Hypothesis

Ferroptotic death of zone 3 hepatocytes in aging MASLD does not merely reflect a local metabolic bottleneck; it actively propagates a 'field of metabolic aging' to upstream zones through the release of oxidized lipid‑derived danger signals that reprogram periportal hepatocytes via TLR4‑MyD88‑NF‑κB signaling, thereby expanding the AHGS beyond its hypoxic origin. This paracrine mechanism explains why AHGS can be detected in zones 1‑2 of advanced MASLD livers even when oxygen gradients remain steep, and why fibrosis progresses centripetally despite the initial pericentral insult.

Predictions

1. Isolating conditioned medium from ferroptotic zone 3 hepatocytes (induced by erastin or hypoxia + iron) will contain elevated levels of oxidized phospholipids (e.g., POVPC) and HMGB1, and will trigger TLR4‑dependent NF‑κB activation and AHGS marker expression (e.g., increased p16^INK4a, reduced CPT1A) in cultured zone 1 hepatocytes.
2. In vivo, liver‑specific TLR4 knockout in zone 1/2 hepatocytes (using Alb‑CreERT2 with a zone‑specific promoter) will markedly reduce the spread of AHGS and fibrosis in aged MASLD mice, despite persistent zone 3 ferroptosis (measured by C11‑BODIPY 581/591 oxidation and 4‑HNE adducts).
3. Pharmacological blockade of TLR4 (e.g., TAK‑242) combined with a ferroptosis inhibitor (Fer‑1) will produce synergistic reduction in AHGS burden and collagen deposition compared with either monotherapy, whereas TLR4 inhibition alone will not affect zone 3 ferroptosis levels.

Mechanistic Rationale The lipid‑rich, hypoxic milieu of zone 3 favors peroxidation of polyunsaturated fatty acids, generating electrophilic oxidized lipids that act as DAMPs. These molecules can diffuse sinusoidally or be packaged in extracellular vesicles, reaching periportal sinusoids where they engage TLR4 on Kupffer cells and hepatocytes. TLR4 signaling drives NF‑κB–mediated transcription of senescence‑associated secretory phenotype (SASP) factors and directly represses PPARα‑coactivator 1α (PGC‑1α), suppressing fatty acid oxidation and reinforcing the AHGS. This creates a feed‑forward loop: more zone 3 ferroptosis → more DAMP release → broader TLR4 activation → metabolic repression in upstream zones → increased lipid spillover back to zone 3, worsening peroxidation.

Experimental Approaches

• Use spatial transcriptomics (10x Visium) on human MASLD livers to map AHGS signatures relative to hypoxia markers (CA9, HIF1A) and TLR4 activity (NF‑κB target genes). Expect AHGS hotspots extending beyond CA9‑high zones correlating with TLR4 activation.
• Deploy a hypoxia‑activated prodrug (e.g., TH‑302) to selectively induce ferroptosis in zone 3, then track DAMP release using mass spectrometry‑based lipidomics of sinusoidal blood.
• Test therapeutic window: administer TLR4 antagonist after early steatosis but before fibrosis stage (F0‑F1) and assess reversal of AHGS and prevention of collagen deposition via Sirius Red staining.

Falsifiability If zone‑specific TLR4 deletion fails to attenuate AHGS spread or fibrosis despite efficient knockout, or if conditioned medium from ferroptotic zone 3 cells does not induce TLR4‑dependent AHGS markers in zone 1 hepatocytes, the hypothesis would be refuted. Likewise, if spatial transcriptomics shows AHGS strictly confined to hypoxia‑positive zones without TLR4 activation in periportal areas, the paracrine field model would not hold.

By integrating ferroptosis, danger‑signal signaling, and zonation, this hypothesis positions TLR4‑mediated metabolic reprogramming as a critical amplifier of aging‑driven MASLD progression, offering a combinatorial strategy that targets both the pericentral trigger (ferroptosis) and the propagating signal (TLR4) to enlarge the therapeutic window before irreversible fibrosis sets in.