National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Chinese Medicine, Nanchang 330006, China.
Key Laboratory for Evaluation on Anti-Tumor Effect of Chinese Medicine by Strengthening Body Resistance to Eliminate Pathogenic Factors, Nanchang 330006, China.
Molecules. 2023 Feb 7;28(4):1611. doi: 10.3390/molecules28041611.
Triple negative breast cancer (TNBC) exhibits the characteristics of strong metastatic ability and a high recurrence rate, and M2-type macrophages play an important role in this process. Previous research data suggested that Anemoside A3 (A3), a monomeric component of Pulsatilla Chinensis, could prevent and treat TNBC by converting M0 macrophages into M1 immunogen phenotypes. This study showed that A3 significantly restrained the lung metastases of 4 T1-Luc cells with bioluminescence imaging in vivo and Hematoxylin and Eosin (H&E) staining. Meanwhile, the percentage of M2-type macrophages (CD206+ labeled cells) in the lung tissues was evidently decreased through immunohistochemical assay. We further proved that A3 markedly prevented M2-type polarization induced by IL-4 in vitro, as illustrated by the down-regulated expression of the cell surface marker CD206 protein by FACS and Arg-1, and of the Fizz1 and Ym1 genes by RT-PCR in M2-type macrophages. Furthermore, the invasion and migration of 4 T1 cells, which was promoted by the conditioned medium from M2-type macrophages, could be suppressed by A3. Luminex assay demonstrated that A3 treatment resulted in a reduction of the levels of CCL2, VEGF, CCL7, and MMP-9 in conditioned medium. Additionally, the expression of phosphorylated-STAT3 protein was inhibited by A3, which resulted in the macrophage M2-type polarization arrest, while no significant difference in JAK2 phosphorylation was detected. SiRNA transfection experiments suggested that STAT3 might be the target of A3 inhibiting M2-type polarization of macrophages. In conclusion, these results indicate that A3 could attenuate the metastasis of TNBC by inhibiting the M2-type polarization of macrophages, which may be related to the STAT3 pathway.
三阴性乳腺癌（TNBC）表现出较强的转移能力和较高的复发率，M2 型巨噬细胞在这一过程中发挥着重要作用。先前的研究数据表明，白头翁皂苷 A3（A3）作为白头翁的单体成分，可通过将 M0 巨噬细胞转化为 M1 免疫表型来预防和治疗 TNBC。本研究通过体内生物发光成像和苏木精和伊红（H&E）染色显示，A3 显著抑制了 4T1-Luc 细胞的肺转移。同时，通过免疫组织化学检测，肺组织中 M2 型巨噬细胞（CD206+标记细胞）的比例明显降低。我们进一步证明，A3 可显著抑制 IL-4 诱导的 M2 型极化，流式细胞术检测到细胞表面标志物 CD206 蛋白表达下调，以及 RT-PCR 检测到 M2 型巨噬细胞中 Arg-1 和 Fizz1、Ym1 基因表达下调。此外，M2 型巨噬细胞条件培养基促进的 4T1 细胞侵袭和迁移可被 A3 抑制。Luminex 检测表明，A3 处理可降低条件培养基中 CCL2、VEGF、CCL7 和 MMP-9 的水平。此外，A3 抑制了 STAT3 蛋白的磷酸化，导致巨噬细胞 M2 型极化阻滞，而 JAK2 磷酸化无明显差异。SiRNA 转染实验表明，STAT3 可能是 A3 抑制巨噬细胞 M2 型极化的靶点。综上所述，这些结果表明，A3 可通过抑制巨噬细胞 M2 型极化来减轻 TNBC 的转移，这可能与 STAT3 通路有关。
