铁死亡是一种新兴的铁依赖性程序性细胞死亡模式，其特征是脂质过氧化和铁积累，与肝细胞癌（HCC）进展密切相关。尽管重楼素 I (PPI)（一种源自重楼的重要生物活性成分）对多种恶性肿瘤的影响已经确定，但 PPI 调节 HCC 铁死亡的具体作用和潜在机制途径仍然难以捉摸。本研究旨在阐明抗铁死亡的机制。 -PPI在HCC中诱导铁死亡和引发线粒体损伤的癌症特性和潜在机制。使用CCK-8测定法评估细胞活力。 EdU 增殖和集落形成测定用于评估细胞增殖。进行伤口愈合测定以评估细胞迁移。 Transwell实验用于评估细胞侵袭。通过使用 FerroOrange 荧光探针、丙二醛 (MDA) 和还原型谷胱甘肽 (GSH) 检测试剂盒、DCFH-DA 荧光探针、蛋白质印迹和透射电子显微镜 (TEM) 分析来评估铁死亡。采用分子对接、免疫荧光和蛋白质印迹来预测和验证 PPI 与 Nrf2、HO-1、xCT 和 GPX4 的结合和相互作用。使用 JC-1 和 Mito Tracker Green 荧光探针评估线粒体结构和膜电位变化。构建裸鼠异种移植模型，测定PPI通过苏木精和伊红（H）对HCC的抑制作用及铁死亡水平。

Ferroptosis is an emerging iron-dependent programmed cell death mode characterized by lipid peroxidation and iron accumulation, closely associated with Hepatocellular Carcinoma (HCC) progression. Although the impact of Polyphyllin I (PPI), a prominent bioactive constituent derived from Paris polyphylla, on diverse malignancies has been established, the specific role and potential mechanistic pathways through which PPI modulates ferroptosis in HCC remain elusive.This study aimed to elucidate the anti-cancer properties and potential mechanisms of PPI in inducing ferroptosis and triggering mitochondrial injury in HCC.Cell viability was assessed using CCK-8 assays. EdU proliferation and colony formation assays were employed to evaluate cell proliferation. A wound-healing assay was performed to assess cell migration. Transwell assay was utilized to evaluate cell invasion. Ferroptosis was evaluated through the utilization of a FerroOrange fluorescent probe, malondialdehyde (MDA) and reduced glutathione (GSH) assay kits, DCFH-DA fluorescent probe, western blotting, and transmission electron microscopy (TEM) analysis. Molecular docking, immunofluorescence, and western blotting were employed to predict and validate the binding and interaction of PPI with Nrf2, HO-1, xCT, and GPX4. Mitochondrial structure and membrane potential changes were evaluated using JC-1 and Mito Tracker Green fluorescent probes. A nude mice xenograft model was constructed to determine the inhibitory effects and the levels of ferroptosis of PPI on HCC through hematoxylin and eosin (H&E), Prussian blue reaction, immunofluorescence staining, immunohistochemistry, and western blotting analysis, in vivo.PPI exhibited dose-dependent inhibitory effects on the proliferation, invasion, and metastasis of HCC cells mediated by increasing reactive oxygen species (ROS) and MDA levels, promoting Fe2+ accumulation, depleting GSH, and suppressing the expression of xCT and GPX4, thereby inducing ferroptosis in HCC. The induction of ferroptosis by PPI was associated with the binding of PPI to Nrf2, HO-1, and GPX4 proteins, modulating the Nrf2/HO-1/GPX4 antioxidant axis. PPI also induced mitochondrial structural damage and decreased mitochondrial membrane potential (MMP). Inhibition of ferroptosis by ferrostatin-1 (Fer-1) mitigated the mitochondrial disruption induced by PPI. In vivo, PPI inhibited Nrf2/HO-1/GPX4 axis-induced ferroptosis, impeding HCC growth similar to the effects of sorafenib.These results demonstrated that PPI intervention can suppress the proliferation, invasion, and metastasis of HCC cells by enhancing mitochondrial disruption and inducing ferroptosis via the Nrf2/HO-1/GPX4 axis. Consequently, our research advances the frontiers of pharmacodynamics and deepens our comprehension of the intricate mechanisms underpinning PPI. Furthermore, it has yielded an innovative treatment stratagem rooted in the tenets of Traditional Chinese Medicine (TCM), thereby furnishing a novel therapeutic avenue for addressing HCC.Copyright © 2023 Elsevier GmbH. All rights reserved.