Toosendanin induced hepatotoxicity via triggering PERK-eIF2α-ATF4 mediated ferroptosis

Toosendanin (TSN) is the main active compound of Melia toosendan Sieb et Zucc with various bioactivities. In this study, we investigated the role of ferroptosis in TSN-induced hepatotoxicity. The characteristic indicators of ferroptosis were detected including reactive oxygen species (ROS), lipid-ROS, glutathione (GSH), ferrous ion and the expression of glutathione peroxidase 4 (GPX4), which showed that TSN caused ferroptosis in hepatocytes. The results of qPCR analysis and western blotting assay showed that TSN-induced activation of protein kinase R-like endoplasmic reticulum kinase (PERK)- eukaryotic initiation factor 2 α subunit (eIF2α)- activation transcription factor 4 (ATF4) signaling pathway resulted in increasing activation transcription factor 3 (ATF3) expression, which upregulated the expression of transferrin receptor 1 (TFRC). Furthermore, TFRC mediated iron accumulation leading to ferroptosis in hepatocytes. To clarify whether TSN triggered ferroptosis in vivo, male Balb/c mice were treated with the different doses of TSN. The results of hematoxylin-eosin (H&E) staining, 4-hydroxynonenal (4-HNE) staining, malondialdehyde (MDA) content and the protein expression of GPX4 showed that ferroptosis contributed to TSN-induced hepatotoxicity. Iron homeostasis relative protein and PERK- eIF2α- ATF4 signaling pathway also involved in hepatotoxicity of TSN in vivo.