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维生素K(VK),又称凝血维生素,是一类具有叶绿醌生物活性的脂溶性维生素。除具有凝血功能外,维生素K还能促进骨代谢,防治骨质疏松症,天然VK1和VK2(主要活性体为MK4和MK7)可单独或协同其他抗骨质疏松药物治疗骨质疏松症[1-4],人工合成的VK3也有少量抗骨质疏松研究报道[5-6],然而,不同维生素K的抗骨质疏松作用的比较研究非常有限。有研究发现维生素K具有促进骨形成和抑制骨吸收的双向调节机制[7],药理研究发现VK1、MK4和MK7能显著促进小鼠成骨细胞MC3T3-E1增殖和碱性磷酸酶(ALP)活性[8],同时报道VK1、MK4和MK7也能显著抑制RANKL诱导骨髓单核细胞分化的破骨细胞抗酒石酸酸性磷酸酶(TRAP)基因和组织蛋白酶K(CTSK) mRNA表达[9]。临床报道发现VK2能显著降低绝经后骨质疏松的TRAP和CTSK表达[10]。CTSK是骨吸收过程中的关键溶骨活性酶[11],与粘多糖(glycosaminoglycan, GAG),如硫酸软骨素(CSA),形成一种高分子量的复合物,可将胶原蛋白降解。目前尚未有VK对CTSK骨胶原降解的研究报道。本研究以新生大鼠颅盖骨分离成骨细胞和巨噬细胞集落刺激因子(M-CSF)和核因子κB受体活化因子配体(RANKL)诱导骨髓单核细胞的破骨细胞为模型[12],系统比较VK1、MK4、MK7和VK3对成骨细胞增殖和ALP活性、破骨细胞分化、TRAP活性和CTSK降解胶原活性的影响。通过比较不同VK抗骨质疏松作用,对临床选用合适的VK营养补充剂、指导人群VK膳食补充,以满足我国人群骨健康需求具有重要意义。
Effects of vitamin K on osteoblastic bone formation and osteoclastic bone absorption
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摘要:
目的 比较维生素K1(VK1)、维生素K2(MK4)、维生素K2(MK7)和维生素K3(VK3)促进骨形成和抑制骨吸收的作用。 方法 采用新生大鼠颅盖骨分离成骨细胞和以核因子κB受体活化因子配体(RANKL)诱导骨髓单核细胞的破骨细胞为模型,用磷酸苯二钠法检测成骨碱性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(TRAP)活性;用CellTilter试剂盒检测破骨细胞代谢活力;用Z-FR-MCA荧光底物和胶原底物降解检测组织蛋白酶K(CTSK)抑制作用。 结果 MK4和MK7在0.1~1 μmol/L时显著促进成骨细胞增殖(P<0.05),1 μmol/L时显著提高ALP活性和骨结节形成面积,VK3抑制了骨结节形成(P<0.05)。VK1、VK3、MK4和MK7在1 μmol/L时对破骨细胞代谢活力均无影响,MK4和MK7在0.1~1 μmol/L时显著抑制TRAP活性(P<0.05),而VK1和VK3无抑制作用。MK4在25 μmol/L可对CTSK与Z-FR-MCA底物结合的抑制率达58.9%,在100 μmol/L对CTSK胶原降解的抑制率达73.2%。 结论 相较于VK1和VK3,MK7和MK4有显著促进成骨细胞活性和抑制破骨细胞骨吸收的作用,MK4能显著抑制破骨细胞CTSK酶活性。 Abstract:Objective To compare the effects of vitamin K1 (VK1), vitamin K2 (MK4), vitamin K2 (MK7) and vitamin K3 (VK3) on bone formation and bone absorption. Methods Osteoblasts were isolated from calvaria of newborn rats and osteoclasts were induced by receptor activator of nuclear factor-κ B ligand (RANKL). ALP and TRAP activity were measured by diphenyl phosphate method. Osteoclast metabolic activity was measured by Celltiter kit. The inhibition of cathepsin K (CTSK) was measured by Z-FR-MCA fluorescent substrate and collagen substrate degradation. Results MK4 and MK7 at 0.1~1 μmol/L significantly increased the proliferation of osteoblasts (P<0.05) and at 1 μmol/L increased ALP activity and bone nodule formation area. VK3 inhibited bone nodule formation (P<0.05). VK1,VK3,MK4 and MK7 at 1 μmol/L had no effect on osteoclastic bone absorption. MK4 and MK7 significantly inhibited TRAP activity at 0.1~1 μmol/L (P<0.05), while VK1 and VK3 did not show the inhibitory effect. The inhibition of MK4 at 25 μmol/L on CTSK binding to Z-FR-MCA substrate activity is 58.9% and the inhibition of MK4 at 100 μmol/L on collagen degradation of CTSK activity is 73.2%. Conclusion Compared with VK1 and VK3, MK7 and MK4 significantly increase osteoblast activity and inhibit osteoclast bone absorption, MK4 inhibits osteoclast CTSK enzyme activity. -
Key words:
- vitamin K /
- osteoporosis /
- osteoblasts /
- osteoclasts
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