Hypothesis: Combined oral supplementation with urolithin A (UA) and spermidine will synergistically lower senescence biomarkers in human immune cells by coupling UA-driven mito-phagy with spermidine-stimulated bulk autophagy, resulting in enhanced lysosomal clearance and attenuated senescence-associated secretory phenotype.

Mechanistic basis: UA activates the AMPK-SIRT1-PGC-1alpha axis, which promotes PINK1/Parkin-mediated removal of damaged mitochondria and stimulates mitochondrial biogenesis[1]. AMPK activation also phosphorylates ULK1, initiating autophagosome formation. Spermidine inhibits the acetyltransferase EP300, leading to increased acetylation of histone H3 at autophagy-gene promoters and to deacetylation of essential autophagy proteins such as ATG5 and ATG7, thereby amplifying general autophagosome synthesis[2]. Both pathways converge on the lysosomal transcription factor TFEB: SIRT1-dependent deacetylation increases TFEB nuclear localization, while EP300 inhibition prevents TFEB acetylation-driven inactivation, together boosting lysosomal biogenesis and acidification. Elevated lysosomal capacity accelerates degradation of mito-phagy cargos and cytosolic protein aggregates, reducing oxidative stress and DNA damage that drive p16^INK4a and p21^CIP1 expression. Lower mitochondrial ROS also dampens NF-kappaB signaling, curbing the secretion of IL-6, TNF-alpha and other SASP factors.

Testable prediction: In a randomized, double-blind, placebo-controlled study of adults aged 65-80, participants receiving UA (1 g/day) plus spermidine (5 mg/day) for 12 weeks will show a >=35% reduction in PBMC mRNA levels of p16^INK4a and p21^CIP1 compared with placebo, whereas monotherapy with either UA or spermidine will yield <=15% reduction. Secondary outcomes will include: (i) increased frequency of naive-like CD8+ T cells and mitochondrial spare respiratory capacity measured by Seahorse assay; (ii) elevated mito-phagy flux assessed by mt-Keima staining; (iii) higher autophagic flux indicated by LC3-II turnover in the presence of bafilomycin A1; (iv) decreased plasma IL-6, TNF-alpha and CRP; and (v) improved lysosomal activity quantified by Lysotracker intensity. If the combination fails to produce a statistically significant greater decline in senescence markers than either agent alone, the hypothesis is falsified.

Additional considerations: Inter-individual variability in gut microbiota influences endogenous UA production; therefore, baseline urinary urolithin levels will be stratified to adjust for differential exposure. Spermidine levels will be monitored via plasma metabolomics to confirm adherence. Dose-response sub-studies can define the minimal effective ratio of UA to spermidine for synergistic lysosomal activation.