An integrated strategy combining network toxicology and feature-based molecular networking for exploring hepatotoxic constituents and mechanism of Epimedii Folium-induced hepatotoxicity in vitro

Epimedii Folium (EF), a commonly used herbal medicine to treat osteoporosis, has caused serious concern due to potential hepatotoxicity. Until now, its intrinsic hepatotoxic mechanism and hepatotoxic ingredients remain unclear. Here, a novel high-throughput approach was designed to investigate the intrinsic hepatotoxic of EF. High-content screen imaging (HCS) and biochemical tests were first performed to obtain the cytotoxicity parameter matrix of 17 batch EF samples. EF-treated alpha mouse liver 12 (AML12) cells showed increased reactive oxygen species (ROS), reduced glutathione (GSH) and mitochondrial membrane potential (MMP), and apoptosis and cholestasis were further observed. Network toxicology predicted that EF-triggered hepatotoxiciy was involved in transcription factor (TF) activity. The FXR expression, screened by a TF PCR array, exhibited down-regulation following EF extract administration. Moreover, EF inhibited bile acid (BA) metabolism pathway in an FXR-dependent manner. Pearson correlation between the cytotoxicity parameter matrix and quantification feature table obtained from UHPLC-QTOF data of EF suggested 7 prenylated flavonoids possessed potent hepatotoxicities and their cytotoxicity order was further summarized. The transcriptional repression effects of them on FXR were also verified. Collectively, our findings indicate that FXR is probably responsible for EF-induced hepatotoxicity and prenylated flavonoids may be a major class of hepatotoxic constituents in EF.