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目的:利用人乳腺癌细胞MCF-7检测淫羊藿苷(icariin,I)与染料木黄酮(genistein,G)雌激素样活性的大小。方法:MCF-7细胞系采用无酚红DMEM培养基(含5%活性炭吸附处理血清CS-FBS)培养48 h后,利用CCK-8试剂盒检测不同浓度的I和G对体外培养MCF-7增殖的影响,筛选出最佳药物浓度;以最佳浓度的I和G处理细胞12,24,36,48 h后提取总RNA,Real-time RT-PCR检测I和G对MCF-7细胞雌激素受体α(ERα)、雌激素受体β(ERβ)、雌激素调节蛋白(PS2)、孕激素受体(PR)mRNA表达水平的影响;以最佳浓度的I和G处理MCF-7细胞48 h后,Western blot检测G和I对MCF-7细胞ERα,ERβ,PS2,PR蛋白表达量的影响。结果:I和G均能显著促进MCF-7的增殖,其中最佳的I和G作用浓度为10μmol·L-1,并且在该浓度下G促MCF-7增殖的能力明显高于I;I和G作用MCF-7细胞24,36 h后,10μmol·L-1的I和G均能显著促进ERα,ERβ,PS2,PR mRNA表达水平,其中G促进ERα,PS2,PR mRNA表达的能力明显强于I,对于ERβmRNA的表达两者没有显著性差异;Western blot检测结果表明,10μmol·L-1的I和G均能促进ERα,ERβ,PS2,PR蛋白的表达,其中G促进ERα,PS2,PR蛋白表达的能力明显强于I,对于ERβ蛋白的表达两者没有显著性差异。结论:10μmol·L-1的G和I都具有较强的雌激素活性,但是该浓度下G的雌激素活性明显高于I,而本课题组前期研究表明,I在促进成骨细胞分化成熟及生物矿化的药理学活性明显强于G,因此I促成骨细胞分化成熟主要不是依赖其雌激素活性。
OBJECTIVE: To determine the estrogen-like activity of icariin (I) and genistein (G) using human breast cancer cell line MCF-7. Methods: MCF-7 cells were cultured in phenol red-free DMEM medium (CS-FBS containing 5% activated charcoal adsorption) for 48 h. The CCK-8 kit was used to detect the effect of different concentrations of I and G on MCF-7 The optimal concentration of I and G was selected to treat the cells at 12, 24, 36, and 48 h after extraction, and the total RNA was extracted. Real-time RT-PCR was used to detect the effect of I and G on MCF-7 cells (ERα), estrogen receptor β (ERβ), estrogen regulatory protein (PS2) and progesterone receptor (PR) mRNA expression levels. The optimal concentrations of I and G were used to treat MCF-7 After 48 h, the expression of ERα, ERβ, PS2 and PR in MCF-7 cells were detected by Western blot. Results: Both I and G significantly promoted the proliferation of MCF-7, and the optimal concentrations of I and G were 10μmol·L-1, and the ability of G to proliferate MCF-7 was significantly higher than that of I After treated with G and MCF-7 cells for 24 and 36 h, the expression of ERα, ERβ, PS2 and PR mRNAs was significantly enhanced by 10μmol·L-1 I and G, respectively. The results of Western blot showed that 10μmol·L-1 I and G could promote the expression of ERα, ERβ, PS2 and PR, and G promoted the expression of ERα, PS2 , PR protein expression was significantly stronger than I, for the expression of ERβ protein there was no significant difference between the two. CONCLUSION: Both G and I at 10μmol·L-1 have strong estrogenic activity, but the estrogenic activity of G at this concentration is significantly higher than that of I. However, previous studies in this group show that I can promote the differentiation and maturation of osteoblasts And the pharmacological activity of biomineralization is significantly stronger than that of G, therefore I promote osteoblast differentiation and maturation is not dependent on its estrogen activity.