Rebamipide Reprograms Hepatic Networks to Prevent and Reverse Metabolic-Dysfunction-Associated Steatotic Liver Disease: Multi-Omics Insights and Histological Validation

Background: Metabolic-dysfunction-associated steatotic liver disease (MASLD) is a growing global health burden, yet no approved pharmacological therapy currently exists. Purpose: The purpose of this study is to investigate the prophylactic and therapeutic potential of Rebamipide, a mucosal-protective and anti-inflammatory drug, in a high-fat diet (MHFD)-induced MASLD rat model, integrating quantitative liver proteomics, network analysis, and histopathology. Methods: Male Wistar rats were fed MHFD for 16 weeks and treated with Rebamipide either prophylactically (Reb T1, co-administered with diet) or therapeutically (Reb T2, administered post-NASH onset). Label-free LC-MS/MS proteomics combined with principal component analysis (PCA), partial squares discriminant analysis (PLS-DA), and enrichment analyses (including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome via g: Profiler, network mapping, and Rat Genome Database (RGD) mining) revealed that MHFD had the following impacts: it induced the profound suppression of mitochondrial chaperones (Hspa9), microsomal triglyceride transfer protein (Mttp), and cytochrome P450 isoforms (Cyp2c6); it disrupted lipid trafficking, oxidative stress defense, and xenobiotic metabolism. Results: Rebamipide prophylaxis preserved lipid-handling proteins, prevented glycogen loss, and maintained antioxidant defenses. In contrast, therapeutic administration reversed established steatosis and remodeled metabolic pathways, enhancing fatty acid β-oxidation, detoxification, and mitochondrial protein import. Nine shared proteins across all comparisons, including MTTP and multiple Stress-70 mitochondrial isoforms, mapped to three core genes (Mttp, Cyp2c6, Hspa9) central to lipid transport, protein import, and metabolic stress adaptation. KEGG and Reactome analyses highlighted Rebamipide’s modulation of bile acid synthesis, ceramide and phosphatidylcholine metabolism, lipoprotein remodeling, and MAPK signaling. Histopathological evaluation confirmed Rebamipide’s efficacy, showing reduced steatosis and the normalization of the hepatocyte structure, with near-complete restoration in the therapeutic (Reb T2) group compared to partial protection in the Reb T1 group. Conclusions: These findings demonstrate Rebamipide’s dual-phase, multi-targeted mechanism: early protection against diet-induced metabolic injury and robust reversal of established MASLD pathology. The identified protein triad (Mttp, Cyp2c6, Hspa9) and associated pathways provide novel biomarker candidates and mechanistic insight supporting Rebamipide’s repurposing as a therapeutic for metabolic liver disease.