大豆苷原对成骨细胞MC3T3-E1甾体激素受体辅激活因子1表达的调节作用及其机制

目的：研究大豆苷原对成骨细胞MC3T3－E1甾体激素受体辅激活因子1（steroid receptor coactivator-1,SRC-1）和核受体辅抑制因子（nuclear receptor corepressor,NcoR）表达的调节作用及其机制。方法：体外培养MC3T3-E1细胞于含29／6胎牛血清（fetal bovine serum,FBS）的α最低必须培养基（α—minimal essential medium,α—MEM）中,给予不同浓度大豆苷原或10^-6 mol／L的雌二醇作用3d后收获细胞,采用蛋白质印迹法观察SRC-1和NcoR蛋白的表达水平。分别应用雌激素受体（estrogen receptor,ER）拮抗剂ICI182780（Faslodex,ICI,10^-7 mol／L）和雌激素受体α（estrogen receptor α,ERα）特异性拮抗剂（methyl—piperidino—pyrazole,MPP,10^-6 mol／L）干预,观察大豆苷原对细胞SRC-1和NcoR蛋白表达水平的影响。结果：大豆苷原可上调MC3T3-E1细胞SRC-1的表达,10^-7mol／L和10smol／L大豆苷原组SRC-1的蛋白水平分别增加至对照组的2．5倍（P〈0．05）和2倍（P〈0．05）。各剂量组NcoR表达水平与对照组比较差异无统计学意义。雌二醇组SRC-1水平与对照组比较未见明显变化,而其NcoR表达水平较对照组下调35％（P〈0．05）。ICI182780可拮抗大豆苷原对SRC-1的上调作用。在ICI182780作用下,各剂量组SRC-1蛋白水平与对照组比较差异无统计学意义;MPP干预下,10^-7mol／L和10^-5 mol／L大豆苷原组SRC-1的蛋白水平分别为对照组的1．8倍（P〈0．05）和2．4倍（P〈0．05）。结论：大豆苷原可增加成骨细胞SRC-1的蛋白表达水平,提高细胞SRC-1／NcoR比值。ER8参与了大豆苷原对SRC-1的调节过程。成骨细胞内SRC-1蛋白的增加和SRC-1／NcoR比值的提高是大豆苷原促进成骨细胞分化的机制之一。

Effects of daidzein on steroid receptor coactivator-1 expression in MC3T3-E1 cells and the mechanism

Objective： To investigate the roles of daidzein in the expressions of steroid receptor coactivator-1 （SRC-1） and nuclear receptor corepressor （NcoR） in MC3T3-E1 osteoblastic cells. Metheds. MC3T3-E1 cells were cultured in e-minimal essential medium （α-MEM） containing 2% fetal bovine serum and treated with various concentrations of daidzein （10^-9 , 10^-7 and 10-s mol/L） or 17β-estradiol at 10^-8 mol/L for 3 d. The protein levels of SRC-1 and NcoR in MC3T3-E1 cells were determined by Western blotting. Estrogen receptor （ER） antagonist ICI182780 at 10^- 7 mol/L or specific ERe antagonist methyl-piperidino- pyrazole （MPP） at 10 6 mol/L were used to block the corresponding receptors, and then MC3T3-E1 cells were treated with daidzein at 10^-7 mol/L or 10-5 mol/L for 3 d. SRC-1 and NcoR protein levels were detected by Western blotting. Results： The protein levels of SRC-1 increased by 2.5 fold （P〈0.05） and 2 fold （P〈0.05） by 10-7 and 10-s mol/L of daidzein respectively, while the NcoR levels were not significantly altered. 1713-Estradiol at dose of 10-8 mol/L did not affect the expression of SRC-1 but decreased NcoR protein expression by 35% （P〈0. 05）. Compared with the control, daidzein at 10^-7 and 10^-5 mol/L did not increase SRC-1 expression when ERs were blocked by antagonist ICI182780. Daidzein at 10^-7 and 10^-5 mol/L up-regulated SRC-1 by 1.8 fold （P〈0. 05） and 2.4 fold （P〈0.05） respectively while ERe was blocked by MPP. Conclusion= Daidzein increases protein level of SRC-1 and the ratio of SRC-1/NcoR. ERI3, instead of ERe, participates in the action of daidzein in regulating SRC-1 expression. Up-regulation of SRC-1 and increase of SRC-1/NcoR are part of the mechanism of the estrogenic effect of daidzein in improving osteogenesis.