Notch1 Inhibition Exacerbates APAP-Induced Liver Injury via β-Catenin and Macrophage Polarization

Background: Notch1 signaling regulates innate immune-mediated inflammation in acute liver injury (ALI). However, the precise mechanism by which Notch1 governs macrophage polarization during ALI remains poorly understood. Methods: Wild-type (WT) mice received DAPT (10 mg/kg) prior to acetaminophen (APAP)-induced ALI. In parallel, bone marrow-derived macrophages (BMMs) were pretreated with either the β-catenin inhibitor XAV939 or the activator SKL2001, exposed to DAPT, and then challenged with lipopolysaccharide (LPS). Liver injury and inflammation were evaluated by hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, immunohistochemistry, immunofluorescence, quantitative real-time PCR (RT-PCR), and western blotting. Results: Unexpectedly, DAPT treatment exacerbated APAP-induced liver injury (AILI), resulting in more severe hepatocellular damage and inflammation than in controls. DAPT-treated macrophages exhibited enhanced pro-inflammatory cytokines expression and a shift toward an M1-like phenotype. Mechanistically, the β-catenin/glycogen synthase kinase 3 beta (GSK3β) signaling pathway emerged as a pivotal regulator of macrophage polarization. Conclusions: Notch1 inhibition unexpectedly worsens AILI by amplifying macrophage-driven pro-inflammatory responses via β-catenin signaling. These findings highlight the Notch1–β-catenin axis as a key regulator of hepatic macrophage function and a potential therapeutic target for sterile liver inflammation.