Abstract
Background and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) affects over 30% of adults globally; its progressive form, metabolic dysfunction-associated steatohepatitis (MASH), has limited pharmacological options beyond the recently approved resmetirom. The statin–ezetimibe combination, widely co-prescribed for dyslipidemia in patients with MASH, significantly reduces hepatic fat versus statin monotherapy in the ESSENTIAL randomized trial, yet macrophage-driven mechanisms linking this lipid-lowering combination to hepatic inflammation and fibrosis remain poorly characterized. We investigated these mechanisms using in vivo and in vitro approaches. Methods: Male C57BL/6 mice (n = 6/group; 6 groups) received a high-fat diet (HFD; 45% kcal) plus thioacetamide (TAA; 300 mg/kg twice-weekly, 8 weeks) to establish MASH with advanced fibrosis. Simvastatin (5 mg/kg) and/or Ezetimibe (10 mg/kg) were added to the diet for 4 weeks. Blinded hepatic histology (H&E, Picrosirius red, Oil Red-O), qPCR, immunohistochemistry, and Western blot were performed. Macrophage polarization and NF-κB nuclear translocation were assessed in RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages by flow cytometry and immunofluorescence. Results: Combination therapy significantly reduced steatosis, inflammation, and fibrosis versus HFD-TAA controls. Pro-inflammatory (IL-1β, IL-6, TNF-α, iNOS) and fibrosis-related (collagen-I, α-SMA, TGF-β1) markers were markedly suppressed (p < 0.01). In vitro, the combination reduced the proportion of iNOS+ macrophages to approximately 50% of the LPS control (normalized to each experiment), although statistical significance was not reached; monotherapies showed weaker and variable trends and inhibited NF-κB p65 nuclear translocation in cultured macrophages (Holm-adjusted p < 0.01); MAPK signaling was not significantly altered under the present conditions. Anti-fibrotic effects were consistent with a macrophage-mediated mechanism, with no direct stellate cell suppression observed by LX-2 assays. Conclusions: Statin–Ezetimibe combination exerts anti-inflammatory and anti-fibrotic effects in a multi-hit murine model of MASH with advanced fibrosis associated with macrophage NF-κB inhibition in vitro and with reduced hepatic inflammatory signaling in vivo. These findings provide mechanistic rationale for the hepatic benefits of this widely used regimen in MASH patients with dyslipidemia, supporting its evaluation as a drug-repurposing strategy.