Genipin rewires endogenous amino acid metabolism and induces hepatocellular necroptosis by activation of the TNF-necrosome signaling axis.

Genipin, mainly derived from the Chinese herbal medicines Gardenia jasminoides Ellis and Eucommia ulmoides Oliv., is an iridoid derivative with multiple pharmacological activities. However, genipin can exert hepatotoxicity, which limits its application. To identify the underlying mechanisms of genipin hepatotoxicity, we integrated various biochemical readouts, transmission electron microscopy, flow cytometry, and targeted metabolomics. In mice, genipin significantly induced alanine aminotransferase (ALT; 53.0 ± 14.4 vs. 37.4 ± 3.1 in controls) and aspartate aminotransferase (AST) levels (146.5 ± 19.7 vs. 115.8 ± 17.4 in controls), which was paralleled by a rewiring of hepatic amino acid metabolism and an induction of oxidative stress. Molecular docking indicated that genipin could directly bind TNFR1 while inhibiting caspase-8. Consequently, phosphorylation of RIPK1, RIPK3, and MLKL was increased by genipin, triggering a hepatocellular necroptotic program both in vitro and in vivo. Administration of GSK-872, a selective RIPK3 inhibitor, attenuated genipin-induced necroptosis. Combined, these results indicate that genipin triggers metabolic stress and activates the necrosome by inhibiting caspase-8, leading to cell death signals being diverted from apoptosis towards a necroptotic program. These findings clarify the molecular mechanisms underlying genipin hepatotoxicity and promote future development of safer genipin derivatives.