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Volume 38 Issue 4
Jul.  2020
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JIANG Yizhong, XIA Tianshuang, XIN Hailiang, JIN Yu'e, JIANG Yiping, XUE Liming. Effects of vitamin K on osteoblastic bone formation and osteoclastic bone absorption[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(4): 340-345. doi: 10.12206/j.issn.1006-0111.202001077
Citation: JIANG Yizhong, XIA Tianshuang, XIN Hailiang, JIN Yu'e, JIANG Yiping, XUE Liming. Effects of vitamin K on osteoblastic bone formation and osteoclastic bone absorption[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(4): 340-345. doi: 10.12206/j.issn.1006-0111.202001077

Effects of vitamin K on osteoblastic bone formation and osteoclastic bone absorption

doi: 10.12206/j.issn.1006-0111.202001077
  • Received Date: 2020-01-17
  • Rev Recd Date: 2020-04-13
  • Available Online: 2020-07-27
  • Publish Date: 2020-07-25
  •   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.
  • [1] NAGURA N, KOMATSU J, IWASE H, et al. Effects of the combination of vitamin K and teriparatide on the bone metabolism in ovariectomized rats[J]. Biomed Rep,2015,3(3):295-300. doi:  10.3892/br.2015.431
    [2] BRAAM L A, KNAPEN M H, GEUSENS P, et al. Vitamin K1 supplementation retards bone loss in postmenopausal women between 50 and 60 years of age[J]. Calcif Tissue Int,2003,73(1):21-26. doi:  10.1007/s00223-002-2084-4
    [3] 罗洪斌, 徐杰, 柯良骏. 钙尔奇D联用维生素K对老年骨质疏松患者骨密度、血清骨钙素及其Ⅰ-型胶原C-末端的影响[J]. 海峡药学, 2012, 24(9):84-86. doi:  10.3969/j.issn.1006-3765.2012.09.036
    [4] 张丽梅. 阿仑膦酸钠联合维生素K治疗2型糖尿病患者骨质疏松的疗效观察[J]. 中国民康医学, 2015, 27(10):28-30. doi:  10.3969/j.issn.1672-0369.2015.10.014
    [5] HONG Y J, LIU S, JIANG N Y, et al. Vitamin K3 increased BMD at 1 and 2 months post-surgery and the maximum stress of the middle femur in the rat[J]. Nutr Res,2015,35(2):155-161. doi:  10.1016/j.nutres.2014.10.008
    [6] FLEMING R H, MCCORMACK H A, MCTEIR L, et al. Effects of dietary particulate limestone, vitamin K3 and fluoride and photostimulation on skeletal morphology and osteoporosis in laying hens[J]. Br Poult Sci,2003,44(5):683-689. doi:  10.1080/00071660310001643688
    [7] 周建烈, 陈杰鹏, 段丽丽, 等. 维生素K2(MK-7)防治骨质疏松的作用机制研究进展[J]. 中国骨质疏松杂志, 2019, 25(4):539-545. doi:  10.3969/j.issn.1006-7108.2019.04.023
    [8] WU W J, GAO H Y, JIN J S, et al. A comparatively study of menaquinone-7 isolated from Cheonggukjang with vitamin K1 and menaquinone-4 on osteoblastic cells differentiation and mineralization[J]. Food Chem Toxicol,2019,131:110540. doi:  10.1016/j.fct.2019.05.048
    [9] WU W J, KIM M S, AHN B Y. The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption[J]. Food Funct,2015,6(10):3351-3358. doi:  10.1039/C5FO00544B
    [10] 庄焕雄, 陈东峰, 徐孟凡, 等. 维生素K2对绝经后骨质疏松症的防治作用及血清组织蛋白酶K影响[J]. 中国骨质疏松杂志, 2017, 23(5):627-630. doi:  10.3969/j.issn.1006-7108.2017.05.014
    [11] 蒋益萍, 吴岩斌, 秦路平, 等. 墨旱莲组分中组织蛋白酶K非活性位点抑制剂研究[J]. 药学学报, 2017, 52(6):936-942.
    [12] 张乃丹, 蒋益萍, 薛黎明, 等. 仙茅酚苷类成分促进成骨细胞骨形成和抑制破骨细胞骨吸收[J]. 第二军医大学学报, 2016, 37(5):562-568.
    [13] 秦路平, 蒋益萍, 薛黎明, 等. 防治骨质疏松的中药制剂及其制备方法: 201210359162.5[P]. 2012-09-17.
    [14] 袁婷婷. 仙茅苷对骨细胞雌激素受体表达的影响及其初步药代动力学研究[D]. 福州: 福建中医药大学, 2014.
    [15] 邹志强, 符诗聪, 刘忠厚. 维生素K2的研究进展[J]. 中国骨质疏松杂志, 2005, 11(3):389-392. doi:  10.3969/j.issn.1006-7108.2005.03.036
    [16] 中华医学会骨质疏松和骨矿盐疾病分会. 糖皮质激素性骨质疏松症诊疗指南(讨论稿)[J]. 中华全科医师杂志, 2006, 5(8):460-461. doi:  10.3760/cma.j.issn.1671-7368.2006.08.005
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Effects of vitamin K on osteoblastic bone formation and osteoclastic bone absorption

doi: 10.12206/j.issn.1006-0111.202001077

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.

JIANG Yizhong, XIA Tianshuang, XIN Hailiang, JIN Yu'e, JIANG Yiping, XUE Liming. Effects of vitamin K on osteoblastic bone formation and osteoclastic bone absorption[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(4): 340-345. doi: 10.12206/j.issn.1006-0111.202001077
Citation: JIANG Yizhong, XIA Tianshuang, XIN Hailiang, JIN Yu'e, JIANG Yiping, XUE Liming. Effects of vitamin K on osteoblastic bone formation and osteoclastic bone absorption[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(4): 340-345. doi: 10.12206/j.issn.1006-0111.202001077
  • 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。

    • 新生3 d的SD大鼠,雌雄不限,购自上海斯莱克实验动物有限公司[SCXK(沪)2012-0002];II型胶原酶、胰蛋白酶、特级胎牛血清、α-MEM培养基(美国Gibco公司);CellTriter-blue试剂(美国Promega™),核刺激因子受体的配体(receptor for activation of nuclear factor kappa B ligand, RANKL,400-30)、巨噬细胞集落刺激因子(macrophage colony-stimulating factor, M-CSF,400-28)购自PEPROTECH公司,淋巴细胞分离液Ficoll-Paque密度梯度液(GE)购自上海生工生物工程技术服务有限公司;TRACP染色试剂盒、Benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA)购自日本WAKO公司;L-3-carboxy-trans-2-3-epoxypropionyl-leucylamido-(4guanidino)-butane(E-64)、硫酸软骨素A (CSA)胃蛋白酶、组织蛋白酶K抑制剂奥当卡替(odanacatib, ODN)、VK1、MK4、MK7和VK3,购自Sigma公司;I型胶原(美国Affymetrix公司);牛血清白蛋白、β-甘油磷酸钠、抗坏血酸、多聚甲醛等试剂均为国产分析纯。

    • 实验室自建原代成骨细胞培养方法[12]。取新生3 d的SD大鼠5只,在无菌条件下取其颅顶盖骨,去除骨膜、血管及结缔组织,用D-Hanks液冲洗干净,用0.25%的胰蛋白酶37 ℃消化30 min,再用0.05%胰蛋白酶及3 mg/ml的II型胶原酶37 ℃消化1 h,经100目滤网过滤,1 000 r/min离心10 min,即为新鲜的成骨细胞,加入10%胎牛血清的α-MEM培养基,置37 ℃,5% CO2培养箱。取第四代大鼠颅盖骨成骨细胞,消化分离出来,以α-MEM培养基配制成浓度为2×104/ml的细胞悬液,以100 μl/孔接种于96孔培养板,设12个药物组,分别为1、0.1和0.01 μmol/L的VK1、MK4、MK7和VK3,每组选取1个药物浓度加入1、0.1和0.01 μmol/L的VK1、MK4、MK7和VK3,培养48 h,用MTT法检测细胞增殖活性。ALP活性需要连续培养6 d后,用100 μl 50 mmol/L的二乙醇胺,50 μl 2.5 mmol/L的对硝基苯酚磷酸二钠,37 ℃反应30 min后,用0.3 mol/L的NaOH终止反应,于波长405 nm处测得吸光度值[12-13]

    • 取成骨细胞第三代细胞,按每孔5×104的细胞数接种于12孔板内,α-MEM培养基培养24 h,换骨结节诱导培养基(0.1%BSA,10 nmol/L地塞米松,10 mmol/L β-甘油磷酸钠和50 μg/ml抗坏血酸以及10%胎牛血清的α-MEM培养基),加入1 μmol/L的VK1、MK4、MK7和VK3后,每3 d换药1次,连续培养观察14 d后,用0.1%茜素红-Tris-Hcl染液(pH 8.3)染色。染色前,用预冷的10%中性甲醛缓冲液固定10 min,PBS冲洗3次。加入0.1%茜素红-Tris-Hcl染液(pH 8.3),37 ℃下染色30 min。蒸馏水冲洗,干燥,封片。倒置相差显微镜(Leica DMI 3000)下进行观察并随机拍照10张,用image-Pro Plus (IPP 6.0)分析图片,得出每张图片骨结节面积[13]

    • 取新生3 d的SD大鼠胫骨,用α-MEM培养基冲洗骨髓腔以收集骨髓细胞,加入等量Ficoll试剂分离骨髓单核细胞,用含有25 ng/ml M-CSF、25 ng/ml RANKL的细胞因子和10%胎牛血清的α-MEM培养基进行培养,每3 d换液1次,6 d后破骨细胞分化成熟。细胞成熟后,以1×105/ml接种于96孔板,加入1、0.1和0.01 μmol/L的VK1、MK4、MK7和VK3。继续培养48 h后,取100 μl培养基,加入20 μl CellTriter-blue试剂,轻轻晃10 s混匀,37 ℃孵育30 min后,将96孔板置于荧光分光光度计,检测细胞悬液荧光强度(发散光波长560 nm,吸收光波长590 nm)。

    • 成熟破骨细胞以1×105/ml接种于96孔板内,加入1、0.1和0.01 μmol/L的VK1、MK4、MK7和VK3,培养48 h后,弃上清液,PBS冲洗2次,20 µl 0.1%Triton X-100室温破碎细胞15 min,加入100 µl反应液(0.4 g对硝基苯基磷酸二钠,去离子水溶解后加入2.0 g酒石酸钾钠,加水溶解至150 ml,HCl调节pH至3.5,再加水定容至200 ml),于37 ℃反应30 min,迅速加入100 µl 1 mol/L的NaOH终止反应,于波长405 nm处测定其吸光度值[14]

    • 25 μmol/L的VK1、MK4、MK7、VK3和1 μmol/L阳性药Odanacatib用100 mmol/L醋酸钠缓冲液(pH5.5,包含2.5 mmol/L DTT,和2.5 mmol/L EDTA)稀释至浓度为25 μmol/L加入96孔板,最终反应容量为0.2 ml,加入终浓度为5 nmol/L的CTSK孵育5 min,加入5 μl底物1 mmol/L的Z-FR-MCA溶液开始反应,检测5 min荧光信号(发散光波长460 nm,吸收光波长355 nm)。实验设阴性对照,阳性对照(1 μmol/L E-64)。

      计算公式:抑制率(%)=100-(1-Vi/V0)。

      Vi和V0分别表示存在和不存在VK情况下,记录荧光信号强度随时间变化斜率slope值[11]

    • 可溶性I型胶原溶解在100 mmol/L醋酸钠缓冲液(pH5.5,包含2.5 mmol/L DTT和2.5 mmol/L EDTA),最终I型胶原浓度为0.6 mg/ml,加入终浓度100 μmol/L的VK1、MK4、MK7和VK3及10 μmol/L阳性对照药Odanacatib,依次将CTSK和CSA(终浓度分别为400和200 nmol/L)加入含有I型胶原蛋白的反应液中,使总反应液容量为50 μl。混匀,28 ℃孵育4 h,取出后加入1 μl的100 μmol/L E64终止反应。采用10%的SDS-PAGE凝胶电泳分离,卡马斯亮蓝染色20 min,用乙酸-甲醇(4∶1)脱色。I型胶原的α1条带的灰度用Gene Snap(Syngene Inc.Frederick,MD)软件进行定量分析[14]

    • 每组实验重复3次。采用SPSS软件经ANOVA方差分析检验(α=0.05),再用SNK对每两组进行两两比较,以P<0.05为差异有统计学意义。

    • VK1和VK3在0.01~1 μmol/L浓度范围内对成骨细胞增殖和ALP活性均未有影响(图1A1B)。MK4在0.01和0.1 μmol/L浓度显著促进了成骨细胞增殖活性,分别提高了15.5%和23.0%(P<0.05),而MK7分别提高了25.1%和18.4%。MK4和MK7在1 μmol/L显著提高了ALP活性(P<0.05),促进率分别为30.2%和25.7%。成骨细胞在骨结节诱导培养基培养14 d后,经茜素红染色液染色,骨结节处形成红色钙化晶体,骨结节的面积代表了成骨细胞的钙化程度(图1C),结果显示MK4和MK7在1 μmol/L浓度时显著提高了骨结节形成面积(P<0.05),分别增加25.2%和34.0%,VK3显著抑制了骨结节的形成。

    • 图2A可得,VK1、VK3、MK4和MK7在0.01~1 μmol/L浓度对破骨细胞代谢活力均无影响,对破骨细胞无毒性。VK1、MK4和MK7在1 μmol/L表现出抑制TRAP活性,抑制率分别为27.4%、54.0%和35.0%,MK4在0.01和0.11 μmol/L浓度显著降低了破骨细胞TRAP活性(P<0.01),分别降低了50%和41%,MK7在0.1 μmol/L浓度时,TRAP活性显著降低26.8%(图2B)。

    • 本研究考察VK1、VK3、MK4和MK7抑制CTSK与Z-FR-MCA底物结合活性。图3A结果显示,1 μmol/L的ODN抑制了CTSK与Z-FR-MCA底物结合效率达98.3%,而25 μmol/L的VK3、MK4、MK7和VK1抑制率分别为0.3%、58.9%、16.6%和9.4%。与空白胶原相比,VK3、MK4、MK7和VK1在100 μmol/L抑制胶原降解效率达30.8%、73.8%、18.4%和19.2% (图3B3C),仅有MK4和ODN超过60%的抑制率,与未加CSA的CTSK阴性对比,抑制率达38.8%、93.0%、23.1%和24.2%。与空白胶原组对比,阳性抑制剂ODN在1 μmol/L的抑制率达77.3%,与未加CSA的CTSK阴性对比,抑制率达到99.0%。

    • 研究报道VK1、MK4和MK7能显著促进成骨细胞活性和抑制破骨细胞活性[9-10]。临床报道服用高含量维生素K1能够降低髋部骨折风险和提高髋部及腰椎部骨密度,可延缓50~60岁绝经后女性的骨丢失[2]。同时药理研究发现VK3显著促进去卵巢大鼠的骨密度和改善骨质疏松相关指标[5]。而本研究并未发现VK1和VK3具有促进成骨细胞和抑制破骨细胞的作用,可能原因是选择的筛选浓度过低所致。MK4和MK7在1 μmol/L能促进成骨细胞增殖和矿化作用,二者并没有显著差异。但在抑制骨吸收作用方面,MK4相较于MK7在相同浓度,有更显著的抑制TRAP活性的作用。

      CTSK是破骨细胞发挥骨吸收作用的关键靶标酶,选择性地大量表达于破骨细胞,其生理作用底物正是在有机骨基质中含量达95%的I型胶原[11]。我们研究发现,在相同浓度,只有MK4能显著抑制其底物骨胶原降解和人工合成底物Z-FR-MCA的活性。

      日本早在1995年首次将MK4作为治疗骨质疏松的药物应用[15],2011年MK4录入我国《原发性骨质疏松症诊疗指南》[16]。因此,我们认为MK4是人体内源性营养物质,安全性高,适用于各年龄人群作为营养补充剂使用。MK7具有促进骨形成作用,在体内也可以分解成MK4发挥作用,适合作为营养食品补充剂。

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