淫羊藿醇提物调节M2样巨噬细胞极化减少乳腺癌细胞迁移和集落形成的研究

Alcohol extract of Epimedium brevicornu reduces migration and colony formation of breast cancer cells by regulating polarization of M2 macrophages

目的探讨淫羊藿Epimedium brevicornu醇提物对体外巨噬细胞极化调控机制及对M2样巨噬细胞促进4T1细胞迁移和集落形成的影响。方法采用CCK-8法检测不同浓度淫羊藿醇提物对骨髓来源巨噬细胞(bonemarrowderived macrophages,BMDM)活力的影响,确定淫羊藿醇提物的安全作用浓度;采用Western blotting和q RT-PCR检测淫羊藿醇提物对M2样巨噬细胞标志物精氨酸酶-1(arginase-1,Arg-1)和CD163表达的影响;采用q RT-PCR检测淫羊藿醇提物对M2-肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)相关基因炎症区域1(found in inflammatory zone 1,Fizz1)、过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)、几丁质酶3样分子3(chitinase 3-like 3,Ym1)、趋化因子24(C-C motif chemokine ligand 24,CCL24)、巨噬细胞半乳糖型C型凝集素2(macrophage galactose-type C-type lectin 2,MGL2)和干扰素调节因子4(interferon regulatory factor 4,IRF4)m RNA表达的影响;划痕实验检测淫羊藿醇提物对M2-TAMs促4T1细胞迁移的影响;集落实验检测淫羊藿醇提物对M2-TAMs促4T1细胞生长的影响。结果淫羊藿醇提物在0.015625~2.000000 mg/mL对BMDM细胞无毒副作用;淫羊藿醇提物显著抑制M2-TAMs相关基因Arg-1、CD163、Fizz1、PPARγ、CCL24、Ym1、MGL2和IRF4表达(P<0.05、0.01、0.001),抑制M2-TAMs促4T1细胞的迁移和集落形成能力(P<0.05、0.001)。结论淫羊藿醇提物减弱M2样巨噬细胞对4T1细胞迁移和集落形成的促进作用,其作用机制可能与抑制巨噬细胞M2型极化有关。

Objective To investigate the effect and regulatory mechanism of alcohol extract of Epimedium brevicornu on macrophage polarization and M2 macrophages in promoting the migration and colony formation of 4T1 cells.Methods CCK-8 assay was used to detect the effect of different concentrations of alcohol extract of E.brevicornu on activity of bone marrow derived macrophages(BMDM),and determine the safe concentration of alcohol extract of E.brevicornu.Western blotting and qRT-PCR were used to detect the expressions of arginase-1(Arg-1)and CD163.qRT-PCR was used to detect the effect of alcohol extract of E.brevicornu on mRNA expressions of M2-tumor-associated macrophages(TAMs)related genes such as found in inflammatory zone 1(Fizz1),peroxisome proliferator-activated receptorγ(PPARγ),chitinase 3-like 3(Ym1),C-C motif chemokine ligand 24(CCL24),macrophage galactosetype C-type lectin 2(MGL2)and interferon regulatory factor 4(IRF4).The effect of alcohol extract of E.brevicornu on M2-TAMs promoting the migration of 4T1 cells was detected by wound healing assay.Colony formation experiment was conducted to detect the effect of alcohol extract of E.brevicornu on the growth of 4T1 cells promoted by M2-TAMs.Results Alcohol extract of E.brevicornu had no adverse effects on BMDM cells at 0.015625—2.000000 mg/mL.Alcohol extract of E.brevicornu significantly inhibited the expression of M2-TAMS related genes Arg-1,CD163,Fizz1,PPARγ,CCL24,Ym1,MGL2 and IRF4(P<0.05,0.01,0.001),and reduced the ability of M2-TAMS promoting the migration and colony formation of 4T1 cells(P<0.05,0.001).Conclusion Alcohol extract of E.brevicornu attenuates the promoting effects of M2 macrophages on migration and colony formation of 4T1 cells,and the mechanism may be related to the inhibition of M2 polarization of macrophages.