Mechanistic insights into cholesterol-mediated MASH pathogenesis: Glycation, lipoxidation, and RAGE pathway

The pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH) is multifactorial, with evidence suggesting a pathogenic role for the AGE/ALE-RAGE axis. Although cholesterol and the AGE/ALE axis have been linked, evidence of their association in MASH remains limited. In this study, we investigated the role of the AGE/ALE-RAGE axis in cholesterol-driven MASH progression. Male C57BL/6 mice were fed either a short- or long-term high-fat, high-carbohydrate, high-cholesterol diet to induce MASH. In a subgroup of mice with MASH, the pharmacological RAGE inhibitor FPS-ZM1 (1 mg/kg/day) was administered. Mechanistic insights were further explored in vitro using HepG2 cells exposed to free fatty acids (FFAs) and oxidized LDL (oxLDL) to mimetize diet-induced MASH. Body and liver weights, adipose tissue distribution, blood glucose, liver histology, inflammatory cytokines, oxidative stress, and AGE/ALE signaling were assessed. In HepG2 cells, inflammation, dicarbonyl stress, and AGE formation were evaluated. Cholesterol supplementation exacerbated MASH progression, increasing steatosis, inflammation, hepatocellular ballooning, and fibrosis, accompanied by activation of the AGE/ALE-RAGE axis, redox imbalance, and inflammation. RAGE blockade improved hepatic lipid accumulation, steatosis, inflammation, hepatocellular ballooning, AGE levels, and restored antioxidant capacity. In vitro, oxLDL induced the inflammatory response and promoted dicarbonyl stress. Collectively, these findings identify dietary cholesterol as a key modulator of MASH. Importantly, pharmacological inhibition of RAGE with FPS-ZM1 effectively reversed key pathological features, supporting a functional contribution of RAGE signaling to cholesterol-associated disease severity.