Found programs: National Natural Science Foundation of China ( No．81973625) ; “Xinglin Scholar” Discipline Talent Research Enhancement Program of Chengdu University of Traditional Chinese Medicine ( No． YYZX2022164) ; Project for the Construction of Studio for Shanghai Famous TCM Expert ( No．SHGZS－202224)
Authors：Xiao Zengyou1，2 ，Yang Zean1，2 ，Chen Caihong3 ，Li Jiaxian2 ，He Yujie2 ，Fu Pinting2 ，Wang Jie1，2
Keywords：icariin; triple-negative breast cancer; TGF-β/Smad; invasion and metastasis; epithelial-mesenchymal transition; molecular mechanisms;
DOI：10.19405/j.cnki.issn1000-1492.2025.09.002
〔Abstract〕 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 with 10 μmol / L TGF-β receptor inhibitor LY2109761; a combination group ( LY2109761 + ICA) treated with both 15 μmol / L ICA and 10 μmol / L LY2109761; and a control group．Cell proliferation，migration，and invasion were as- sessed 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 ex- pression 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．