海参皂苷对去卵巢小鼠骨密度的改善作用及机制
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摘要
目的:研究海参皂苷对双侧去卵巢小鼠骨密度的影响及作用机制。方法:雌性健康C57BL/6J小鼠采用去卵巢术建立骨质疏松症模型,术后4周,随机分为假手术组(生理盐水)、模型组(生理盐水)和低、高剂量海参皂苷组(分别为7.5、15.0 mg/kg m_b)。连续灌胃90 d后,检测尿钙浓度、尿磷浓度、骨密度和骨矿化沉积率,评价海参皂苷对模型小鼠骨矿物质的影响;进一步通过测定ALP等成骨标志指标的mRNA表达情况,以及调控其表达水平的Wnt/β-catenin通路关键基因的mRNA表达情况,探究海参皂苷改善骨密度的分子机制。结果:尿液检测显示,海参皂苷能够显著降低尿钙、磷浓度(P<0.05),减少骨矿流失;双能X射线扫描及荧光双标记结果显示,海参皂苷能显著增加去卵巢小鼠骨密度,提高骨矿化沉积率(P<0.05);实时荧光定量聚合酶链式反应结果表明,海参皂苷显著降低Wnt/β-catenin通路中关键基因的水平(P<0.05),下调骨生成标志基因ALP、OCN、Col1a和BMP2的mRNA表达水平(P<0.05)。结论:海参皂苷能通过抑制Wnt/β-catenin通路降低去卵巢模型小鼠代偿性增加的骨生成,并能够抑制骨矿流失,提高骨密度和骨矿化沉积,改善骨质疏松症。
Objective: To investigate the effect and underlying mechanism of sea cucumber saponin(SCS) on bone mineral density in bilaterally ovariectomized mice. Methods: Female C57 BL/6 J mice were ovariectomized to establish an osteoporotic animal model. After 4 weeks, the mice were randomly divided into four groups: sham operation(normal saline), model(normal saline), low-dose SCS(7.5 mg/kg mb) and high-dose SCS(15.0 mg/kg m_b) groups. After administration for 90 days,the urinary concentrations of Ca and P, bone mineral density(BMD) and mineral apposition rate(MAR) were determined to evaluate the effect of SCS on bone minerals. Furthermore, the relative mRNA expression of biochemical parameters of bone formation(ALP, OCN, Col1 a, BMP2) and key genes involved in the Wnt/β-catenin pathway were detected to elucidate the underlying molecular mechanism. Results: SCS signi?cantly inhibited the urinary levels of Ca and P(P < 0.05),and decreased the loss of bone mineral. Dual energy X-ray scanning and ?uorescence double labeling results showed that SCS significantly increased BMD and bone mineralization in ovariectomized mice(P < 0.05). Quantitative real-time polymerase chain reaction(qPCR) showed that SCS could decrease the relative mRNA expression of ALP, OCN, Col1 a and BMP2 by down-regulating the relative mRNA expression of Wnt10 b, LRP5, GSK-3β,β-catenin, Runx2 and OSX, the key genes involved in the Wnt/β-catenin pathway(P < 0.05). Conclusion: SCS can improve osteoporosis by inhibiting the loss of bone mineral, increasing BMD and bone mineralization, inhibiting the activation of the Wnt/β-catenin pathway and consequently limiting the compensatory increase in bone formation.
Objective: To investigate the effect and underlying mechanism of sea cucumber saponin(SCS) on bone mineral density in bilaterally ovariectomized mice. Methods: Female C57 BL/6 J mice were ovariectomized to establish an osteoporotic animal model. After 4 weeks, the mice were randomly divided into four groups: sham operation(normal saline), model(normal saline), low-dose SCS(7.5 mg/kg mb) and high-dose SCS(15.0 mg/kg m_b) groups. After administration for 90 days,the urinary concentrations of Ca and P, bone mineral density(BMD) and mineral apposition rate(MAR) were determined to evaluate the effect of SCS on bone minerals. Furthermore, the relative mRNA expression of biochemical parameters of bone formation(ALP, OCN, Col1 a, BMP2) and key genes involved in the Wnt/β-catenin pathway were detected to elucidate the underlying molecular mechanism. Results: SCS signi?cantly inhibited the urinary levels of Ca and P(P < 0.05),and decreased the loss of bone mineral. Dual energy X-ray scanning and ?uorescence double labeling results showed that SCS significantly increased BMD and bone mineralization in ovariectomized mice(P < 0.05). Quantitative real-time polymerase chain reaction(qPCR) showed that SCS could decrease the relative mRNA expression of ALP, OCN, Col1 a and BMP2 by down-regulating the relative mRNA expression of Wnt10 b, LRP5, GSK-3β,β-catenin, Runx2 and OSX, the key genes involved in the Wnt/β-catenin pathway(P < 0.05). Conclusion: SCS can improve osteoporosis by inhibiting the loss of bone mineral, increasing BMD and bone mineralization, inhibiting the activation of the Wnt/β-catenin pathway and consequently limiting the compensatory increase in bone formation.
引文
[1]KANIS J A,BURLET N,COOPER C,et al.European guidance for the diagnosis and management of osteoporosis in postmenopausal women[J].Osteoporosis International,2008,19(4):399-428.DOI:10.1007/s00198-012-2074-y.
[2]LI W,WANG K,LIU Z,et al.HIF-1alpha change in serum and callus during fracture healing in ovariectomized mice[J].International Journal of Clinical and Experimental Pathology,2015,8(1):117-126.
[3]KEEN R W.Burden of osteoporosis and fractures[J].Current Osteoporosis Reports,2003,1(2):66-70.
[4]MIGLIORATI C A,SCHUBERT M M,PETERSON D E.Bisphosphonate osteonecrosis(BON):unanswered questions and research possibilities[J].Reviews on Recent Clinical Trials,2009,4(2):99-109.
[5]王柄棋,孙雨晴,陈翔,等.绝经后骨质疏松症药物治疗的现状与思考[J].中国骨质疏松杂志,2017,23(6):818-823.DOI:10.3969/j.issn.1006-7108.
[6]张伟伟,陆茵.海参的抗肿瘤作用研究进展[J].中华中医中药杂志,2010,25(1):105-108.
[7]王静,张京楼,王铎喜,等.海参多肽的抗氧化性能研究[J].食品与机械,2010,26(2):67-71.DOI:10.13652/j.issn.1003-5788.2010.02.043.
[8]张梅秀,王锡昌,刘源.海参生物活性研究进展[J].天然产物研究与开发,2012,24(8):1151-1159.DOI:10.16333/j.1001-6880.2012.08.029.
[9]郭盈莹,丁燕,徐飞飞,等.海参中主要生物活性成分研究进展[J].食品科学,2014,35(15):335-344.DOI:10.7506/spkx1002-6630-201415066.
[10]董平.革皮氏海参皂苷化合物的分离鉴定、结构修饰及活性研究[D].青岛: 中国海洋大学,2009:54-86.
[11]GU Y Q,ZHOU J C,WANG Q,et al.Ginsenoside Rg1 promotes osteogenic differentiation of rBMSCs and healing of rat tibial fractures through regulation of GR-dependent BMP-2/SMAD signaling[J].Scientific Reports,2016,12(5):1-13.DOI:10.1038/srep25282.
[12]CHENG B B,LI J,DU J,et al.Ginsenoside Rb1 inhibits osteoclastogenesis by modulating NF-κB and MAPKs pathways[J].Food and Chemical Toxicology,2012,50(5):1610-1615.DOI:10.1016/j.fct.2012.02.019.
[13]AMININ D L,CHAYKINA E L,AGAFONOVA I G,et al.Antitumor activity of the immunomodulatory lead Cumaside[J].International Immunopharmacology,2010,10(6):648-654.DOI:10.1016/j.intimp.2010.03.003.
[14]胡晓倩.海参皂苷对脂质代谢的影响及其机制研究[D].青岛: 中国海洋大学,2010:36-93.
[15]黄公怡.骨质疏松性骨折的特点及临床与研究进展[J].基础医学与临床,2007,10(4):1088-1092.
[16]黄火强.骨质疏松症发病机理及临床药物治疗[J].标记免疫分析与临床,2010,17(3):205-208.
[17]王冰梅,赵娜,于洪宇,等.骨质疏松的中西医研究进展[J].中医药信息,2016,33(6):128-131.
[18]KARNER C M,LONG F X.Wnt signaling and cellular metabolism in osteoblasts[J].Cellular and Molecular Life Science,2017,74(9):1649-1657.DOI:10.1007/s00018-016-2425-5.
[19]王一名,王静凤,毛美霖,等.南极磷虾肽对去卵巢大鼠骨质疏松症的改善作用[J].中国海洋药物,2015,34(4):19-25.DOI:10.13400/j.cnki.cjmd.2015.04.004.
[20]王姗姗,周晓春,李秀秀,等.鲫鱼卵唾液酸糖蛋白对去卵巢大鼠骨质疏松症的改善作用[J].食品科学,2014,35(13):182-185.DOI:10.7506/spkx1002-6630-201413035.
[21]NJEH C F,BOIVIN C M,LANGTON C M,et al.The role of ultrasound in the assessnment of osteoporosis:a review[J].Osteoporosis International,1997,7(1):7-22.
[22]王冰.骨代谢生化指标在绝经后骨质疏松症患者长得应用价值[J].现代妇产科进展,2014,23(2):136-138.DOI:10.13283/j.cnki.xdfckjz.2014.02.065.
[23]HUANG Qiang,GAO Bo,JIE Qiang,et al.Ginsenoside-Rb2 displays anti-osteoporosis effects through reducing oxidative damage and boneresorbing cytokines during osteogenesis[J].Bone,2014,66:306-314.DOI:10.1016/j.bone.2014.06.010.
[24]HE Long,LEE J,JANG J H,et al.Ginsenoside Rh2 inhibits osteoclastogenesis through down-regulation of NF-κB,NFATc1and c-Fos[J].Bone,2012,50(6):1207-1213.DOI:10.1016/j.bone.2012.03.022.
[25]张珂.鸡蛋壳复合抗菌支架材料的制备及骨缺损修复的实验研究[D].上海: 复旦大学,2012:24-39.
[26]丁婧文.紫外线处理微弧氧化纯钛种植体早期成骨作用的组织学探讨[D].广州: 南方医科大学,2013:60-62.
[27]刘忠厚.骨矿与临床[M].北京:中国科学技术出版社,2006:300-350.
[28]王智存,施婕,邹远妩,等.骨代谢生化指标对骨质疏松的诊断价值[J].实用医技杂志,2016,23(1):70-72.
[29]唐三元,谭文成,杨辉,等.多种骨代谢生化指标联合预测老年骨质疏松性髋部骨折风险的意义[J].中国矫形外科杂志,2015,23(18):1653-1656.DOI:10.3977/j.issn.1005-8478.2015.18.05.
[30]张楠,章秋.骨代谢生化指标在骨质疏松症中的应用[J].安徽医药,2011,15(4):401-404.
[31]KOBAYASHI Y,UEHARA S,UDAGAWA N,et al.Regulation of bone metabolism by Wnt signals[J].Journal of Biochemistry,2016,159(4):387-392.DOI:10.1093/jb/mvv124.
[32]BARON R,KNEISSEL M.WNT signaling in bone homeostasis and disease:from human mutations to treatments[J].Nature Medicine,2013,19(2):179-192.DOI:10.1038/nm.3074.
[33]CANALIS E.Wnt signalling in osteoporosis:mechanisms and novel therapeutic approaches[J].Natture Reviews Endocrinology,2013,9(10):575-583.DOI:10.1038/nrendo.2013.154.
[34]WANG Fei,WANG Yiming,ZHAO Yanlei,et al.Sialoglycoprotein isolated from eggs of Carassius auratus ameliorates osteoporosis:an effect associated with regulation of the Wnt/β-catenin pathway in rodents[J].Journal of Agricultural and Food Chemistry,2016,64(14):2875-2882.DOI:10.1021/acs.jafc.5b06132.
[2]LI W,WANG K,LIU Z,et al.HIF-1alpha change in serum and callus during fracture healing in ovariectomized mice[J].International Journal of Clinical and Experimental Pathology,2015,8(1):117-126.
[3]KEEN R W.Burden of osteoporosis and fractures[J].Current Osteoporosis Reports,2003,1(2):66-70.
[4]MIGLIORATI C A,SCHUBERT M M,PETERSON D E.Bisphosphonate osteonecrosis(BON):unanswered questions and research possibilities[J].Reviews on Recent Clinical Trials,2009,4(2):99-109.
[5]王柄棋,孙雨晴,陈翔,等.绝经后骨质疏松症药物治疗的现状与思考[J].中国骨质疏松杂志,2017,23(6):818-823.DOI:10.3969/j.issn.1006-7108.
[6]张伟伟,陆茵.海参的抗肿瘤作用研究进展[J].中华中医中药杂志,2010,25(1):105-108.
[7]王静,张京楼,王铎喜,等.海参多肽的抗氧化性能研究[J].食品与机械,2010,26(2):67-71.DOI:10.13652/j.issn.1003-5788.2010.02.043.
[8]张梅秀,王锡昌,刘源.海参生物活性研究进展[J].天然产物研究与开发,2012,24(8):1151-1159.DOI:10.16333/j.1001-6880.2012.08.029.
[9]郭盈莹,丁燕,徐飞飞,等.海参中主要生物活性成分研究进展[J].食品科学,2014,35(15):335-344.DOI:10.7506/spkx1002-6630-201415066.
[10]董平.革皮氏海参皂苷化合物的分离鉴定、结构修饰及活性研究[D].青岛: 中国海洋大学,2009:54-86.
[11]GU Y Q,ZHOU J C,WANG Q,et al.Ginsenoside Rg1 promotes osteogenic differentiation of rBMSCs and healing of rat tibial fractures through regulation of GR-dependent BMP-2/SMAD signaling[J].Scientific Reports,2016,12(5):1-13.DOI:10.1038/srep25282.
[12]CHENG B B,LI J,DU J,et al.Ginsenoside Rb1 inhibits osteoclastogenesis by modulating NF-κB and MAPKs pathways[J].Food and Chemical Toxicology,2012,50(5):1610-1615.DOI:10.1016/j.fct.2012.02.019.
[13]AMININ D L,CHAYKINA E L,AGAFONOVA I G,et al.Antitumor activity of the immunomodulatory lead Cumaside[J].International Immunopharmacology,2010,10(6):648-654.DOI:10.1016/j.intimp.2010.03.003.
[14]胡晓倩.海参皂苷对脂质代谢的影响及其机制研究[D].青岛: 中国海洋大学,2010:36-93.
[15]黄公怡.骨质疏松性骨折的特点及临床与研究进展[J].基础医学与临床,2007,10(4):1088-1092.
[16]黄火强.骨质疏松症发病机理及临床药物治疗[J].标记免疫分析与临床,2010,17(3):205-208.
[17]王冰梅,赵娜,于洪宇,等.骨质疏松的中西医研究进展[J].中医药信息,2016,33(6):128-131.
[18]KARNER C M,LONG F X.Wnt signaling and cellular metabolism in osteoblasts[J].Cellular and Molecular Life Science,2017,74(9):1649-1657.DOI:10.1007/s00018-016-2425-5.
[19]王一名,王静凤,毛美霖,等.南极磷虾肽对去卵巢大鼠骨质疏松症的改善作用[J].中国海洋药物,2015,34(4):19-25.DOI:10.13400/j.cnki.cjmd.2015.04.004.
[20]王姗姗,周晓春,李秀秀,等.鲫鱼卵唾液酸糖蛋白对去卵巢大鼠骨质疏松症的改善作用[J].食品科学,2014,35(13):182-185.DOI:10.7506/spkx1002-6630-201413035.
[21]NJEH C F,BOIVIN C M,LANGTON C M,et al.The role of ultrasound in the assessnment of osteoporosis:a review[J].Osteoporosis International,1997,7(1):7-22.
[22]王冰.骨代谢生化指标在绝经后骨质疏松症患者长得应用价值[J].现代妇产科进展,2014,23(2):136-138.DOI:10.13283/j.cnki.xdfckjz.2014.02.065.
[23]HUANG Qiang,GAO Bo,JIE Qiang,et al.Ginsenoside-Rb2 displays anti-osteoporosis effects through reducing oxidative damage and boneresorbing cytokines during osteogenesis[J].Bone,2014,66:306-314.DOI:10.1016/j.bone.2014.06.010.
[24]HE Long,LEE J,JANG J H,et al.Ginsenoside Rh2 inhibits osteoclastogenesis through down-regulation of NF-κB,NFATc1and c-Fos[J].Bone,2012,50(6):1207-1213.DOI:10.1016/j.bone.2012.03.022.
[25]张珂.鸡蛋壳复合抗菌支架材料的制备及骨缺损修复的实验研究[D].上海: 复旦大学,2012:24-39.
[26]丁婧文.紫外线处理微弧氧化纯钛种植体早期成骨作用的组织学探讨[D].广州: 南方医科大学,2013:60-62.
[27]刘忠厚.骨矿与临床[M].北京:中国科学技术出版社,2006:300-350.
[28]王智存,施婕,邹远妩,等.骨代谢生化指标对骨质疏松的诊断价值[J].实用医技杂志,2016,23(1):70-72.
[29]唐三元,谭文成,杨辉,等.多种骨代谢生化指标联合预测老年骨质疏松性髋部骨折风险的意义[J].中国矫形外科杂志,2015,23(18):1653-1656.DOI:10.3977/j.issn.1005-8478.2015.18.05.
[30]张楠,章秋.骨代谢生化指标在骨质疏松症中的应用[J].安徽医药,2011,15(4):401-404.
[31]KOBAYASHI Y,UEHARA S,UDAGAWA N,et al.Regulation of bone metabolism by Wnt signals[J].Journal of Biochemistry,2016,159(4):387-392.DOI:10.1093/jb/mvv124.
[32]BARON R,KNEISSEL M.WNT signaling in bone homeostasis and disease:from human mutations to treatments[J].Nature Medicine,2013,19(2):179-192.DOI:10.1038/nm.3074.
[33]CANALIS E.Wnt signalling in osteoporosis:mechanisms and novel therapeutic approaches[J].Natture Reviews Endocrinology,2013,9(10):575-583.DOI:10.1038/nrendo.2013.154.
[34]WANG Fei,WANG Yiming,ZHAO Yanlei,et al.Sialoglycoprotein isolated from eggs of Carassius auratus ameliorates osteoporosis:an effect associated with regulation of the Wnt/β-catenin pathway in rodents[J].Journal of Agricultural and Food Chemistry,2016,64(14):2875-2882.DOI:10.1021/acs.jafc.5b06132.