目的 探讨桔梗皂苷D（PD）诱导非小细胞肺癌（NSCLC）铁死亡的作用及机制。方法 体外实验采用A549细胞，添加PD（5、10、20 μmol·L^-1）作用6 h，通过MTT法及克隆形成实验检测细胞增殖能力；利用流式细胞术结合特异性荧光探针检测细胞内活性氧（ROS）；采用试剂盒测定丙二醛（MDA）含量；利用免疫荧光法检测细胞内Fe²⁺和脂质过氧化物水平；通过Western blotting法检测铁死亡相关蛋白谷胱甘肽过氧化物酶4（GPX4）、长链酰基辅酶A合成酶4（ACSL4）、铁蛋白重链（FTH1）、核受体共激活因子4（NCOA4）的表达；并使用铁死亡抑制剂Ferrostatin-1（Fer-1）和铁螯剂DFO验证PD的作用途径。体内实验采用Lewis肺癌（LLC）荷瘤C57BL/6J小鼠模型，观察PD（2、4、8 mg·kg^-1）对荷瘤小鼠肿瘤生长、体质量及血液学指标的影响，并对肿瘤组织进行病理学分析，同时通过蛋白质免疫印迹法检测瘤组织中铁死亡相关蛋白的表达。结果 PD抑制A549细胞活力与增殖（P＜0.05、0.01、0.001），诱导细胞内ROS积累、MDA含量升高、Fe²⁺水平升高、脂质过氧化物增加（P＜0.05、0.01、0.001），Fer-1和DFO可逆转PD的抑制作用（P＜0.05、0.01、0.001）。此外，PD显著下调GPX4与FTH1蛋白表达，上调ACSL4与NCOA4蛋白表达（P＜0.05、0.01、0.001）。体内实验中，PD各剂量组（2、4、8 mg·kg^-1）均能显著抑制LLC荷瘤小鼠肿瘤生长，且未引起明显不良反应；肿瘤组织中铁死亡相关蛋白表达趋势与体外一致。结论 PD可通过诱导铁死亡抑制NSCLC细胞体内外生长，其机制可能与调节GPX4/FTH1抗氧化轴及ACSL4/NCOA4介导的脂质代谢、铁存储蛋白降解有关。

Objective To investigate the effect and mechanism of platycodin D (PD) in inducing ferroptosis in non-small cell lung cancer (NSCLC). Methods In vitro experiments were conducted using A549 cells, with PD (5, 10, and 20 μmol·L^-1) added and incubated for 6 h. Cell proliferation was detected by MTT assay and colony formation assay. Intracellular reactive oxygen species (ROS) were detected by flow cytometry combined with specific fluorescent probes. Malondialdehyde (MDA) content was measured using a kit. Intracellular Fe²⁺ and lipid peroxide levels were detected by immunofluorescence. The expression of ferroptosis-related proteins, glutathione peroxidase 4 (GPX4), longS-chain acyl-CoA synthetase 4 (ACSL4), ferritin heavy chain (FTH1), and nuclear receptor coactivator 4 (NCOA4), was detected by Western blotting. The iron death inhibitor Ferrostatin-1 (Fer-1) and the iron chelator DFO were used to verify the action pathway of PD. In vivo experiments were conducted using a Lewis lung cancer (LLC) tumor- bearing C57BL/6J mouse model. The effects of PD (2, 4, 8 mg·kg^-1) on tumor growth, body weight, and hematological parameters of tumor-bearing mice were observed, and pathological analysis of tumor tissues was performed. The expression of ferroptosis-related proteins in tumor tissues was detected by Western blotting. Results PD inhibited the viability and proliferation of A549 cells (P<0.05, 0.01, 0.001), induced the accumulation of intracellular ROS, increased MDA content, elevated Fe²⁺ levels, and increased lipid peroxides (P<0.05, 0.01, 0.001). Fer-1 and DFO could reverse the inhibitory effect of PD (P<0.05, 0.01, 0.001). Additionally, PD significantly downregulated the expression of GPX4 and FTH1 proteins and upregulated the expression of ACSL4 and NCOA4 proteins (P<0.05, 0.01, 0.001). In the in vivo experiments, all doses of PD (2, 4, 8 mg·kg^-1) significantly inhibited tumor growth in LLC tumorbearing mice without causing obvious toxic side effects. The expression trend of ferroptosis-related proteins in tumor tissues was consistent with that in vitro. Conclusion PD inhibits the growth of NSCLC cells both in vitro and in vivo by inducing ferroptosis. The mechanism may involve the regulation of the GPX4/FTH1 antioxidant axis and ACSL4/NCOA4 mediated lipid metabolism and iron storage protein degradation.

R965

广西青年岐黄学者培育项目（GXQH202408）；广西青年科技人才托举工程项目（GXYESS2025031）