目的 基于蛋白组学方法探讨17-羟-岩大戟内酯B（HJB）通过System xc^--谷胱甘肽（GSH）-谷胱甘肽过氧化物酶4（GPX4）轴诱导乳腺癌MCF-7细胞铁死亡与线粒体凋亡的双重作用及协同机制，明确其抗乳腺癌的核心调控靶点。方法 采用MTT法检测HJB（5、10、20、40、80 μmol·L^-1）对乳腺癌MCF-7细胞活力的影响；通过流式细胞术与激光共聚焦显微镜检测HJB对MCF-7细胞凋亡、线粒体膜电位（JC-1）、活性氧（ROS）的影响；采用串联质谱蛋白组学技术获取HJB作用MCF-7细胞的亚细胞定位和差异表达蛋白；通过eggNOG和KAAS数据库进行基因本体（GO）以及京都基因与基因组百科全书（KEGG）富集；通过STRING数据库构建蛋白质-蛋白质相互作用（PPI）网络；基于DTNB显色法与GSH还原酶循环法，测定GSH/GSH/氧化型谷胱甘肽（GSSG）含量；采用RhoNox-6与CM-H₂DCFDA探针检测亚铁离子（Fe²⁺）与ROS水平；显色法检测总铁离子与Fe²⁺含量； WST-8法检测NAD⁺/还原型辅酶Ⅰ（NADH）含量，明确铁死亡相关表型；Western blotting实验检测相关蛋白的表达；分子对接技术验证HJB对相关核心靶点蛋白的结合能力。结果 MTT结果显示，与对照组比较，HJB可呈浓度与时间相关性抑制MCF-7细胞增殖，作用24、48、72 h的半数抑制浓度（IC₅₀）分别为30.49、23.93、19.30 μmol·L^-1； HJB显著诱导细胞凋亡，降低线粒体膜电位，上调MCF-7细胞内ROS表达水平（P＜0.01、0.001）。铁死亡相关指标检测显示，HJB组细胞内GSH含量降低、GSSG水平升高，GSH/GSSG比值下降； Fe²⁺特异性积累而总铁含量无明显变化，NAD⁺/NADH比值降低，细胞氧化还原稳态遭到破坏（P＜0.05、0.01、0.001） ； Western blotting证实HJB可下调溶质载体家族7成员11（SLC7A11）、谷氨酸半胱氨酸连接酶催化亚基（GCLC）、GR、谷胱甘肽过氧化物酶4（GPX4）蛋白表达，显著抑制System xc^--GSH-GPX4轴功能（P＜0.01、0.001） ；同时，HJB上调Bcl-2相关X蛋白（Bax）抗体、细胞色素C（Cyt C）、cleaved Caspase-7蛋白表达，下调Bcl-2蛋白表达，激活线粒体凋亡通路（P＜0.01、0.001）。蛋白组学分析共鉴定出2 136个差异表达蛋白，主要富集于GSH代谢与肿瘤相关通路，线粒体定位蛋白差异显著，与双通路调控的实验结果相互印证。分子对接结果显示，HJB可与GSH、GSSG、GPX4蛋白稳定结合。结论 HJB可通过抑制System xc^--GSH-GPX4轴触发细胞铁死亡，同时激活线粒体凋亡通路，双重途径协同杀伤乳腺癌MCF-7细胞。

Objective To investigate the dual effects and synergistic mechanism of 17-hydroxy jolkinolide B (HJB) on inducing ferroptosis and mitochondrial apoptosis in breast cancer MCF-7 cells via the System xc^--GSH-GPX4 axis based on proteomic approaches, and to identify its core regulatory targets against breast cancer. Methods The effect of HJB (5, 10, 20, 40, and 80 μmol·L^-1) on the viability of breast cancer MCF-7 cells was detected by MTT assay. Flow cytometry and laser confocal microscopy were applied to measure cell apoptosis, mitochondrial membrane potential (JC-1) and reactive oxygen species (ROS) levels. Tandem mass spectrometry-based proteomics was performed to analyze the subcellular localization and differentially expressed proteins (DEPs) in MCF-7 cells upon HJB treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the eggNOG and KAAS databases, and a protein-protein interaction (PPI) network was constructed via the STRING database. The contents of glutathione (GSH/GSSG) were determined by DTNB colorimetric assay and glutathione reductase cycling assay. The levels of intracellular ferrous iron (Fe²⁺) and ROS were detected using RhoNox-6 and CM-H2DCFDA fluorescent probes, respectively. Total iron and ferrous iron contents were measured by colorimetric assay, and the NAD⁺/NADH ratio was detected by WST-8 assay to clarify ferroptosis-related phenotypes. The expression levels of target proteins were detected by Western blotting. Molecular docking was performed to verify the binding affinity of HJB to core target proteins. Results MTT results showed that HJB significantly inhibited MCF-7 cell proliferation in a concentration- and time-dependent manner compared to the control group, with half-maximal inhibitory concentrations (IC₅₀) of 30.49, 23.93, and 19.30 μmol·L^-1 at 24, 48, and 72 h, respectively. HJB markedly induced apoptosis, reduced mitochondrial membrane potential, and upregulated intracellular ROS levels in MCF-7 cells (P<0.01, 0.001). Analysis of ferroptosis-related indicators revealed that HJB decreased intracellular GSH content while increasing GSSG levels, leading to a reduced GSH/GSSG ratio; Fe²⁺ accumulated specifically without significant changes in total iron content, and the NAD⁺/NADH ratio decreased, indicating disruption of cellular redox homeostasis (P<0.05, 0.01, 0.001). Western blotting confirmed that HJB downregulated the expression of SLC7A11, GCLC, GR, and GPX4 proteins, significantly suppressing the function of the System xc^--GSH-GPX4 axis (P<0.01, 0.001). Meanwhile, HJB upregulated the expression of Bax, cytochrome c (Cyt C), and cleaved Caspase-7 proteins, while downregulating Bcl-2 protein expression, thereby activating the mitochondrial apoptotic pathway (P<0.01, 0.001). Proteomic analysis identified 2 136 differentially expressed proteins, primarily enriched in GSH metabolism and tumor-related pathways, with significant differences observed in mitochondrion-localized proteins, consistent with the dual-pathway regulation demonstrated experimentally. Molecular docking results indicated that HJB could stably bind to GSH, GSSG, and GPX4 proteins. Conclusion HJB triggers cellular ferroptosis by inhibiting the System xc^--GSH-GPX4 axis and concurrently activates the mitochondrial apoptosis pathway. The two pathways exert synergistic cytotoxic effects on breast cancer MCF-7 cells.

R965

黑龙江省自然科学基金项目（LH2024H077）；黑龙江省博士后科研启动基金项目（LBH-QY22003）；黑龙江省省属本科高校基本科研业务费科研项目（2023-KYYWF-0861）；齐齐哈尔医学院重点学科建设资助项目（QYZDXK-008）