菝葜抗肿瘤活性部位筛选及其对肿瘤相关巨噬细胞的调控作用和作用机制研究

Screening of Anti-tumor Active Sites of Smilax china L. and its Regulation and Mechanism on Tumor-associated Macrophages

关键词:  网络药理学  巨噬细胞极化  菝葜  乳腺癌  PI3K/Akt信号通路

Key words:  networkpharmacology  macrophage polarization  Smilax china L.  breastcancer  PI3K/Akt signaling pathway

摘要:

摘要：目的 筛选菝葜醇提物不同极性部位的抗肿瘤活性，并探讨其对巨噬细胞的调控作用机制。方法 体内构建小鼠黑色素瘤皮下移植模型，通过肿瘤体积和肿瘤重量评价菝葜不同极性部位的抗肿瘤作用，并采用MMTV-PyMT乳腺癌小鼠进一步验证，结合流式细胞术探讨活性部位对肿瘤免疫微环境的影响。通过IL-4刺激小鼠腹腔原代巨噬细胞体外构建M2极化模型，流式细胞术检测M1和M2比例。通过网络药理学方法找到菝葜抗乳腺癌的主要信号通路，并采用蛋白免疫印迹法进行验证。结果 体内实验结果显示，与B16-F10模型组相比，菝葜醇提物和乙酸乙酯部位组小鼠肿瘤重量显著减轻（P<0.01、P<0.05），菝葜正丁醇部位和水部位无抑制肿瘤作用。在MMTV-PyMT小鼠中验证了此结果，并发现与模型组相比，菝葜醇提物和乙酸乙酯部位组小鼠肿瘤组织中M2型肿瘤相关巨噬细胞浸润显著降低（P<0.05）。体外实验结果显示，与模型组相比，菝葜醇提物（100 μg/mL）和乙酸乙酯部位能够显著抑制IL-4诱导的M2极化（P<0.001）。网络药理学结果显示PI3K/Akt信号通路可能是菝葜抗乳腺癌的重要途径。Western Blot 结果显示，与模型组相比，菝葜乙酸乙酯部位50 μg/mL和25 μg/mL组P-PI3K/PI3K 蛋白相对表达量显著降低（P<0.001），P-Akt/Akt 蛋白相对表达量也显著降低（P<0.01，P<0.001）。结论 菝葜的抗肿瘤活性部位为乙酸乙酯部位，其抗肿瘤作用与抑制肿瘤相关巨噬细胞M2极化有关，其作用机制可能与抑制PI3K/Akt信号通路有关。

Abstract:

ABSTRACT: OBJECTIVE To screen the anti-tumor activity of different polar fractions of the alcoholic extract of Smilax china L. and explore its regulatory mechanisms on macrophages. METHODS A subcutaneous melanoma transplantation model was established in mice to evaluate the anti-tumor effects of different polar fractions of Smilax china L., based on tumor volume and weight. The findings were further validated in MMTV-PyMT breast cancer mice, and the effects of the active fractions on the tumor immune microenvironment were analyzed using flow cytometry. An M2 polarization model was constructed in vitro by stimulating primary mouse peritoneal macrophages with IL-4, and the M1/M2 ratio was measured by flow cytometry. The main signaling pathways responsible for the anti-breast cancer effects of Smilax china L. were identified using network pharmacology, and validated via Western blotting. RESULTS In vivo experiments showed that, compared to the B16-F10 model group, the tumor weight of mice treated with the ethanol extract and ethyl acetate fraction of Smilax china L. was significantly reduced (P<0.01, P<0.05), while the n-butanol and water fractions exhibited no anti-tumor effects. These findings were confirmed in MMTV-PyMT mice, where the ethanol and ethyl acetate fractions significantly reduced the infiltration of M2 tumor-associated macrophages compared to the model group (P<0.05). In vitro experiments showed that, compared to the model group, the ethanol extract (100 μg/mL) and ethyl acetate fraction significantly inhibited IL-4-induced M2 polarization (P<0.001). Network pharmacology results suggested that the PI3K/Akt signaling pathway might be a key mechanism for the anti-breast cancer effects of Smilax china L.. Western blot analysis revealed that, compared to the model group, the relative expression levels of P-PI3K/PI3K protein in the 50 μg/mL and 25 μg/mL ethyl acetate groups were significantly reduced (P<0.001). Similarly, the expression levels of P-Akt/Akt protein were also significantly decreased (P<0.01, P<0.001). CONCLUSION The ethyl acetate fraction of Smilax china L. is the active anti-tumor fraction, and its anti-tumor effect is related to the inhibition of M2 polarization in tumor-associated macrophages, potentially via suppression of the PI3K/Akt signaling pathway.