Genipin induces liver injury by disrupting the CPS1-regulated urea cycle and redox homeostasis

Genipin (GP) is the key metabolite of geniposide (GE), the primary active component of the traditional Chinese medicine Gardeniae Fructus. Its hepatotoxicity poses a potential risk to safe clinical administration, while its core pathophysiological mechanism remains unclear. This study systematically elucidated the molecular mechanisms of GP-induced hepatotoxicity through integrated multi-omics and functional validation. GP treatment induced significant liver damage and oxidative stress in rats, characterized by elevated serum transaminases (alanine aminotransferase (ALT) increased by approximately 63.5-fold; aspartate aminotransferase (AST) by 18.6-fold), an approximately 1.8-fold decrease in superoxide dismutase (SOD) activity, and an approximately 10-fold accumulation of malondialdehyde (MDA) (all P < 0.001). Metabolomic and proteomic analyses revealed that GP binds to carbamoyl phosphate synthetase 1 (CPS1), triggering urea cycle dysfunction marked by citrulline depletion and significant downregulation of its downstream product fumarate. In vitro experiments confirmed that disrupting CPS1 impairs the urea cycle and induces oxidative stress, while CPS1 overexpression or exogenous fumarate supplementation significantly reversed oxidative stress. Clinical sample analysis revealed similar urea cycle metabolic disturbances, characterized by citrulline depletion and reduced fumarate levels, in plasma from patients with liver injury due to inappropriate use of Gardeniae Fructus-containing formulas, confirming clinical relevance. This study reveals a novel hepatotoxic mechanism whereby GP targets CPS1 to disrupt the urea cycle, reducing fumarate production and impairing nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant defense, thereby inducing oxidative stress. These findings provide a scientific basis for improving the safety evaluation and rational clinical use of Gardeniae Fructus-containing preparations.