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目的 探讨淫羊藿苷(ICA)下调转化生长因子-β/Smad(TGF-β/Smad)信号通路抑制人三阴性乳腺癌(TNBC)细胞侵袭转移的作用机制。方法 体外培养TNBC细胞(MDA-MB-231和MDA-MB-468)，分为4组，加入ICA(15μmol/L)设为实验组;加入10μmol/L TGF-β受体抑制剂LY2109761设为模型组;在模型组基础上，加入15μmol/L ICA设为LY2109761+ICA组;另设对照组。CCK-8、平板克隆、5-乙炔基-2'脱氧尿嘧啶核苷(Ed U)、细胞划痕实验和Transwell实验检测细胞增殖、迁移和侵袭能力;免疫荧光和Western blot实验检测上皮-间质转化(EMT)相关蛋白以及转化生长因子β1(TGF-β1)、Smad2和磷酸化Smad2(P-Smad2)通路蛋白表达水平。结果 CCK-8结果显示随着ICA浓度增加，细胞的增殖能力逐渐减弱(P<0.001)。平板克隆与Ed U法结果显示，与对照组相比，实验组细胞增殖能力减弱(P<0.05)。划痕实验和Transwell实验显示，与对照组相比，实验组细胞迁移和侵袭能力下降(P<0.05)。与模型组相比，LY2109761+ICA组细胞侵袭能力进一步下降(P<0.05)。免疫荧光结果显示，与对照组相比，实验组细胞波形蛋白(Vimentin)表达下降(P<0.05)。与模型组相比，LY2109761+ICA组细胞Vimentin蛋白表达进一步下降(P<0.01)。Western blot结果显示，与对照组相比，实验组细胞神经钙黏蛋白(N-cadherin)、基质金属蛋白酶-9(MMP9 )、Vimentin以及TGF-β1、Smad2、P-Smad2蛋白表达量下降(P<0.05 )。与模型组相比，LY2109761+ICA组细胞N-cadherin、MMP9、Vimentin以及TGF-β1、Smad2、P-Smad2蛋白表达量进一步下降(P<0.05)。结论 ICA通过下调TGF-β/Smad信号通路抑制TNBC细胞增殖、侵袭转移和EMT。
Abstract：Objective To investigate the mechanism by which icariin(ICA) inhibits the invasion and metastasis of human triple-negative breast cancer(TNBC) cells via downregulation of the transforming growth factor-β/Smad(TGF-β/Smad) signaling pathway. Methods TNBC cells(MDA-MB-231 and MDA-MB-468) were cultured in vitro and divided into four groups: an experimental group treated with 15 μmol/L ICA; a model group treated with10 μmol/L TGF-β receptor inhibitor LY2109761; a combination group(LY2109761 + ICA) treated with both 15μmol/L ICA and 10 μmol/L LY2109761; and a control group. Cell proliferation,migration,and invasion were assessed using CCK-8,colony formation,5-ethynyl-2'-deoxyuridine(EdU),wound healing,and Transwell assays.The expression levels of epithelial-mesenchymal transition(EMT)-related proteins,as well as TGF-β1,Smad2,and phosphorylated Smad2(P-Smad2) were detected by immunofluorescence and Western blot. Results CCK-8 results showed that cell proliferation decreased gradually with increasing concentrations of ICA(P < 0. 05). Colony formation and EdU assays indicated significantly inhibited proliferation in the ICA-treated group compared to the control(P < 0. 05). Wound healing and Transwell assays demonstrated reduced migration and invasion capabilities in the experimental group relative to the control(P < 0. 05). Compared to the model group,the LY2109761 + ICA group exhibited further suppression of invasion(P < 0. 05). Immunofluorescence revealed decreased Vimentin expression in the experimental group(P < 0. 05),with an even more pronounced reduction in the LY2109761 + ICA group(P < 0. 01). Western blot analysis showed that the protein levels of N-cadherin,matrix metalloproteinase-9(MMP9),Vimentin,TGF-β1,Smad2,and P-Smad2 were downregulated in the experimental group compared to the control(P < 0. 05). These proteins were further suppressed in the LY2109761 + ICA group compared to the model group(P < 0. 05). Conclusion ICA inhibits TNBC cells proliferation,invasion,metastasis,and EMT by downregulating the TGF-β/Smad signaling pathway.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2025.09.002
中图分类号:R285
引用信息:
[1]肖增友,杨泽安,陈彩红,等.淫羊藿苷下调TGF-β/Smad信号通路抑制三阴性乳腺癌侵袭转移[J].安徽医科大学学报,2025,60(09):1574-1582.DOI:10.19405/j.cnki.issn1000-1492.2025.09.002.
基金信息:
国家自然科学基金项目(编号：81973625); 成都中医药大学“杏林学者”学科人才科研提升计划项目(编号：YYZX2022164); 上海市名老中医工作室建设项目(编号：SHGZS-202224)~~