骨碎补经骨髓间充质干细胞调节OPG/RANKL/RANK通路抑制破骨细胞的实验研究
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摘要
目的研究骨碎补作用绝经后骨质疏松大鼠骨髓间充质干细胞(bone marrow stromal cells,BMSCs)调节OPG/RANKL/RANK通路及对破骨细胞分化成熟的影响并探讨其可能的作用机制。方法实验大鼠去双侧卵巢造模,分为实验组(OVXDF,造模+骨碎补水煎液灌胃)、模型组(OVX,造模+0.9%生理盐水灌胃)、假手术组(SHAM,假手术+0.9%生理盐水灌胃),造模成功后提取BMSCs,将BMSCs和骨髓单核细胞共培养于Transwell小室的上室和下室,分为实验组+破骨细胞(OVXDF+OC)、模型组+破骨细胞(OVX+OC)、假手术组+破骨细胞(SHAM+OC)。下室加入破骨细胞诱导剂,倒置相差显微镜观察破骨细胞的分化成熟情况并计数,酶联免疫吸附剂测定(ELISA)检测下室培养液中骨保护素(osteoprotegerin,OPG)、RANKL的含量,实时荧光定量PCR和蛋白质印迹法(Western blot)检测BMSCs中Wnt10b、β-catenin、RANKL、OPG mRAN及蛋白表达并计算RANKL/OPG。结果在共培养系统中,与去卵巢灌胃大鼠BMSCs共培养的破骨细胞(OVXDF+OC)数量较单纯模型组+破骨细胞(OVX+OC)明显减少(P<0.05)。下室培养液OPG含量及共培养BMSCs中Wnt10b、β-catenin、OPG mRAN及蛋白表达模型组+破骨细胞(OVX+OC)最低,RANKL及RANKL/OPG最高,经骨碎补灌胃后(OVXDF+OC)培养液中OPG含量及BMSCs细胞中Wnt10b、β-catenin、OPG mRAN及蛋白表达明显升高,培养液及BMSCs细胞中RANKL及RANKL/OPG明显降低(P<0.05)。结论骨碎补可调节BMSCs细胞OPG、RANKL的表达,激活OPG/RANKL/RANK信号通路抑制破骨细胞的分化和成熟,此作用可能与BMSCs的Wnt/β-catenin信号通路的激活有关。
Objective To study the effect of rhizoma drynariae on OPG/RANKL/RANK signal path and osteoclast differentiation by bone marrow stromal cells(BMSCs) in postmenopausal osteoporotic rats, and to explore its possible mechanism. Methods Experimental rats were divided into experimental group(OVXDF, ovariectomized and rhizoma drynariae gavage), model group(OVX, ovariectomized and 0.9% NaCl gavage), and sham operation group(SHAM, sham surgery and 0.9% NaCl gavage). After successful modeling, BMSCs were extracted. BMSC and bone marrow mononuclear cells were co-cultured in the upper and lower chambers of the Transwell chamber, respectively. The co-cultured cells were divided into experimental group+osteoclasts(OVXDF+OC), model group+osteoclasts(OVX+OC), and sham operation group + osteoclasts(SHAM+OC). The differentiation and maturation of osteoclasts were observed and counted with a phase-contract microscope. OPG and RANKL in the culture medium of the lower chamber were detected using ELISA method. mRNA and protein expressions of Wnt10 b, β-Catenin, RANKL, and OPG in BMSCs were detected using PCR and Western blotting. Results In the co-culture systems, the number of osteoclasts in OVXDF+OC group was significantly less than in OVX+OC group(P<0.05). OPG content in the culture medium of the lower chamber and the expression of wnt10 b, β-Catenin, and OPG in the co-culture systems were the lowest, but RANKL and RANKL/OPG were the highest, in OVX+OC group. After rhizoma drynariae gavage, OPG content in OVXDF+OC medium and mRNA and protein expressions of Wnt10 b, β-Catenin, and OPG in BMSCs increased significantly(P<0.05), but RANKL content and RANKL/OPG decreased significantly(P<0.05). Conclusion Rhizoma drynariae regulates the expression of OPG and RANKL, and activates OPG/RANKL/RANK signaling pathway to inhibit the differentiation and maturation of osteoclasts in BMSCs. This effect may be related to the activation of Wnt/β-Catenin signaling pathway in BMSCs.
Objective To study the effect of rhizoma drynariae on OPG/RANKL/RANK signal path and osteoclast differentiation by bone marrow stromal cells(BMSCs) in postmenopausal osteoporotic rats, and to explore its possible mechanism. Methods Experimental rats were divided into experimental group(OVXDF, ovariectomized and rhizoma drynariae gavage), model group(OVX, ovariectomized and 0.9% NaCl gavage), and sham operation group(SHAM, sham surgery and 0.9% NaCl gavage). After successful modeling, BMSCs were extracted. BMSC and bone marrow mononuclear cells were co-cultured in the upper and lower chambers of the Transwell chamber, respectively. The co-cultured cells were divided into experimental group+osteoclasts(OVXDF+OC), model group+osteoclasts(OVX+OC), and sham operation group + osteoclasts(SHAM+OC). The differentiation and maturation of osteoclasts were observed and counted with a phase-contract microscope. OPG and RANKL in the culture medium of the lower chamber were detected using ELISA method. mRNA and protein expressions of Wnt10 b, β-Catenin, RANKL, and OPG in BMSCs were detected using PCR and Western blotting. Results In the co-culture systems, the number of osteoclasts in OVXDF+OC group was significantly less than in OVX+OC group(P<0.05). OPG content in the culture medium of the lower chamber and the expression of wnt10 b, β-Catenin, and OPG in the co-culture systems were the lowest, but RANKL and RANKL/OPG were the highest, in OVX+OC group. After rhizoma drynariae gavage, OPG content in OVXDF+OC medium and mRNA and protein expressions of Wnt10 b, β-Catenin, and OPG in BMSCs increased significantly(P<0.05), but RANKL content and RANKL/OPG decreased significantly(P<0.05). Conclusion Rhizoma drynariae regulates the expression of OPG and RANKL, and activates OPG/RANKL/RANK signaling pathway to inhibit the differentiation and maturation of osteoclasts in BMSCs. This effect may be related to the activation of Wnt/β-Catenin signaling pathway in BMSCs.
引文
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[22] Guo D,Wang J,Wang X,et al.Double directional adjusting estrogenic effect of naringin from Rhizoma drynariae (Gusuibu) [J].J Ethnopharmacol,2011,138(2):451-457.
[23] 尹文哲,张小玲,叶义杰,等.骨碎补对微重力下共培养骨细胞中成骨细胞分化的影响[J].中医药学报,2017,45(4):16-20.
[24] 李洋,康倩,荣婵,等.骨碎补总黄酮对MLO-Y4细胞增殖、分化、矿化和凋亡影响的探究[J].中国骨质疏松杂志,2015,21(5):592-598.
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[27] 刘康,吴风晴,吴连国,等.骨碎补总黄酮对骨质疏松模型大鼠OPG/RANKL/RANK 轴系统的影响[J].中国现代应用药学,2015,32(6):652-656.
[28] 刘钟,郭梁,吴亚东,等.骨碎补总黄酮作用于opg/rankl/rank轴系统的相关研究[J].中华中医药学刊,2010,28(2):271-274.
[29] Kobayashi Y,Uehara S,Koide M,et al.The regulation of osteoclast differentiation by wnt signals[J].Bonekey Rep,2015,4:713.
[30] Zhang L,Liu W,Zhao J,et al.Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway[J].Biochim Biophys Acta,2016,1860(10):2211-2219.
[31] Glass DA,Bialek P,Ahn JD,et al.Canonical wnt signaling in differentiated osteoblasts controls osteoclast differentiation[J].Developmental Cell,2005,8(5):751-764.
[2] 宋渊,李盛华,何志军.骨碎补含药血清对成骨细胞增殖、成骨的影响[J].中国骨质疏松杂志,2014,20(2):125-128,170.
[3] 薛海鹏,刘国岩,吴燕,等.骨碎补促进骨髓间充质干细胞增殖及成骨分化[J].中国矫形外科杂志,2018,26(11):1035-1040.
[4] 李风波,孙晓雷,马剑雄,等.柚皮苷对破骨细胞分化的影响[J].中国中药杂志,2015,40(2):308-312.
[5] 李明,吕厚辰,尹鹏滨,等.两种卵巢切除术式建立大鼠骨质疏松模型的优势比较[J].解放军医学院学报,2015(04):383-387.
[6] Simon D,Derer A,Andes FT,et al.Galectin-3 as a novel regulator of osteoblast-osteoclast interaction and bone homeostasis[J].Bone,2017,105:35-41.
[7] 徐丽丽,胥方元,万永鲜.人bmscs自噬水平和成骨分化能力比较[J].中国骨质疏松杂志,2018,24(11):1446-1450,1463.
[8] 任磊,代光明,林枭,等.骨细胞wnt/β-catenin通过notch信号促进bmscs成骨分化[J].中国骨质疏松杂志,2018,24(5):600-605.
[9] 张丽媛,纳青青,吴天秀,等.Orexin-1受体抑制剂通过wnt通路促进骨髓间充质干细胞的成骨分化[J].中国骨质疏松杂志,2017,23(5):606-611.
[10] Wang L,Wang L,Cong X,et al.Human umbilical cord mesenchymal stem cell therapy for patients with active rheumatoid arthritis:Safety and efficacy[J].Stem Cells Dev,2013,22(24):3192-3202.
[11] Gaur T,Lengner CJ,Hovhannisyan H,et al.Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression[J].J Biol Chem,2005,280:33132-33140.
[12] Cai T,Sun D,Duan Y,et al.WNT/β-catenin signaling promotes VSMCs to osteogenic transdifferentiation and calcification through directly modulating Runx2 gene expression[J].Exper Cell Res,2016,345(2):206-217.
[13] Ardeshirylajimi A,Golchin A,Khojasteh A,et al.Increased osteogenic differentiation potential of MSCs cultured on nanofibrous structure through activation of Wnt/β-catenin signalling by inorganic polyphosphate[J].Artif Cells Nanomed Biotechnol,2018,29:1-7.
[14] Takano T,Li YJ,Kukita A,et al.Mesenchymal stem cells markedly suppress inflammatory bone destruction in rats with adjuvant-induced arthritis[J].Lab Invest,2014,94(3):286-296.
[15] Varin A,Pontikoglou C,Labat E,et al.CD200R/CD200 inhibits osteoclastogenesis:new mechanism of osteoclast control by mesenchymal stem cells in human[J].PLoS One,2013,8(8):e72831.
[16] Ma L,Aijima R,Hoshino Y,et al.Transplantation of mesenchymal stem cells ameliorates secondary osteoporosis through interleukin-17-impaired functions of recipient bone marrow mesenchymal stem cells in MRL/lpr mice[J].Stem Cell Res Ther,2015,6:104.
[17] Galli C,Fu Q,Wang W,et al.Commitment to the osteoblast lineage is not required for RANKL gene expression[J].J Biol Chem,2009,284(19):12654-12662.
[18] Galli C,Fu Q,Wang W,et al.Human mesenchymal stem cells inhibit osteoclastogenesis through osteoprotegerin production[J].Arthritis Rheum,2011,63(6):1658-1667.
[19] 张峻玮,李朝辉,陈玲玲,等.基于数据挖掘的中医药治疗原发性骨质疏松症用药规律分析[J].河北中医,2018,40(6):933-937.
[20] 李晋玉,赵学千,孙旗,等.骨碎补总黄酮的实验及临床研究概况[J].中国骨质疏松杂志,2018,24(10):1357-1364.
[21] 刘剑刚,谢雁鸣,徐哲,等.骨碎补总黄酮的活血化瘀作用及对实验性微循环障碍和骨质疏松症的影响[J].中国骨质疏松杂志,2006,12(1):46-49.
[22] Guo D,Wang J,Wang X,et al.Double directional adjusting estrogenic effect of naringin from Rhizoma drynariae (Gusuibu) [J].J Ethnopharmacol,2011,138(2):451-457.
[23] 尹文哲,张小玲,叶义杰,等.骨碎补对微重力下共培养骨细胞中成骨细胞分化的影响[J].中医药学报,2017,45(4):16-20.
[24] 李洋,康倩,荣婵,等.骨碎补总黄酮对MLO-Y4细胞增殖、分化、矿化和凋亡影响的探究[J].中国骨质疏松杂志,2015,21(5):592-598.
[25] 陈云刚,谭国庆,任维龙,等.骨碎补含药血清经wnt/β-catenin信号通路对大鼠骨髓间充质干细胞成骨分化的影响[J].中国药理学通报,2017,33(6):830-836.
[26] 高俊,胡继红,张曦.骨碎补总黄酮对废用性骨质疏松大鼠BMSCs成脂分化的影响及机制探讨[J].山东医药,2015,55(35):28-30.
[27] 刘康,吴风晴,吴连国,等.骨碎补总黄酮对骨质疏松模型大鼠OPG/RANKL/RANK 轴系统的影响[J].中国现代应用药学,2015,32(6):652-656.
[28] 刘钟,郭梁,吴亚东,等.骨碎补总黄酮作用于opg/rankl/rank轴系统的相关研究[J].中华中医药学刊,2010,28(2):271-274.
[29] Kobayashi Y,Uehara S,Koide M,et al.The regulation of osteoclast differentiation by wnt signals[J].Bonekey Rep,2015,4:713.
[30] Zhang L,Liu W,Zhao J,et al.Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway[J].Biochim Biophys Acta,2016,1860(10):2211-2219.
[31] Glass DA,Bialek P,Ahn JD,et al.Canonical wnt signaling in differentiated osteoblasts controls osteoclast differentiation[J].Developmental Cell,2005,8(5):751-764.