Integrated Transcriptomic and Metabolomic Analyses Reveal the Protective Mechanism of Icaritin Against High‐Fat Diet‐Induced Metabolic Dysfunction‐Associated Steatotic Liver Disease in Mice

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent chronic liver disease. Icaritin (ICT) has demonstrated potential hepatoprotective effects, while its protective mechanisms on MASLD are still unclear. This study aims to investigate the therapeutic efficacy of ICT against MASLD and elucidate its underlying molecular mechanisms. A MASLD mouse model was established via a high-fat diet (HFD) for 12 weeks, with or without gavage of ICT for 4 weeks. Palmitic acid (PA) was used to induce an in vitro model in AML12 hepatocytes. Histological, biochemical, transcriptomic (RNA-Seq), metabolomic, and lipidomic analyses were employed. Key targets were validated using molecular docking, cellular thermal shift assay (CETSA), and gene knockdown approaches. ICT treatment ameliorated HFD-induced hepatic steatosis, dyslipidemia, and reversed the suppression of reverse cholesterol transport genes. The expression of key genes identified by RNA sequencing was verified by RT-qPCR. Integration of transcriptomics and metabolomics revealed that ICT reshaped transcriptomic and metabolomic profiles, highlighting key pathways in glycogen metabolism, lipid metabolism, and antioxidant responses. Both in vivo and in vitro, ICT reversed the downregulation of GSTA1 expression. Molecular docking and CETSA confirmed a direct binding interaction between ICT and the GSTA1 protein. GSTA1 knockdown in AML12 cells abolished the protective effects of ICT. ICT alleviates MASLD progression by targeting GSTA1-mediated metabolic reprogramming, providing a novel mechanistic foundation for ICT as a promising candidate for MASLD treatment.