ER/NO/cGMP通路介导雌马酚对成骨细胞的促分化作用

Effect of Equol on stimulating osteoblastic differentiation via ER/NO/cGMP

目的探讨植物雌激素雌马酚(Equol)诱导小鼠骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)增殖及向成骨细胞分化的作用机制。方法以无酚红α-MEM(含活性碳吸附过的10%胎牛血清)培养小鼠BMSCs,同时分别给予雌马酚(10-6mol·L-1)、雌马酚(10-6mol·L-1)合用雌激素受体(estrogen receptor,ER)拮抗剂ICI182,780(10-7mol·L-1)、雌马酚(10-6mol·L-1)合用非选择性一氧化氮合酶(nitric oxide synthase,NOS)抑制剂L-硝基精氨酸甲酯(L-NAME(6×10-3mol·L-1));测定[3H]-甲基胸腺嘧啶掺入率反映细胞增殖情况;测定细胞内碱性磷酸酶活性与钙沉积量反映细胞向成骨细胞分化状态;用一氧化氮(nitric oxide,NO)及环磷酸鸟苷(cyclic GMP,cGMP)试剂盒分别测定细胞培养液中NO产量与细胞内cGMP含量。结果合用ICI 182,780(10-7mol·L-1)或L-NAME(6×10-3mol·L-1)可拮抗雌马酚(10-6mol·L-1)引起的培养液中NO产量与细胞内cGMP含量的增加,并同时取消雌马酚(10-6mol·L-1)刺激BMSCs增殖及向成骨细胞分化的作用。结论雌马酚可通过ER/NO/cGMP信号通路而促进BMSCs增殖及向成骨细胞分化。

Objective The purpose of this study was to investigate the mechanism of Equal on the proliferation and osteoblastic differentiation of mouse bone marrow-derived mesenchymal stem cells （BMSCs） cultures. Methods Mouse BMSCs were cultured in phenol red-free a-MEM supplemented with 10% charcoal-stripped fetal bovine serum and treated with 10^-6 mol·L^-1 Equol with or without ICI 182,780 （10^-7 mol· L^-1） used as an ER antagonist or N-nitro-L-arginine methyl ester （L-NAME, 6 × 10^-3 mol· L^-1 ） used as a non-selective inhibitor of nitric oxide synthase （NOS）; The cell proliferation was measured by [3H]-thymidine incorporation. The osteoblastic differentiation and mineralization of extracellular matrix in BMSCs was assessed by measuring alkaline phosphatase （ALP） activity and calcium deposition, respectively. Nitric oxide （NO） production in the conditioned media and cyclic GMP （cGMP） content in BMSCs were determined by using commercial NO and cGMP kit, respectively. Results Concurrent treatment with ICI 182,780 （ 10^-7 mol·L^-1 ） or L-NAME（6×10^-3 mol·L^-1 ） abolished the Equol （10^-6 mol·L^-1）-mediated increase in NO production and cGMP content and eliminated the stimulation of BMSCs on the proliferation and osteoblastic differentiation by Equol. Conclusion Equol may be through ER/NO/cGMP pathway to induce the proliferation and osteoblastic differentiation of BMSCs.