Single-nucleus RNA Sequencing and multi-omics reveal Uncaria-derived indole alkaloids induce hepatocyte injury by mediating CAR/PPARα axis.

Risk assessment and management of endogenous potentially toxic components are critical for promoting the rational clinical use of herbal medicines. However, most herbal medicines lack sufficient safety data in clinical, especially for those with multiple botanical sources. As a commonly used multi-botanical source herbal medicine, Uncariae Ramulus Cum Uncis-derived indole alkaloids (IA-URCU) are primarily responsible for its potential hepatotoxicity in clinical practice. Nevertheless, the underlying mechanism of URCU-induced hepatocyte injury remains unclear. This study aimed to investigate the mechanism of IA-URCU-induced hepatocyte injury using a multidisciplinary approach, thereby providing a critical foundation for its safety assessment. In this study, we employed an "Integrated Toxicology" strategy-incorporating analyses of toxic effects, toxic substances, mechanisms, and compound interactions-combined with single-nucleus RNA sequencing (snRNA-seq) and multi-omics to characterize the cellular response heterogeneity to IA-URCU -induced liver injury at the single-cell level. IA-URCU could disrupt hepatic lipid homeostasis by activating CAR/NR1I3, which promoted their proliferation and transdifferentiation from the periportal hepatocyte region to the pericentral hepatocyte region. Concurrently, the activation of CAR could antagonize the expression of PPARα with binding to RXRA. This disruption leads to the accumulation of specific metabolites, including LysoPC (20:0) and PC (18:1/18:1), which in turn stimulated the release of IL-6 and IL-8 and ultimately provoked liver inflammation. This study advances the understanding of how IA-URCU induces hepatocyte injury at the single-cell level by promoting hepatocyte proliferation and differentiation, which subsequently disrupts lipid metabolism and triggers inflammatory responses. These findings not only provide the guidance for the rational clinical application of URCU but also offer new insights and methodologies for establishing a scientific supervision system based on risk assessment.