Repurposed metformin-apigenin combination therapy for metabolic dysfunction-associated steatohepatitis: from integrative multi-omics discovery to in vivo validation.

BACKGROUND: Metabolic dysfunction-associated steatohepatitis (MASH) is a chronic liver disease. Although recent drugs have been approved, the complex pathophysiology of MASH limits monotherapy efficacy and generates considerable inter-patient heterogeneity. The purpose of this research was to identify new molecular targets and develop a rational combination therapeutic approach for MASH. METHODS: To determine the molecular signatures of MASH mouse models and healthy controls, we conducted bulk RNA sequencing on liver tissues and then analyzed the data with a variety of Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway enrichment, weighted gene co-expression network analysis, and so on. The role of lipidomic profiling was to determine the relation between genes and lipids. The human MASH liver specimens were used to test clinical relevance. Causal relationships were measured using Mendelian randomization (MR). Direct interaction between the drugs and the target was predicted by molecular docking and molecular dynamics simulations. The efficacy of metformin and apigenin as single therapy and in combination was assessed in vivo using MASH mice. RESULTS: We discovered a five-gene signature (Gpd2, Cybb, Clec12a, Ifi211, and Tifab), which was highly expressed by MASH and was strongly connected to lipid metabolism and inflammatory events. Integrative lipidomic correlation analysis showed that these genes were specifically linked with certain lipotoxic species. Molecular docking and molecular dynamics simulation studies have shown that metformin and apigenin, respectively, bind to Gpd2 and Cybb directly. Both agents alleviated the MASH pathology, and combined therapy was more effective than monotherapy. CONCLUSION: This study provides a new five-gene signature transcending hepatic lipid dysregulation and inflammation associated with MASH. The demonstration that metformin targets Gpd2 and apigenin targets Cybb provides a mechanistic basis for combination therapy, which exhibited superior efficacy over monotherapy. This repurposed oral drug combination offers a promising multi-target strategy for MASH treatment.