Urolithin A preserves regenerative senescence by coupling mitophagy to SASP reprogramming in fibroblast hostage‑negotiators

Hypothesis

Urolithin A (UA) selectively enhances mitophagy in senescent fibroblasts, shifting their SASP from a chronic, inflammatory profile to an acute, regenerative state that maintains tissue‑repair negotiations without triggering premature clearance.

Mechanistic Rationale

Senescent fibroblasts in young wounds express p16+/p21+ and secrete PDGF‑AA, IL‑6 and chemokines that recruit immune cells for timely removal [1, 2]. Persistent senescence in aged or chronic wounds switches SASP toward IL‑1β, TNF‑α and MMP‑3, driving fibrosis [3]. Impaired mitophagy sustains mitochondrial damage, which fuels the chronic SASP loop [4]. UA is a known mitophagy inducer that restores mitochondrial quality in multiple cell types [4]. We propose that UA‑driven mitophagy reduces mitochondrial ROS and NAD⁺ depletion, thereby lowering NF‑κB activity and favoring transcription of regenerative SASP components via FOXO3‑mediated gene expression. This metabolic reprogramming would keep senescent fibroblasts in a “hostage‑negotiator” mode: they continue to signal for coordinated repair and immune recruitment while avoiding the inflammatory shift that leads to pathological persistence.

Novel Insight Beyond Existing Data

Current literature treats senescent cells as a binary target for removal or retention. Our hypothesis introduces a metabolic checkpoint: mitophagy status dictates SASP quality. By linking UA‑mediated mitochondrial clearance to a specific SASP fingerprint, we suggest a therapeutic window where senescent cells are neither indiscriminately cleared nor allowed to become detrimental. This reframes the hostage‑negotiator metaphor: the negotiator’s tone (peaceful vs. hostile) is tunable through mitochondrial health, not merely its presence.

Testable Predictions

1. In vitro: Human fibroblasts induced to senesce via irradiation will show increased mitochondrial mass and ROS. Treatment with 10 µM UA for 48 h will decrease MitoTracker signal and restore NAD⁺/NADH ratio [4]. Concurrently, RNA‑seq will reveal upregulated PDGF‑AA, IL‑6 and CXCL12, and downregulated IL‑1β, TNF‑α, MMP‑3 compared with untreated senescent cells.
2. Ex vivo: Full‑thickness murine wounds treated topically with UA will retain a higher proportion of p16+/PDGF‑AA+ fibroblasts at day 4 post‑injury, while exhibiting reduced p16+/IL‑1β+ cells relative to vehicle. Wound closure rates will improve, and histology will show organized collagen deposition without excess fibrosis.
3. In vivo: Aged mice receiving chronic UA supplementation will display attenuated accumulation of IL‑1β‑high senescent fibroblasts in skin and gut, measured by flow cytometry for p16+IL‑1β+ cells. Despite lower inflammatory SASP, tissue‑specific repair assays (e.g., epidermal thickness, mucus layer integrity) will remain comparable to young controls, indicating preserved negotiator function.
4. Falsification: If UA fails to shift SASP toward regenerative factors or does not reduce mitochondrial ROS in senescent fibroblasts, the hypothesis is refuted. Likewise, if UA‑treated wounds show accelerated clearance of all p16+ cells without improved repair outcomes, the negotiator‑preservation mechanism is invalid.

Experimental Approach

• Use CRISPR‑KO of PINK1 or Parkin to block mitophagy in fibroblasts; UA should lose its SASP‑shifting effect, confirming mitophagy dependence.
• Employ Seahorse flux analysis to quantify OCR/ECAR changes post‑UA treatment.
• Apply SASP‑specific ELISA multiplex panels to supernatants from treated cultures.

By targeting the mitochondrial‑SASP axis, UA could enable precision modulation of senescence—keeping the hostage negotiator at the table while preventing the takeover of hostile demands.