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背景:长期服用环孢素可抑制成骨细胞分化,导致骨质疏松,而植物雌激素雌马酚可促进成骨细胞增殖与分化。目的:探讨雌马酚对环孢素处理的小鼠骨髓间充质干细胞增殖及向成骨细胞分化的影响,分析其可能存在的信号通路。方法:贴壁法分离培养小鼠骨髓间充质干细胞,分为5组,分别给予雌马酚、环孢素、雌马酚+环孢素、雌马酚+环孢素+ICI182780、雌马酚+环孢素+L-NAME。倒置显微镜下观察骨髓间充质干细胞的形态学变化及矿化能力,通过检测[3H]-甲基胸腺嘧啶掺入率反映细胞增殖情况,通过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能力,测定培养液中一氧化氮产量与细胞内环磷酸鸟苷含量。结果与结论:合用雌马酚与环孢素可完全逆转单用环孢素引起[3H]-甲基胸腺嘧啶掺入率、细胞内碱性磷酸酶活性、钙沉积量的减少(P<0.05或0.01),并伴随一氧化氮产量与细胞内环磷酸鸟苷含量的恢复(P<0.01),同时倒置显微镜下可观察到干预12d时较高的细胞生长密度和矿化结节形成,以上作用可被雌激素受体拮抗剂ICI182780、一氧化氮合酶抑制剂L-NAME取消。提示雌马酚可通过雌激素受体/一氧化氮/环磷酸鸟苷信号通路逆转环孢素抑制骨髓间充质干细胞的增殖及向成骨细胞的分化。
Background: Long-term use of cyclosporine can inhibit osteoblast differentiation, leading to osteoporosis, while phytoestrogen equol can promote osteoblast proliferation and differentiation. Objective: To investigate the effect of equol on the proliferation and differentiation of mouse bone marrow mesenchymal stem cells treated with cyclosporine, and to analyze the possible signaling pathways. Methods: Bone marrow-derived mesenchymal stem cells (MSCs) were isolated and cultured by adherence method and divided into 5 groups: equol, cyclosporine, equol + cyclosporine, equol + cyclosporin + ICI182780, Phenol + Cyclosporine + L-NAME. Morphology and mineralization ability of bone marrow mesenchymal stem cells were observed under inverted microscope. The proliferation of bone marrow mesenchymal stem cells was observed by measuring the incorporation rate of [3H] -methyl thymidine. The intracellular alkaline phosphatase activity and calcium deposition were measured The ability of cells to differentiate into osteoblasts was assayed for nitric oxide production and intracellular cyclic guanosine monophosphate content in culture medium. RESULTS AND CONCLUSION: Combined use of equol and cyclosporin completely reversed the incorporation of [3H] -methylthymidine and the decrease of intracellular alkaline phosphatase activity and calcium deposition by cyclosporine alone (P <0.05 Or 0.01), accompanied by the recovery of nitric oxide production and intracellular cyclic guanosine monophosphate (P <0.01). At the same time, higher cell growth density and mineralized nodule formation were observed at 12 d after intervention The effect can be estrogen receptor antagonist ICI182780, nitric oxide synthase inhibitor L-NAME canceled. These results suggest that equol may reverse cyclosporine’s proliferation and differentiation into osteoblasts through estrogen receptor / nitric oxide / guanosine monophosphate signal pathway.