D-limonene ameliorates metabolic dysfunction-associated steatotic liver disease by inhibiting the PPARγ/SCD-1 pathway and improving lipid metabolism disorders.

Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a highly prevalent chronic liver disorder characterized by intrahepatic lipid accumulation, oxidative stress, and inflammatory responses, underscoring the urgent need for effective therapeutic strategies. D-limonene, a cyclic monoterpenoid primarily derived from citrus fruits, possesses well-documented antioxidant, anti-fibrotic, and wound-healing properties. However, its therapeutic potential in MASLD remains largely unexplored. Methods: To evaluate the therapeutic efficacy of D-limonene, a rat model of MASLD was established using a high-fat diet (HFD). An integrated approach combining metabolomics, transcriptomics, and network analysis was employed to identify key signaling pathways and molecular targets. The expression levels of candidate targets were validated by RT-qPCR and Western blotting. Molecular docking simulations were performed to predict the binding affinity between D-limonene and the target proteins. Furthermore, a cellular thermal shift assay (CETSA) was conducted to confirm direct binding and protein stabilization. In parallel, cellular experiments using BRL-3A and HepG2 cells were carried out to assess the effect of D-limonene on lipid accumulation. Results: D-limonene treatment significantly ameliorated liver injury, reduced excessive lipid deposition, attenuated inflammation, and suppressed oxidative stress in HFD-induced rats. Integrated multi-omics and network analyses identified the PPAR signaling pathway as the core pathway mediating the regulatory effects of D-limonene on lipid metabolism, with PPARγ and SCD-1 pinpointed as key downstream targets. RT-qPCR and Western blot analyses confirmed that D-limonene significantly downregulated the expression of both PPARγ and SCD-1. Molecular docking simulations revealed stable binding of D-limonene to PPARγ and SCD-1. Importantly, CETSA further demonstrated that D-limonene directly binds to and stabilizes these two proteins. Consistently, cellular assays showed that D-limonene significantly reduced lipid accumulation in BRL-3A and HepG2 cells by inhibiting the PPARγ/SCD-1 axis. Conclusion: Collectively, these findings suggest that D-limonene alleviates HFD-induced MASLD, and its protective effects may be mediated, at least in part, through inhibition of the PPARγ/SCD-1 axis. This study provides preliminary evidence supporting the potential development of D-limonene as a targeted therapeutic agent for MASLD. Further studies are warranted to validate these findings and elucidate additional underlying mechanisms.