雌激素受体和过氧化物酶体增殖物激活受体γ介导大豆苷原对骨形成的量效作用

Dose-dependent effects of daidzein in regulating bone formation through estrogen receptors and peroxisome proliferator-activated receptor γ

目的:研究不同剂量大豆苷原(daidzein,DAI)对成骨细胞骨形成的作用及雌激素受体(estrogen receptor,ER)和过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptorγ,PPARγ)对DAI促进骨形成的调节作用。方法:以培养的未经处理的人类胎儿成骨细胞(human fetal osteoblast,hFOB)为对照组,用17β-戊酸雌二醇(β-estradiol-17-valerate,E2)和不同浓度DAI分别处理hFOB72h后,噻唑蓝(methyl thiazolyl tetrazolium,MTT)法测定细胞增殖情况,4-硝基苯基磷酸二钠盐(4-nitrophenylphosphate disodiumsalt,PNPP)偶氮法测定成骨细胞碱性磷酸酶(alkaline phosphatase,ALP)活性。用ER拮抗剂ICI182780、ERα拮抗剂MPP(methyl-piperidino-pyrazole)、PPARγ拮抗剂GW9662分别阻断受体ER、ERα和PPARγ后,再给予E2及不同浓度的DAI处理,MTT法检测成骨细胞增殖情况并计算细胞增殖率,PNPP偶氮法测定ALP水平。结果:随着DAI剂量的增加,成骨细胞增殖率递减,DAI10-9mol/L组较对照组显著升高,DAI10-5mol/L组较对照组显著降低(P<0.05)。随着DAI剂量的增加,成骨细胞ALP水平递减,但差异无统计学意义(P>0.05)。ICI182780阻滞ER后,E2组,DAI10-9、10-7及10-5mol/L组的细胞增殖率分别为88.16%、76.30%、81.18%、83.19%,均显著低于阻滞前(P<0.05);MPP单纯阻滞ERα后,E2组,DAI10-9、10-7及10-5mol/L组的细胞增殖率分别为69.78%、63.31%、70.71%、78.43%,均显著低于阻滞前(P<0.05)。MPP阻滞后,DAI10-9mol/L组的ALP水平显著低于阻滞前(P<0.05)。GW9662阻滞PPARγ后,E2组,DAI10-9、10-7及10-5mol/L组的细胞增殖率分别为103.14%、96.99%、112.88%和122.22%,其中DAI10-7及10-5mol/L组均较阻滞前显著升高(P<0.05),DAI10-9mol/L组轻度降低但差异无统计学意义。DAI10-5mol/L组的ALP水平较阻滞前显著升高(P<0.05)。结论:DAI的骨保护作用具有剂量依赖性的双相作用特点,即低剂量DAI促进成骨细胞增殖,高剂量DAI抑制成骨细胞增殖。低剂量DAI主要通过作用于ER促进成骨细胞增殖和分化,对PPARγ的作用不明显;高剂量DAI主要通过作用于PPARγ抑制成骨细胞增殖和分化,同时也可通过作用于ER产生一定促进成骨细胞增殖的作用。

Objective：To investigate different doses of daidzein（DAI）in regulating bone formation of osteoblasts,and the regulating mechanisms of estrogen receptors（ERs）and peroxisome proliferator-activated receptor γ（PPARγ）in bone formation.Methods：Human fetal osteoblasts（hFOBs）incubated without any treatment were served as controls（control group）.The hFOBs were exposed to DAI of 10-9,10-7 and 10-5 mol/L for 72 h,and to β-estradiol-17-valerate（E2）of 10-8 mol/L as positive control,respectively.Methyl thiazolyl tetrazolium assay was employed to determine the proliferation status of osteoblasts,and 4-nitrophenyl phosphate disodium salt（PNPP）method was employed to determine the activity of alkaline phosphatase（ALP）.ER antagonist ICI 182780（ICI）,ERα-selective antagonist methyl-piperidino-pyrazole（MPP）and irreversible PPARγ antagonist GW9662（GW）were used to block the corresponding receptor,while hFOBs were exposed to E2 or different concentrations of DAI for 48 h.MTT assay and PNPP method were used respectively to determine the proliferation status and ALP activity of osteoblasts cultured in vitro.Results：The osteoblast proliferation rate decreased progressively as the dose of DAI increased.Compared with the controls,the osteblast proliferation rate in the DAI 10-9 mol/L group increased significantly,while DAI 10-5 mol/L group decreased significantly（P0.05）.ALP level decreased progressively as the dose of DAI increased,but there was no significant difference between groups（P0.05）.When ERs were blocked by ICI,proliferation rates in the E2 group and DAI 10-9,10-7 and 10-5 mol/L groups were 88.16%,76.30%,81.18% and 83.19% respectively,which were all significantly lower than before（P 0.05）.After ERα was blocked by MPP alone,proliferation rates in E2 group and DAI 10-9,10-7 and 10-5 mol/L groups were 69.78%,63.31%,70.71% and 78.43%,respectively,which were also significantly lower than before（P0.05）.ALP level in the DAI 10-9 mol/L group decreased significantly when ERα was blocked alone.When PPARγ inhibitor GW was added to the culture system,proliferation rates in E2 group and DAI 10-9,10-7 and 10-5 mol/L groups were 103.14%,96.99%,112.88% and 122.22%,respectively.Compared with before,proliferation rates in DAI 10-7 and 10-5 mol/L groups increased significantly（P0.05）,and ALP level increased significantly（P0.05）in the DAI 10-5mol/L group.Conclusion：DAI shows a biphasic effect on osteoporosis,whereby the effect is dose-dependent;a low-dose DAI stimulates proliferation of osteoblasts,while a high-dose DAI inhibits proliferation of osteoblasts.Low-dose DAI mainly acts on ERs,whereas high-dose DAI mainly acts on PPARγ to inhibit proliferation of osteoblasts and to some extent,acts on ERs to promote the proliferation of osteoblasts.