目的 基于蛋白组学、分子对接和实验验证探究羽扇豆醇抗乳腺癌MCF-7细胞增殖的作用机制。方法 采用MTT法检测羽扇豆醇(5、10、20、40、80、160μmol·L^-1)对乳腺癌MCF-7细胞活力的影响；流式细胞术检测细胞凋亡率和活性氧水平的变化情况；蛋白组学预测靶点通路，通过AutoDockTools软件对羽扇豆醇与核糖核苷酸还原酶调节亚基M2（RRM2）蛋白进行分子对接；酶标仪检测细胞内Ca²⁺、丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）浓度以及铁含量；Western blotting法检测钙失衡、凋亡、铁死亡相关蛋白表达水平；羽扇豆醇联用铁死亡抑制剂Ferrostatin-1（Fer-1），检测细胞中的MDA、SOD、GSH和铁水平以及铁死亡相关蛋白表达水平。结果 羽扇豆醇作用MCF-7细胞24、48、72 h的半数抑制浓度(IC₅₀)值分别为（93.43±1.87）、（63.02±1.56）、（41.58±1.23）μmol·L^-1，且与对照组比较，可显著诱导细胞凋亡、升高活性氧水平（P<0.01、0.001）；蛋白组学富集分析结果在铁死亡高度富集，羽扇豆醇与RRM2蛋白的结合自由能为-33.64 kJ·mol^-1。与对照组比较，羽扇豆醇组MCF-7细胞内Ca²⁺浓度显著升高（P<0.01、0.001）,2型兰尼碱受体（Ry R2）蛋白表达显著上调（P<0.01、0.001），三磷酸肌醇受体1（IP3R1）蛋白表达呈上调趋势，从而引发钙稳态失衡；高钙环境进一步激活凋亡通路，Bcl-2相关X蛋白（Bax）、细胞色素C（Cyt C）蛋白表达显著上调（P<0.01、0.001）,B细胞淋巴瘤2（Bcl-2）蛋白表达显著下降（P<0.001）;MDA含量显著升高（P<0.01、0.001）,SOD活性显著降低（P<0.01、0.001），同时铁水平显著增加（P<0.01、0.001）,GSH水平显著降低（P<0.01、0.001）；酰基辅酶A合成酶长链家族成员4（ACSL4）蛋白表达显著升高（P<0.001）,RRM2、溶质载体家族7成员11（SLC7A11）、谷胱甘肽过氧化物酶4（GPX4）蛋白表达显著下降（P<0.001）；与羽扇豆醇组比较，Fer-1+羽扇豆醇组的MDA和铁含量显著降低（P<0.001）,SOD和GSH水平显著升高（P<0.001）,RRM2、SLC7A11、GPX4蛋白表达显著升高（P<0.001）,ALSL4蛋白表达显著降低（P<0.001），几乎恢复至对照组水平。结论 羽扇豆醇可抑制乳腺癌MCF-7细胞增殖，诱导细胞凋亡，同时能够诱导乳腺癌MCF-7细胞发生铁死亡，其作用机制可能与RRM2低表达及SLC7A11/GPX4通路被抑制密切相关。

Objective To explore the mechanism of lupeol against the proliferation of breast cancer MCF-7 cells based on proteomics, molecular docking, and experimental verification. Methods The effects of lupeol(5, 10, 20, 40, 80, 160 μmol·L^-1) on the viability of breast cancer MCF-7 cells were detected by MTT assay; The apoptosis rate and reactive oxygen species（ROS） levels were detected by flow cytometry; The target pathways were predicted by proteomics, and the molecular docking of lupeol with the regulatory subunit M2（RRM2） of ribonucleotide reductase was performed by AutoDockTools software; The intracellular Ca²⁺, malondialdehyde（MDA）, superoxide dismutase（SOD）, glutathione（GSH） concentrations and iron content were detected by microplate reader; the expression levels of proteins related to calcium imbalance, apoptosis and ferroptosis were detected by Western blotting; lupeol was combined with the ferroptosis inhibitor Ferrostatin-1（Fer-1）, and the levels of MDA, SOD, GSH and iron in cells and the expression levels of proteins related to ferroptosis were detected. Results The half-maximal inhibitory concentration(IC₅₀) values of lupeol on MCF-7 cells for 24, 48 and 72 h were（93.43 ±1.87）,（63.02 ±1.56） and（41.58 ±1.23） μmol·L^-1, respectively. Compared with the control group, lupeol could significantly induce apoptosis and increase ROS levels（P ＜ 0.01, 0.001）. The enrichment analysis of proteomics was highly enriched in ferroptosis, and the binding free energy of lupeol with RRM2 protein was-33.64 kJ·mol^-1. Compared with the control group, the intracellular Ca²⁺ concentration in the lupeol group was significantly increased（P ＜ 0.01, 0.001）, and the expression of type 2 ryanodine receptor（RyR2） protein was significantly upregulated（P ＜ 0.01, 0.001）, while the expression of inositol 1,4,5-trisphosphate receptor 1（IP3R1） protein showed an upward trend, thereby causing calcium homeostasis imbalance. The high calcium environment further activated the apoptosis pathway, and the expression of Bcl-2 associated X protein（Bax） and cytochrome C（Cyt C） proteins was significantly upregulated（P ＜ 0.01, 0.001）, while the expression of B-cell lymphoma 2（Bcl-2） protein was significantly downregulated（P ＜ 0.001）. The MDA content was significantly increased（P ＜ 0.01, 0.001）, the SOD activity was significantly decreased（P ＜ 0.01, 0.001）, and the iron level was significantly increased（P ＜ 0.01, 0.001）, while the GSH level was significantly decreased（P ＜ 0.01, 0.001）. The expression of acyl-CoA synthetase long-chain family member 4（ACSL4） protein was significantly increased（P ＜ 0.001）, while the expression of RRM2, solute carrier family 7 member 11（SLC7A11）, and glutathione peroxidase 4（GPX4） proteins was significantly decreased（P ＜ 0.001）. Compared with the lupeol group, the MDA and iron content in the Fer-1 + lupeol group were significantly decreased（P ＜ 0.001）, the SOD and GSH levels were significantly increased（P ＜ 0.001）, the expression of RRM2, SLC7A11, and GPX4 proteins was significantly increased（P ＜ 0.001）, and the expression of ACSL4 protein was significantly decreased（P ＜ 0.001）, almost returning to the level of the control group. Conclusion Lupeol can inhibit the proliferation of breast cancer MCF-7 cells and induce cell apoptosis; meanwhile, it can also induce ferroptosis in breast cancer MCF-7 cells, and its mechanism may be closely related to the downregulated expression of RRM2 and the inhibition of the SLC7A11/GPX4 pathway.

R285.5

黑龙江省博士后科研启动金资助项目(LBH-QY24003); 齐齐哈尔医学院重点学科建设资助项目(QYZDXK-008)