6‑month follow‑up. Conversely, mice receiving OSK induction without prior senolytic clearance will show modest epigenetic reversal but higher SASP levels and a modest increase in atypical hyperplasia.

To test this, we will use four groups (n=15 per group): (A) vehicle control; (B) D+Q only; (C) OSK induction only (doxycycline for 2 days weekly for 4 weeks); (D) D+Q pretreatment followed by OSK induction. Primary outcomes include epigenetic clock readouts from bisulfite sequencing, SASP cytokine profiling (IL‑6, IL‑1β, TNF‑α) via multiplex ELISA, and tissue‑specific functional assays. Secondary outcomes involve tumorigenicity surveillance and senescence‑associated β‑galactosidase staining to confirm senescent cell depletion.

If the hypothesis holds, group D will demonstrate synergistic benefits: epigenetic age regression exceeding the sum of individual interventions, functional improvement surpassing either monotherapy, and no increase in neoplastic lesions. Falsification would occur if group D shows no additional epigenetic or functional gain over group C, or if senolytic pretreatment exacerbates tumorigenic risk, indicating that senescent cell removal does not create a permissive niche for reprogramming.