目的 从体内外水平探究白头翁皂苷B₄（AB₄）与5-氟尿嘧啶（5-Fu）联合用药对小鼠4T1乳腺癌细胞的作用及其分子机制。方法 体外分别采用细胞增殖抑制实验、细胞创伤愈合实验、Transwell小室侵袭实验、流式细胞分析以及实时荧光定量PCR法（qRT-PCR）检测AB₄与5-Fu联合对4T1细胞功能和凋亡的影响；体内借助4T1乳腺癌荷瘤小鼠模型，观察AB₄与5-Fu联合对移植瘤生长的影响；通过苏木精-伊红（HE）染色法分析联合用药对肿瘤组织形态学和微血管密度的影响；流式细胞术检测AB₄联合5-Fu对骨髓源性细胞（BMDCs）动员的影响；最后，通过qRT-PCR和Western blotting技术分析对肿瘤组织中相关因子mRNA及蛋白表达的影响。结果 体外实验证实AB₄联合5-Fu显著降低4T1乳腺癌细胞活力，并且抑制其迁移和侵袭能力，同时可诱导其凋亡（P＜0.01）；体内实验表明AB₄联合5-Fu显著抑制4T1移植瘤的生长（P＜0.01），减少肿瘤组织内微血管密度以及外周循环血液中促血管新生BMDCs的细胞计数（P＜0.01），还能显著上调肿瘤组织中促凋亡因子的mRNA水平及降低抗凋亡因子的mRNA水平（P＜0.01），同时抑制肿瘤血管新生相关因子蛋白的表达（P＜0.01）。结论 AB₄与5-Fu联合使用可以协同抑制4T1肿瘤细胞的生长。机制可能与其对肿瘤细胞功能的直接抑制作用有关，同时也与其对BMDCs动员的抑制相关，这间接减少了肿瘤血管新生。

Objective To examine the combined impact of Anemoside B₄ (AB₄) and 5-fluorouracil (5-Fu) on murine 4T1 breast cancer cells, both in vitro and in vivo, while also investigating the underlying molecular mechanisms. Methods A series of experiments, including cell proliferation, wound healing, matrigel invasion assays, and flow cytometry, were conducted to assess how the combination of AB 4 and 5-Fu influences 4T1 cell function and apoptosis in vitro. In vivo, a tumor model was created by subcutaneously injecting 4T1 cells into BALB/c mice to observe the combined effects of AB₄ and 5-Fu on tumor growth. HE staining was used to examine changes in tumor tissue microstructure, while flow cytometry analyzed the impact of AB₄ and 5-Fu on the mobilization of bone marrow-derived cells (BMDCs). Additionally, qRT-PCR and Western blotting were employed to measure mRNA levels of apoptosis-related factors and the protein expression of angiogenesis-related factors in tumors. Results The combination of AB 4 and 5-Fu significantly reduced the viability and the migration and invasion capacity of 4T1 breast cancer cells (P < 0.01). Moreover, the proportion of cells undergoing apoptosis was significantly increased when treated with AB₄ and 5-Fu together (P < 0.01). Besides, the combined treatment of AB₄ and 5-Fu led to a more significant tumor growth inhibition in 4T1 xenograft mouse model (P < 0.01). The intratumoral microvascular density in 4T1 xenograft was dramatically reduced according to the HE staining results (P < 0.01) and the cell count of BMDCs in peripheral blood of tumor-bearing mice was also reduced (P < 0.01). qRT-PCR analysis shown that AB₄ combined with 5-Fu significantly increased the mRNA expression of pro-apoptotic factors and decreased the mRNA levels of anti-apoptotic factors in tumor tissues (P < 0.01), and the protein expression of tumor angiogenesis-related factors was inhibited. Conclusions The combined use of AB₄ and 5-Fu effectively inhibited the growth of 4T1 tumor cells in a synergistic manner. This effect is likely due to both the direct inhibition of tumor cell function and the suppression of BMDC mobilization, which in turn reduces tumor angiogenesis indirectly.

R285.5

江苏医药职业学院自然科学基金研究一般项目(20214107);江苏高校哲学社会科学研究一般项目资助(2022SJYB2075);江苏省盐城市卫生健康委员会2023年度医学科研立项项目(YK2023032，YK2023097)