Circadian restoration coupled with senolysis reverses Zone 3 hepatocyte senescence and fibrosis in NAFLD

Circadian disruption drives Zone 3 hepatocyte senescence by uncoupling mitophagy from metabolic demand, leading to mitochondrial damage, mtDNA‑cGAS‑STING activation and a SASP that fuels fibrosis. Restoring hepatic clock amplitude with a timed REV‑ERB agonist re‑synchronizes autophagy flux, while a senolytic clears the resulting senescent cells; together they should attenuate steatosis and fibrosis more effectively than either approach alone.

The core mechanism links the clock to mitochondrial quality control. REV‑ERBα peaks at ZT8‑10 and directly represses lipogenic genes, but it also activates transcription of autophagy regulators such as LC3 and PINK1. When REV‑ERBα rhythm is blunted—as occurs with high‑fat feeding or irregular eating—mitophagy falls, damaged mitochondria accumulate, and oxidized mtDNA escapes to the cytosol. Cytosolic mtDNA engages the cGAS‑STING pathway, triggering type I interferon signaling and a senescence‑associated secretory phenotype (SASP) marked by p16^Ink4a^ elevation and IL‑6 release. This SASP perpetuates inflammation, activates stellate cells, and accelerates pericentral fibrosis, consistent with the observation that severe Zone 3 steatosis predicts fibrosis progression with an odds ratio of 2.7.

We hypothesize that boosting NAD+‑SIRT1‑AMPK signaling—already shown to reduce lipogenesis—will further enhance REV‑ERBα‑dependent mitophagy, creating a feed‑forward loop that limits mitochondrial damage. Simultaneously, removing senescent hepatocytes with a senolytic (e.g., navitoclax) will blunt the SASP, breaking the fibrotic cascade. The combined intervention should therefore reduce Zone 3 lipid accumulation, lower senescence biomarkers, and diminish collagen deposition beyond what chronotherapy or senolysis achieves singly.

Testable prediction: In male C57BL/6J mice fed a high‑fat, high‑fructose diet for 16 weeks, four groups will be compared for 8 weeks—(1) vehicle, (2) timed REV‑ERB agonist (SR9009) administered at ZT6, (3) senolytic (navitoclax) given twice weekly, and (4) combined therapy. Primary outcomes will be hepatic triglyceride content (biochemical assay), Zone 3‑specific lipid staining (Oil‑Red O), senescence burden (p16 immunostaining and SA‑β‑gal activity), and fibrosis severity (Sirius Red area and hydroxyproline assay). Serum ALT and cytokines (IL‑6, TNF‑α) will serve as secondary readouts. We expect the combination group to show at least a 40 % greater reduction in triglyceride levels and a 50 % greater decrease in fibrosis score relative to monotherapies (p < 0.05). Failure to observe a synergistic effect would falsify the hypothesis that circadian‑driven mitophagy and senescence are jointly responsible for Zone 3‑specific pathology.

This framework directly extends prior work showing circadian disruption promotes steatosis, SIRT1‑AMPK activation mitigates lipid accumulation, and zona‑specific vulnerability predicts fibrosis. By integrating mitophagy, mtDNA‑cGAS‑STING signaling, and senolysis, it offers a falsifiable, mechanism‑driven route to test whether clock repair plus senescent‑cell clearance constitutes a potent geroprotective strategy for NAFLD.