目的 從體內(nèi)外水平探究白頭翁皂苷B₄（AB₄）與5-氟尿嘧啶（5-Fu）聯(lián)合用藥對小鼠4T1乳腺癌細胞的作用及其分子機制。方法 體外分別采用細胞增殖抑制實驗、細胞創(chuàng)傷愈合實驗、Transwell小室侵襲實驗、流式細胞分析以及實時熒光定量PCR法（qRT-PCR）檢測AB₄與5-Fu聯(lián)合對4T1細胞功能和凋亡的影響；體內(nèi)借助4T1乳腺癌荷瘤小鼠模型，觀察AB₄與5-Fu聯(lián)合對移植瘤生長的影響；通過蘇木精-伊紅（HE）染色法分析聯(lián)合用藥對腫瘤組織形態(tài)學(xué)和微血管密度的影響；流式細胞術(shù)檢測AB₄聯(lián)合5-Fu對骨髓源性細胞（BMDCs）動員的影響；最后，通過qRT-PCR和Western blotting技術(shù)分析對腫瘤組織中相關(guān)因子mRNA及蛋白表達的影響。結(jié)果 體外實驗證實AB₄聯(lián)合5-Fu顯著降低4T1乳腺癌細胞活力，并且抑制其遷移和侵襲能力，同時可誘導(dǎo)其凋亡（P＜0.01）；體內(nèi)實驗表明AB₄聯(lián)合5-Fu顯著抑制4T1移植瘤的生長（P＜0.01），減少腫瘤組織內(nèi)微血管密度以及外周循環(huán)血液中促血管新生BMDCs的細胞計數(shù)（P＜0.01），還能顯著上調(diào)腫瘤組織中促凋亡因子的mRNA水平及降低抗凋亡因子的mRNA水平（P＜0.01），同時抑制腫瘤血管新生相關(guān)因子蛋白的表達（P＜0.01）。結(jié)論 AB₄與5-Fu聯(lián)合使用可以協(xié)同抑制4T1腫瘤細胞的生長。機制可能與其對腫瘤細胞功能的直接抑制作用有關(guān)，同時也與其對BMDCs動員的抑制相關(guān)，這間接減少了腫瘤血管新生。

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

江蘇醫(yī)藥職業(yè)學(xué)院自然科學(xué)基金研究一般項目(20214107);江蘇高校哲學(xué)社會科學(xué)研究一般項目資助(2022SJYB2075);江蘇省鹽城市衛(wèi)生健康委員會2023年度醫(yī)學(xué)科研立項項目(YK2023032，YK2023097)