淫羊藿苷促成骨细胞分化成熟主要不依赖其雌激素活性

Icariin promote maturation of osteoblasts in vitro by an estrogen-independent mechanism

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 investigate the estrogenic activity of icariin and genistein with estrogen-dependent human breast cancer（ MCF-7） cells. Method： MCF-7 cells were incubated with media containing 5% charcoal dextran-treated FBS in phenol redfree media for 48 h. CCK-8 kit was used to study the impact of defferent concentration of icariin and genistein on MCF-7 proliferation in vitro. Optimal concentration icariin and genistein were added into medium and total RNA was isolated after 12,24,36,48 h. The gene expression of ERα,ERβ,PS2,and PR were investigated by Real-time RT-PCR. Total protein was also isolated and secretion of ERα,ERβ,PS2,and PR were examined by Western blot. Result： 10 μmol·L-1icariin and genistein could promote the proliferation of MCF-7 evidently. However,the ability of genistein to promote the proliferation was better than icariin. With the concentration of 10μmol·L-1,genistein group had a stronger expression of ERα,PS2 and PR mRNA levels than icariin while ERβ expression had no significant difference in two group. The same effects were detected by western blotting. Conclusion： Both genistein and icariin have a strong estrogen-like effect,but the estrogenic activity of genistein is stronger than icariin. It showed that the activity of icariin is stronger than genistein to promote ROB maturation. So it must be that icariin promotes the maturation of osteoblasts in vitro by a estogen-independent mechanism.