Hypothesis

Pharmacologic activation of the farnesoid X receptor (FXR) in middle‑aged hepatocytes will reestablish normal metabolic zonation, inhibit CYP2E1‑dependent iron accumulation, and prevent ferroptosis‑induced ferrosenescence, thereby halting the progression from steatosis to fibrosis in aged MASLD.

Mechanistic Rationale

Aging drives a convergence of lipogenic flux, CYP2E1‑mediated oxidative stress, and iron loading in zone 3 hepatocytes, creating a permissive environment for ferroptosis (Zone 3 hepatocytes are uniquely vulnerable...). Ferroptotic cells adopt a ferrosenescent phenotype, secreting TNFα and IL‑6 that activate macrophage‑mediated fibrosis (ferroptotic stress drives steatosis-to-fibrosis progression). Concurrently, Wnt/β‑catenin signaling becomes dysregulated, blurring zonation boundaries and generating atypical ASS1⁺GS⁺ "bi‑zonal" cells (aging disrupts hepatocyte zonation homeostasis).

FXR activation is known to suppress CYP2E1 transcription, upregulate iron export proteins (e.g., ferroportin), and enhance antioxidant defenses such as GPX4. Moreover, FXR signaling antagonizes β‑catenin transcriptional activity by inducing expression of Axin2 and promoting β‑catenin degradation, thereby limiting ectopic Wnt signaling that underlies zonation loss. Importantly, FXR activity declines with age and iron accumulation, forming a vicious loop where iron‑mediated FXR inhibition further exacerbates CYP2E1 expression and iron uptake (FXR signaling—suppressed by iron accumulation—also emerges as a target).

We hypothesize that restoring FXR signaling will break this loop: reduced CYP2E1 lowers lipid peroxidation and ROS, restored iron efflux limits the labile iron pool, and normalized Wnt/β‑catenin activity reestablishes sharp zonation borders, preventing the emergence of ferrosenescent hepatocytes.

Testable Predictions

1. In aged mice with established MASLD, treatment with an FXR agonist (e.g., obeticholic acid) will decrease zone 3 CYP2E1 protein levels and hepatic labile iron by ≥30% compared with vehicle.
2. FXR agonism will increase the expression of canonical zone 1 (Cyp2e1 low, Glu2 high) and zone 2 (Cyp2a5, Aldh1a1) markers while reducing the ectopic ASS1⁺GS⁺ bi‑zonal population, as quantified by immunofluorescence and single‑nucleus RNA‑seq.
3. Lipid peroxidation (C11‑BODIPY 581/591) and ferroptotic cell death (PTGS2/ACSL4) in zone 3 will be reduced by ≥40%, accompanied by a drop in ferrosenescent SASP cytokines (TNFα, IL‑6) in liver homogenates.
4. Fibrosis severity (Sirius Red area, collagen‑1α1 mRNA) will be attenuated by ≥35% after 8 weeks of treatment, correlating with improvements in the Aging Hepatocyte Gene Signature (AHGS) score moving from >4.0 to <3.0.
5. Genetic hepatocyte‑specific deletion of Fxr will abolish the protective effects of the agonist, confirming cell‑autonomous action.

Experimental Design

• Model: C57BL/6J mice fed a methionine‑choline deficient diet for 16 weeks to induce MASLD, then aged to 80 weeks (biological age approximated by AHGS 3.0–4.0).
• Intervention: Daily oral FXR agonist (10 mg/kg) or vehicle for 8 weeks; include a ferrostatin‑1 arm as a positive control for ferroptosis inhibition.
• Readouts:
  • HPLC‑based measurement of hepatic labile iron.
  • Western blot/qPCR for CYP2E1, ferroportin, GPX4, Axin2, β‑catenin targets.
  • Multiplex immunofluorescence for zonation markers (HNF4A, GLUL, ASS1, GS) and 4‑HNE.
  • snRNA‑seq (10x) to quantify zone‑specific transcriptomes and bi‑zonal cell fractions.
  • ELISA for TNFα, IL‑6 in liver lysates.
  • Histological fibrosis scoring and hydroxyproline assay.
• Analysis: Two‑way ANOVA with post‑hoc Tukey; significance set at p<0.05.

Potential Pitfalls and Alternatives

If FXR agonism fails to reduce iron or CYP2E1, the hypothesis would be falsified, suggesting that iron‑mediated FXR suppression is epistatic to the ferroptosis pathway in aged livers. An alternative would be to combine FXR activation with direct iron chelators (e.g., deferoxamine) to test additive effects. Conversely, if zonation is restored without affecting ferroptosis markers, it would imply that zonation loss is a downstream consequence rather than a driver of ferrosenescence, redirecting focus toward Wnt/β‑catenin modulation alone.