Single-nucleus RNA sequencing reveals that THR-β agonists ameliorate MASH by regulating hepatic parenchymal and immune cell transcriptomes

Therapies targeting thyroid hormone receptor-beta (THR-β) have shown clinical efficacy in metabolic dysfunction-associated steatohepatitis (MASH), but a comprehensive, cell-type-resolved understanding of their molecular mechanisms within the hepatic microenvironment is lacking. This study employed single-nucleus RNA sequencing (snRNA-seq) to dissect the therapeutic effects of the THR-β agonist MGL-3196 (10 mg/kg/day, 8 weeks) in a murine MASH model induced by high-fat diet combined with carbon tetrachloride (HFD/CCl₄) administration. Treatment robustly improved steatosis, fibrosis, and metabolic serum markers. Analysis of 71,786 single-cell transcriptomes revealed that agonist treatment attenuated MASH-induced hepatocyte loss and hepatic stellate cell expansion. We identified a metabolically enriched hepatocyte subpopulation (HEP1), characterized by high expression of detoxification genes (Cyp3a25, Slco1b2) and enriched for bile acid and fatty acid metabolism pathways, which showed reduced representation in MASH and was partially restored after treatment. At the molecular level, the agonist treatment was associated with the downregulation of pro-fibrotic gene networks—including ECM components predominantly expressed in hepatic stellate cells (Col1a1, Col3a1), as well as genes associated with PI3K-Akt signaling and integrin adhesion pathways in hepatocytes. Concurrently, RES treatment was associated with attenuated transcriptional programs in Kupffer cells related to DNA repair, cytoskeletal organization, and stress-responsive pathways. A pivotal novel finding was the synchronized, pan-cellular downregulation of hepatoprotective Major Urinary Protein (MUP) family genes in MASH, which was significantly rescued by THR-β agonist at both the transcript and protein levels. Our results delineate a potential multi-faceted mechanism whereby THR-β agonists restore hepatic ecosystem homeostasis by rescuing a metabolically enriched hepatocyte subpopulation, associated with the attenuation of pro-fibrotic transcriptional programs, modulating macrophage activation, and identifying the MUP/Lipocalin family as a candidate protective axis warranting further investigation, highlighting novel molecular targets for therapeutic intervention.