Background: Maslinic acid (MA), a pentacyclic triterpenoid found in plants, has beneficial effects at low doses, but its impact on liver damage and cholesterol gallstone (CGS) formation at high doses remains unclear. This study explored the effects of high-dose MA on liver damage and CGS formation in C57BL/6J mice.

Methods: This study used animal model experiments, network pharmacology, RNA transcriptome sequencing, molecular docking, and molecular dynamics simulation to explore the molecular mechanism by which high-dose MA promotes CGS formation and cholestatic liver injury (CLI).

Results: High-dose MA significantly promoted CGS formation (p < 0.05) and induced CLI in mice fed a lithogenic diet. Gallstone severity progressed with MA administration, accompanied by significant alterations in biliary lipid composition: increased cholesterol levels alongside decreased phospholipid and bile acid concentrations (p < 0.05), collectively leading to an elevated cholesterol saturation index (CSI, p < 0.05). Serum biochemical analysis confirmed hepatobiliary injury, showing significantly elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bile acids (p < 0.05). Histopathological examination revealed hepatic inflammatory infiltration and structural damage. Mechanistically, MA downregulated key proteins involved in bile acid synthesis and biliary excretion, while modulating regulators of cholesterol and drug metabolism. Transcriptomic profiling revealed that MA significantly disrupted pathways related to bile acid, cholesterol, and drug metabolism, particularly the Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway and bile secretion. Integrated network pharmacology, molecular docking, and dynamics simulations identified PPARα and PPARγ as core targets, with MA showing stable binding to multiple gallstone-related proteins. Subsequent validation confirmed that MA increased PPARα expression and decreased PPARγ expression (p < 0.05), supporting the conclusion that MA promotes gallstone formation by interfering with the PPAR signaling pathway and bile acid homeostasis.

Conclusion: Our findings indicate that high-dose MA promotes CGS formation via the PPAR signaling pathway, highlighting the potential risks of MA-induced CLI and providing a basis for assessing its safety and developing targeted interventions.