最常见的恶性骨肿瘤是骨肉瘤，其预后不理想，治疗效果也不理想。铁死亡有望成为一种有效的 OS 疗法。从紫草中提取的物质紫草素 (SHK) 可抑制多种肿瘤，包括卵巢癌、胃癌和肺癌。然而，SHK是否会引起OS铁死亡及其机制尚不清楚。本研究旨在探讨SHK是否会导致OS铁死亡并阐明其分子机制。采用细胞计数Kit-8、细胞周期和细胞凋亡检测来评估SHK的治疗效果反对操作系统。使用正常细胞（包括 H9C2、AML-12 和 HK-2）、苏木精和伊红染色以及小鼠血清生物标志物测试来评估 SHK 的生物安全性。丙二醛 (MDA) 水平、谷胱甘肽 (GSH)/氧化型谷胱甘肽 (GSSG) 比值、活性氧 (ROS)、脂质过氧化物 (LPO) 和细胞内 Fe2 检测、定量逆转录 PCR (qRT-PCR)、蛋白质印迹 (采用WB)和救援实验来确认SHK是否诱导OS细胞中的铁死亡。采用分子对接、细胞热位移测定（CETSA）和药物亲和力响应靶稳定性（DARTS）测定来评估SHK与缺氧诱导因子-1α（HIF-1α）之间的直接结合。利用蛋白质稳定性和降解分析、小RNA干扰、流式细胞术、qRT-PCR和WB来研究铁死亡的分子机制。采用裸鼠体内异种移植物研究SHK的抗OS作用。SHK显着降低OS细胞活力并诱导细胞凋亡和G2/M阻滞。 SHK 增加细胞内 MDA、ROS、LPO 和 Fe2 水平，同时降低 GSH/GSSG 比率以及 GPX4 和 SLC7A11 表达。 CETSA 和 DARTS 结果表明 SHK 并未靶向结合 HIF-1α。相反，线粒体 ROS (MitoROS) 促进 HIF-1α 表达，导致 HO-1 过度表达、Fe2 生成过多、ROS 积累和 GPX 耗尽以及铁死亡。此外，使用 Mito-TEMPO 抑制 MitoROS 下调 HIF-1α/HO-1 轴并减轻 SHK 诱导的铁死亡。在体内，SHK 有效抑制 OS 生长，具有良好的生物安全性，证实了 SHK 诱导 OS 铁死亡的分子机制。我们观察到 HIF-1α/HO-1 轴是 SHK 诱导 OS 铁死亡的关键因素。重要的是，我们证明 HIF-1α 是由 MitoROS 间接调节的，而不是 SHK 直接与 HIF-1α 结合。我们的研究表明，SHK 是 OS 治疗的潜在候选药物，可能有助于确定 OS 的新治疗靶点。版权所有 © 2024 Elsevier GmbH。版权所有。

The most common malignant bone tumour is osteosarcoma, which has an unsatisfactory prognosis and unsatisfactory treatment. Ferroptosis shows promise as an effective OS therapy. A substance extracted from the Lithospermum erythrorhizon, Shikonin (SHK), inhibits a number of tumours, including ovarian, gastric, and lung cancers. However, whether SHK induces OS ferroptosis and its mechanisms are not clear.Our study is aimed at investigating whether SHK causes ferroptosis and elucidating its molecular mechanism.Cell counting Kit-8, cell cycle and cell apoptosis assay were utilised to assess therapeutic effect of SHK against OS. Normal cells, including H9C2, AML-12 and HK-2, haematoxylin and eosin staining and mice serum biomarker tests were used to assess SHK biosafety. Malondialdehyde (MDA) levels, the glutathione (GSH)/oxidized glutathione (GSSG) ratio, reactive oxygen species (ROS), lipid peroxide (LPO), and intracellular Fe2+ detection, quantitative reverse transcription PCR (qRT-PCR), Western blotting (WB) and rescue experiments were employed to confirm whether SHK induced ferroptosis in OS cells. Molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay were used to evaluate the direct binding between SHK and hypoxia-inducible factor-1α (HIF-1α). Protein stability and degradation analysis, small RNA interference, flow cytometry, qRT-PCR, and WB were used to investigate the molecular mechanism of ferroptosis. In vivo xenografts of nude mouse was used to study the anti-OS effect of SHK.SHK significantly reduced OS cell viability and induced apoptosis and G2/M arrest. SHK increased intracellular levels of MDA, ROS, LPO, and Fe2+ while simultaneously reducing the GSH/GSSG ratio and GPX4 and SLC7A11 expression. CETSA and DARTS results demonstrated that SHK did not bind targetly to HIF-1α. Instead, mitochondrial ROS (MitoROS) promoted HIF-1α expression, resulting in HO-1 overexpression, excess Fe2+ production, ROS accumulation and GPX depletion, and ferroptosis. Furthermore, inhibition of MitoROS using Mito-TEMPO downregulated HIF-1α/HO-1 axis and mitigated the SHK-induced ferroptosis. In vivo, SHK effectively suppressed OS growth with favourable biosafety, confirming the molecular mechanism underlying SHK-induced ferroptosis in OS.We observe that HIF-1α/HO-1 axis is the crucial factor in SHK-induced OS ferroptosis. Importantly, we demonstrate that HIF-1α is indirectly regulated by MitoROS rather than SHK bound directly to HIF-1α. Our research suggest that SHK is a potential drug candidate for OS treatment and may help in identifying novel therapeutic targets for OS.Copyright © 2024 Elsevier GmbH. All rights reserved.