Hypothesis

In MASLD, a critical ferroptosis‑sensitive threshold exists in zone 3 hepatocytes where elevated labile iron pools (LIP) trigger lipid peroxidation, initiating the Aging Hepatocyte Gene Signature (AHGS) and subsequent senescence‑associated secretory phenotype (SASP). When AHGS‑positive hepatocytes exceed ~17 % of the total hepatocyte population, paracrine SASP drives fibroblast activation and collagen deposition, accelerating fibrosis. Reducing zone‑3 LIP below this threshold—via targeted iron chelation or enhanced ferroportin export—will prevent AHGS induction, limit SASP spread, and attenuate fibrosis, even in the presence of PNPLA3 I148M or MET loss‑of‑function alleles.

Rationale

• Ferroptosis is iron‑dependent; hepatocyte LIP varies across the acinus, with zone 3 showing lower oxygen and higher susceptibility to lipid peroxidation [2].
• AHGS captures ferroptosis‑driven loss of metabolic function and gain of senescence/ECM pathways, predicting advanced disease (AUC > 0.80) [1].
• Zone‑3 steatosis confers 1.8‑2.7‑fold increased fibrosis odds [3], aligning with a spatial threshold concept.
• Genetic hits (PNPLA3 I148M, MET mutations) exacerbate lipid handling but do not directly alter iron chemistry; they may raise the LIP needed to cross the ferroptosis threshold [4, 5].
• Blocking ferroptosis rejuvenates aged livers under metabolic stress [1], suggesting iron modulation is therapeutic.

Predictions

1. Spatial LIP Gradient: Quantitative iron staining (e.g., Perl’s‑Prussian blue + calcein‑AM) will show a steep LIP increase from zone 1 to zone 3 in human MASLD livers, correlating with AHGS marker (ACSL4, PTGS2) expression.
2. Threshold Effect: Livers with ≤15 % AHGS‑positive hepatocytes will exhibit minimal SASP (IL‑6, CXCL1) and low collagen‑1 staining, whereas those ≥20 % AHGS‑positive will show robust SASP and fibrosis, independent of overall steatosis grade.
3. Intervention Impact: Targeted delivery of a ferroportin‑activating nanoparticle (or liposomal deferoxamine) to zone 3 will reduce LIP by ≥30 %, lower AHGS‑positive hepatocytes below the 17 % cut‑point, decrease SASP cytokines, and attenuate fibrosis scores by ≥40 % compared with vehicle.
4. Genetic Interaction: In PNPLA3 I148M or MET‑variant hepatocytes, the same LIP reduction will yield a proportionally greater drop in AHGS burden because basal lipid peroxidation is higher, confirming a synergistic risk model.

Experimental Design

• Human Tissue Validation: Obtain consented MASLD explant biopsies (n=30) stratified by fibrosis stage. Perform multiplexed immunofluorescence for ferritin‑H, ACSL4, p16, and collagen‑1. Quantify zone‑specific LIP and AHGS density; apply logistic regression to test the 17 % AHGS threshold as a predictor of advanced fibrosis (F3‑F4).
• Murine Intervention: Use diet‑induced MASLD mice (high‑fat, high‑fructose, with PNPLA3 I148K knock‑in). Administer zone‑3‑targeted liposomal deferoxamine (mannose‑6‑phosphate receptor ligand) twice weekly for 8 weeks. Control groups receive empty liposome or systemic deferoxamine.
  • Measure hepatic LIP (calcein‑AM quenching), lipid peroxidation (C11‑BODIPY), AHGS‑positive hepatocytes (RNAscope for ACSL4/PTGS2), SASP (ELISA for IL‑6, CXCL1), and fibrosis (Sirius Red, hydroxyproline).
  • Primary outcome: change in fibrosis stage; secondary: AHGS burden and SASP levels.
• In Vitro Mechanistic Assay: Primary mouse hepatocytes isolated per zone via perfusion gradient. Treat with ferric ammonium citrate to titrate LIP, assess ACSL4 translocation and cell death (liproxstatin‑1 sensitivity). Determine LIP concentration that yields 50 % AHGS induction; test whether ferroportin over‑expression shifts this curve rightward.

Potential Outcomes and Falsifiability

• Supportive: Zone‑3‑targeted iron reduction lowers AHGS below 17 %, reduces SASP, and improves fibrosis despite persistent steatosis; threshold analysis shows AHGS > 17 % predicts fibrosis with high specificity.
• Refutatory: Iron reduction fails to decrease AHGS or SASP, or fibrosis improves without changes in AHGS burden, indicating that ferroptosis‑driven senescence is not the dominant driver. Similarly, if AHGS burden remains below threshold yet fibrosis progresses, alternative pathways (e.g., inflammasome activation) would be implicated.

This hypothesis is directly testable with existing spatial omics, iron‑sensing probes, and targeted drug delivery platforms. It links the molecular signature of ferroptosis‑mediated aging hepatocytes to a zonated iron threshold, offering a precise biomarker‑guided therapeutic window for MASLD intervention.