目的 探究异甘草素联合阿霉素对三阴性乳腺癌细胞MDA-MB-231侵袭和转移的作用机制。方法 MTT实验检测异甘草素（10、20、30、40 μmol·L^-1）联合阿霉素（0.5、1.0、2.0 μmol·L^-1）对细胞活力的影响；通过流式细胞术、划痕及Transwell侵袭实验分别分析异甘草素（20 μmol·L^-1）、阿霉素（1.0 μmol·L^-1）、异甘草素（20 μmol·L^-1）联合阿霉素（1.0 μmol·L^-1）对细胞周期及侵袭能力的影响； Western blotting检测细胞周期关键蛋白表达；应用转录组学筛选异甘草素联合阿霉素作用于TNBC细胞的信号通路，并通过分子对接及Western blotting对筛选结果进行验证。结果 与对照组相比，异甘草素和阿霉素呈时间和浓度相关性抑制MDA-MB-231细胞的增殖（P＜0.05、0.01），所选浓度组合均表现出协同作用，其对应的联合指数（CI）值均小于1；当阿霉素浓度为1 μmol·L^-1、异甘草素浓度为20 μmol·L^-1时，CI值达到最低（0.62），协同效应最强。流式细胞术显示异甘草素＋阿霉素处理可将细胞阻滞于S期，且对P53蛋白的上调及CCND1蛋白的下调作用显著（P＜0.05、0.01），效果优于单药组。划痕及Transwell实验表明，联合用药组抑制细胞迁移及侵袭的抑制作用较单药组更为明显（P＜0.01）。Western blotting显示，与对照组比较，联合用药组Vimentin、β-catenin表达减少，Ecadherin表达升高（P＜0.05、0.01），效果优于单药组。转录组分析发现PI3K-Akt通路调节上皮-间质转化可能参与联合用药组对MDA-MB-231细胞的调节作用；分子对接实验验证，异甘草素＋阿霉素组与PI3K、Akt靶蛋白的结合能均显著低于单独用药组； Western blotting结果表明，与对照组比较，异甘草素、阿霉素单独或联合处理MDA-MB-231细胞后可抑制pPI3K、p-Akt的表达，联合用药组和异甘草素组差异显著（P＜0.05、0.01），联合给药组作用最明显。结论 异甘草素能通过抑制PI3K-Akt信号通路，调控EMT相关蛋白，从而增效阿霉素抑制MDA-MB-231细胞增殖与迁移的作用。

Objective To investigate the mechanism by which isoliquiritigenin combined with doxorubicin affects invasion and metastasis of triple-negative breast cancer (TNBC) MDA-MB-231 cells. Methods The effect of isoliquiritigenin (10, 20, 30, and 40 μmol·L^-1) combined with doxorubicin (0.5, 1.0, and 2.0 μmol·L^-1) on cell viability was assessed using the MTT assay. Flow cytometry, scratch, and Transwell invasion assays were used to analyze the effects of isoliquiritigenin (20 μmol·L^-1), doxorubicin (1.0 μmol·L^-1), and their combination (20 μmol·L^-1 isoliquiritigenin + 1.0 μmol·L^-1 doxorubicin) on cell cycle progression and invasive capacity. Western blotting was performed to detect expression of key cell cycle proteins. Transcriptomics was applied to screen signaling pathways involved in the action of isoliquiritigenin plus doxorubicin on TNBC cells, and molecular docking and Western blotting were used to validate the results. Results Compared with the control group, both isoliquiritigenin and doxorubicin inhibited proliferation of MDAMB-231 cells in a time- and concentration-dependent manner (P<0.05, 0.01). All selected concentration combinations showed synergistic effects, with corresponding combination index (CI) values all less than 1. The lowest CI value (0.62), indicating the strongest synergistic effect, was observed at 1 μmol·L^-1 doxorubicin and 20 μmol·L^-1 isoliquiritigenin. Flow cytometry revealed that treatment with isoliquiritigenin plus doxorubicin arrested cells in the S phase and significantly upregulated P53 protein while downregulating CCND1 protein (P<0.05, 0.01), with effects superior to those of single-agent treatments. Scratch and Transwell assays demonstrated that the combination therapy more effectively suppressed cell migration and invasion compared to monotherapy (P<0.01). Western blotting showed that, compared with the control group, the combination group exhibited reduced expression of Vimentin, β-catenin and increased expression of E-cadherin (P<0.05, 0.01), with better outcomes than either single agent. Transcriptomic analysis suggested that the PI3K-Akt pathway regulating epithelial-mesenchymal transition (EMT) may be involved in the regulatory effects of the combination on MDA-MB-231 cells. Molecular docking confirmed that the binding energies of isoliquiritigenin and doxorubicin to PI3K and Akt target proteins were significantly lower in the combination group than in the single-agent groups. Western blotting results indicated that, compared with the control group, isoliquiritigenin, doxorubicin, or their combination significantly suppressed pPI3K and p-Akt expression, with the most pronounced differences observed between the combination group and the control group (P<0.05, 0.01), showing the strongest effect in the combination group. Conclusion Isoliquiritigenin enhances the anti-proliferative and anti-migratory effects of doxorubicin on MDA-MB-231 cells by inhibiting the PI3K-Akt signaling pathway and modulating EMTrelated proteins.

R965

宁夏自然科学基金资助项目（2023AAC03479）