补肾活血汤通过Runx2/Osterix促进骨质疏松模型大鼠的骨折愈合
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摘要
背景:Runx2/Osterix是促进成骨细胞分化的重要的转录因子,研究发现中药能通过上调Runx2/Osterix促进骨折愈合。补肾活血汤是临床上治疗骨折的经验方药,能促进骨形成,通过探究其作用机制更好服务临床。目的:通过慢病毒载体介导Runx2基因沉默,研究补肾活血汤对骨质疏松模型大鼠骨折愈合影响及分子机制。方法:将广州中医院药大学动物实验中心提供的40只4月龄成年雌性SD大鼠随机等为5组,模型组、补肾活血汤组、Runx2沉默组、Runx2沉默+补肾活血汤组及对照组,对照组只建立股骨骨折模型,其他各组切除双侧卵巢并造成股骨骨折制作骨质疏松骨折大鼠模型。建模后补肾活血汤组、Runx2沉默+补肾活血汤组予以补肾活血汤灌胃,模型组、对照组及Runx2沉默组予以等体积蒸馏水灌胃;Runx2沉默组、Runx2沉默+补肾活血汤组于骨折处注射Runx2重组慢病毒,模型组、对照组及补肾活血汤组于相同部位处注射等体积PBS。建模4周后,Micro CT测定股骨标本骨密度、组织矿物质密度及骨体积分数(BV/TV),q PCR、Western blot测定骨痂成骨标志基因Runx2及Osterix mRNA及蛋白的表达水平。结果与结论:(1)与对照组相比,模型组骨密度、组织矿物质密度、Runx2和Osterixm RNA及蛋白表达量显著降低(P <0.01);(2)与模型组相比,补肾活血汤组骨体积分数(BV/TV)、Runx2、Osterix mRNA表达量升高(P<0.05),而骨密度、组织矿物质密度、Runx2及Osterix蛋白表达量显著升高(P<0.01);Runx2沉默组骨体积分数和Osterix mRNA表达量降低(P <0.05),骨密度、组织矿物质密度、Runx2 mRNA及蛋白表达量、Osterix蛋白表达量显著降低(P <0.01);(3)与Runx2沉默组比较,Runx2沉默+补肾活血汤组上述全部指标的m RNA及蛋白表达均显著升高(P <0.01);(4)结果表明,慢病毒介导Runx2基因沉默使大鼠骨质疏松骨折愈合延缓,补肾活血汤能上调Runx2/Osterix表达,逆转Runx2基因沉默的效应,具有明显促进骨折愈合的作用,对骨质疏松性骨折具有良好的治疗效果。
BACKGROUND: Runx2/Osterix is an important transcription factor promoting osteoblast differentiation. Chinese herbs have been shown to promote fracture healing through up-regulation of Runx2/Osterix. Bushen Huoxue Decoction is a prescription used to treat fractures and it can promote bone formation. Exploring underlying mechanism is helpful for its clinical application. OBJECTIVE: To study the effect of Bushen Huoxue Decoction on osteoporotic fracture healing in rats and the underlying molecular mechanism. METHODS: Forty 4-month-old adult female Sprague-Dawley rats provided by Laboratory Animal Centre of Guangzhou University of Chinese Medicine were randomly divided into five groups: model, Bushen Huoxue Decoction, Runx2 gene silencing, Runx2 gene silencing + Bushen Huoxue Decoction, and control groups. The femoral fracture model was established in the control group, while the osteoporotic femoral fracture model was established by ovariectomy in the other groups. Rats in the Bushen Huoxue Decoction and Runx2 gene silencing + Bushen Huoxue Decoction groups were treated with Bushen Huoxue Decoction, while rats in the model, control and Runx2 gene silencing groups were treated with equal volume of distilled water. Rats in the Runx2 gene silencing and Runx2 gene silencing + Bushen Huoxue Decoction groups were injected with Runx2 recombinant lentivirus, while rats in the model, control, and Bushen Huoxue Decoction groups were injected with equal volume of PBS at the same site. After 4 weeks, bone mineral density, tissue mineral density and bone volume fraction were measured by Micro CT. The mRNA and protein expression levels of the callus osteogenesis markers Runx2 and Osterix were detected by qPCR and western blot assay, respectively. RESULTS AND CONCLUSION: Compared with the control group, bone mineral density, tissue mineral density, and expression levels of Runx2 and Osterix mRNA and protein in the model group were significantly decreased(P < 0.01). Compared with the model group, bone volume fraction and Runx2 and Osterix mRNA expression levels were significantly increased(P < 0.05), and Runx2 and Osterix protein expression levels were significantly increased(P < 0.01) in the Bushen Huoxue Decoction group. Compared with the model group, Osterix mRNA expression level and bone volume fraction were significantly decreased(P < 0.05), bone mineral density, tissue mineral density, Runx2 mRNA and protein expression levels, and Osterix protein expression level were significantly decreased(P < 0.01) in the Runx2 gene silencing group. Compared with the Runx2 gene silencing group, Runx2 and Osterix mRNA and protein expression levels, bone mineral density, tissue mineral density and bone volume fraction in the Runx2 gene silencing + Bushen Huoxue Decoction group were significantly increased(P < 0.01). To conclude, lentivirus-mediated Runx2 gene silencing can delay the healing of osteoporotic fracture in rats. Bushen Huoxue Decoction can up-regulate the expression of Runx2/Osterix and reverse the effect of Runx2 gene silencing, which can obviously promote the healing of fracture and has good therapeutic effect on osteoporotic fracture.
BACKGROUND: Runx2/Osterix is an important transcription factor promoting osteoblast differentiation. Chinese herbs have been shown to promote fracture healing through up-regulation of Runx2/Osterix. Bushen Huoxue Decoction is a prescription used to treat fractures and it can promote bone formation. Exploring underlying mechanism is helpful for its clinical application. OBJECTIVE: To study the effect of Bushen Huoxue Decoction on osteoporotic fracture healing in rats and the underlying molecular mechanism. METHODS: Forty 4-month-old adult female Sprague-Dawley rats provided by Laboratory Animal Centre of Guangzhou University of Chinese Medicine were randomly divided into five groups: model, Bushen Huoxue Decoction, Runx2 gene silencing, Runx2 gene silencing + Bushen Huoxue Decoction, and control groups. The femoral fracture model was established in the control group, while the osteoporotic femoral fracture model was established by ovariectomy in the other groups. Rats in the Bushen Huoxue Decoction and Runx2 gene silencing + Bushen Huoxue Decoction groups were treated with Bushen Huoxue Decoction, while rats in the model, control and Runx2 gene silencing groups were treated with equal volume of distilled water. Rats in the Runx2 gene silencing and Runx2 gene silencing + Bushen Huoxue Decoction groups were injected with Runx2 recombinant lentivirus, while rats in the model, control, and Bushen Huoxue Decoction groups were injected with equal volume of PBS at the same site. After 4 weeks, bone mineral density, tissue mineral density and bone volume fraction were measured by Micro CT. The mRNA and protein expression levels of the callus osteogenesis markers Runx2 and Osterix were detected by qPCR and western blot assay, respectively. RESULTS AND CONCLUSION: Compared with the control group, bone mineral density, tissue mineral density, and expression levels of Runx2 and Osterix mRNA and protein in the model group were significantly decreased(P < 0.01). Compared with the model group, bone volume fraction and Runx2 and Osterix mRNA expression levels were significantly increased(P < 0.05), and Runx2 and Osterix protein expression levels were significantly increased(P < 0.01) in the Bushen Huoxue Decoction group. Compared with the model group, Osterix mRNA expression level and bone volume fraction were significantly decreased(P < 0.05), bone mineral density, tissue mineral density, Runx2 mRNA and protein expression levels, and Osterix protein expression level were significantly decreased(P < 0.01) in the Runx2 gene silencing group. Compared with the Runx2 gene silencing group, Runx2 and Osterix mRNA and protein expression levels, bone mineral density, tissue mineral density and bone volume fraction in the Runx2 gene silencing + Bushen Huoxue Decoction group were significantly increased(P < 0.01). To conclude, lentivirus-mediated Runx2 gene silencing can delay the healing of osteoporotic fracture in rats. Bushen Huoxue Decoction can up-regulate the expression of Runx2/Osterix and reverse the effect of Runx2 gene silencing, which can obviously promote the healing of fracture and has good therapeutic effect on osteoporotic fracture.
引文
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[11]许兵,金红婷,刘慧,等.补肾活血颗粒对去势大鼠骨组织Wnt/β-Catenin通路的影响研究[J].中华中医药杂志,2013,28(11):3400-3405.
[12]闵文,黄桂成,华永庆,等.补肾通络方对去卵巢骨质疏松模型大鼠骨组织RANKL/OPG基因表达的影响[J].中国实验方剂学杂志,2013,19(15):258-261.
[13]庞坚,王翔,陈元川,等.中药治疗骨质疏松性骨折的组方用药研究[J].时珍国医国药,2015,26(1):238-239.
[14]黄永铨,罗毅文,王斌,等.补肾活血汤治疗老年桡骨远端骨折的临床疗效观察[J].中国中医骨伤科杂志,2015,23(3):5-8.
[15]王羿,党兴.补肾活血法对SD大鼠骨折模型愈合影响的实验研究[J].时珍国医国药,2012,23(12):3150-3151.
[16]Tsai LK,Leng Y,Wang Z,et al.The mood stabilizers valproic acid and lithium enhance mesenchymal stem cell migration via distinct mechanisms.Neuropsychopharmacology.2010;35(11):2225-2237.
[17]高建清,庄素新,黄小敬,等.淫羊藿对大鼠骨量及骨髓基质干细胞增殖、分化过程中Cbfa1和Osterix表达的影响[J].中华中医药学刊,2016,34(2):384-386.
[18]高璐,郑洪新,陈谊敬,等.补肾中药成分配伍调控Runx2、OSX对大鼠BMSCs成骨分化的影响[J].世界中西医结合杂志,2014,9(4):425-429.
[19]Komori T.Regulation of skeletal development by the Runx family of transcription factors.J Cell Biochem.2010;95(3):445-453.
[20]李丹,范哲,李广生,等.Runx2与骨生长发育[J].中华地方病学杂志,2004,23(6):620-623.
[21]杨光正,张文杰,丁迅,等.Runx2、Osterix转录因子过表达驱动内皮细胞成骨分化的机制探讨[J].上海口腔医学,2017,26(4):353-357.
[22]Kobayashi H,Gao Y,Ueta C,et al.Multilineage differentiation of Cbfa1-deficient calvarial cells in vitro.Biochem Biophys Res Commun.2000;273(2):630-636.
[23]Galindo M,Kahler RA,Teplyuk NM,et al.Cell cycle related modulations in Runx2 protein levels are independent of lymphocyte enhancer-binding factor 1(Lef1)in proliferating osteoblasts.J Mol Histol.2007;38(5):501-506.
[24]解光越,侯晓华,张志勇,等.老年骨质疏松患者股骨Runt相关转录因子2水平与骨密度的关系[J].中国老年学杂志,2016,36(8):1962-1963.
[25]Nakashima K,Zhou X,Kunkel G,et al.The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.Cell.2002;108(1):17-29.
[26]Sinha KM,Zhou X.Genetic and molecular control of osterix in skeletal formation.J Cell Biochem.2013;114(5):975-984.
[27]张驰.成骨细胞特异性转录因子Osterix对骨形成作用的分子机制(英文)[J].北京大学学报(医学版),2012,44(5):659-665.
[28]Cao Z,Liu R,Zhang H.Osterix controls cementoblast differentiation through downregulation of Wnt-signaling via enhancing DKK1 expression.International J Biol Sci.2015;11(3):335-344.
[29]Chen S,Feng J,Zhang H,et al.Key role for the transcriptional factor,osterix,in spine development.Spine J.2014;14(4):683-694.
[30]Lai QG,Yuan KF,Xu X,et al.Transcription factor osterix modified bone marrow mesenchymal stem cells enhance callus formation during distraction osteogenesis.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2011;111(4):412-419.
[31]Nishio Y,Dong Y,Paris M,et al.Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene.Gene.2006;372(1-2):62-70.
[32]Komori T.Roles of Runx2 in skeletal development.Adv Exp Med Biol.2017;962:83-93.
[33]Rashid H,Ma C,Chen H,et al.Sp7 and Runx2 molecular complex synergistically regulate expression of target genes.Connect Tissue Res.2014;55 Suppl 1(Supp 1):83-87.
[34]Komori T.Regulation of osteoblast differentiation by transcription factors.J Cell Biochem.2010;99(5):1233-1239.
[2]Cosman F,de Beur SJ,LeBoff MS,et al.Clinician's guide to prevention and treatment of osteoporosis.Osteoporos Int.2014;25(10):2359-2381.
[3]罗毅文,王斌,吴志方,等.补肾活血汤提取物促进大鼠骨髓间充质干细胞体外迁移及CXCR4表达的研究[J].中药新药与临床药理,2016,27(3):356-361.
[4]黄琛,黄浩,艾志,等.补肾活血汤联合经皮锥体成形术对老年骨质疏松性椎体压缩性骨折的疗效及其安全性观察[J].中华中医药学刊,2018,36(3):209-214.
[5]Karp JM,Leng Teo GS.Mesenchymal stem cell homing:the devil is in the details.Cell Stem Cell.2009;4(3):206-216.
[6]程英雄,罗毅文,王斌,等.补肾活血汤水提物调控Cbfal/Runx2基因沉默骨髓间充质干细胞SP7/Osterix及碱性磷酸酶的表达[J].中国组织工程研究,2018,22(13):1987-1992.
[7]Utv?g SE,Korsnes L,Rindal DB,et al.Influence of flexible nailing in the later phase of fracture healing:strength and mineralization in rat femora.J Orthop Sci.2001;6(6):576-584.
[8]刘建文.药理实验方法学[M].北京:化学工业出版社,2008.
[9]Larsson S,Fazzalari NL.Anti-osteoporosis therapy and fracture healing.Arch Orthop Trauma Sur.2014;134(2):291-297.
[10]孙亚奇,卞艳芳,周春娜,等.中药治疗骨质疏松症研究进展[J].中国临床医学,2018,25(2):307-313.
[11]许兵,金红婷,刘慧,等.补肾活血颗粒对去势大鼠骨组织Wnt/β-Catenin通路的影响研究[J].中华中医药杂志,2013,28(11):3400-3405.
[12]闵文,黄桂成,华永庆,等.补肾通络方对去卵巢骨质疏松模型大鼠骨组织RANKL/OPG基因表达的影响[J].中国实验方剂学杂志,2013,19(15):258-261.
[13]庞坚,王翔,陈元川,等.中药治疗骨质疏松性骨折的组方用药研究[J].时珍国医国药,2015,26(1):238-239.
[14]黄永铨,罗毅文,王斌,等.补肾活血汤治疗老年桡骨远端骨折的临床疗效观察[J].中国中医骨伤科杂志,2015,23(3):5-8.
[15]王羿,党兴.补肾活血法对SD大鼠骨折模型愈合影响的实验研究[J].时珍国医国药,2012,23(12):3150-3151.
[16]Tsai LK,Leng Y,Wang Z,et al.The mood stabilizers valproic acid and lithium enhance mesenchymal stem cell migration via distinct mechanisms.Neuropsychopharmacology.2010;35(11):2225-2237.
[17]高建清,庄素新,黄小敬,等.淫羊藿对大鼠骨量及骨髓基质干细胞增殖、分化过程中Cbfa1和Osterix表达的影响[J].中华中医药学刊,2016,34(2):384-386.
[18]高璐,郑洪新,陈谊敬,等.补肾中药成分配伍调控Runx2、OSX对大鼠BMSCs成骨分化的影响[J].世界中西医结合杂志,2014,9(4):425-429.
[19]Komori T.Regulation of skeletal development by the Runx family of transcription factors.J Cell Biochem.2010;95(3):445-453.
[20]李丹,范哲,李广生,等.Runx2与骨生长发育[J].中华地方病学杂志,2004,23(6):620-623.
[21]杨光正,张文杰,丁迅,等.Runx2、Osterix转录因子过表达驱动内皮细胞成骨分化的机制探讨[J].上海口腔医学,2017,26(4):353-357.
[22]Kobayashi H,Gao Y,Ueta C,et al.Multilineage differentiation of Cbfa1-deficient calvarial cells in vitro.Biochem Biophys Res Commun.2000;273(2):630-636.
[23]Galindo M,Kahler RA,Teplyuk NM,et al.Cell cycle related modulations in Runx2 protein levels are independent of lymphocyte enhancer-binding factor 1(Lef1)in proliferating osteoblasts.J Mol Histol.2007;38(5):501-506.
[24]解光越,侯晓华,张志勇,等.老年骨质疏松患者股骨Runt相关转录因子2水平与骨密度的关系[J].中国老年学杂志,2016,36(8):1962-1963.
[25]Nakashima K,Zhou X,Kunkel G,et al.The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.Cell.2002;108(1):17-29.
[26]Sinha KM,Zhou X.Genetic and molecular control of osterix in skeletal formation.J Cell Biochem.2013;114(5):975-984.
[27]张驰.成骨细胞特异性转录因子Osterix对骨形成作用的分子机制(英文)[J].北京大学学报(医学版),2012,44(5):659-665.
[28]Cao Z,Liu R,Zhang H.Osterix controls cementoblast differentiation through downregulation of Wnt-signaling via enhancing DKK1 expression.International J Biol Sci.2015;11(3):335-344.
[29]Chen S,Feng J,Zhang H,et al.Key role for the transcriptional factor,osterix,in spine development.Spine J.2014;14(4):683-694.
[30]Lai QG,Yuan KF,Xu X,et al.Transcription factor osterix modified bone marrow mesenchymal stem cells enhance callus formation during distraction osteogenesis.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2011;111(4):412-419.
[31]Nishio Y,Dong Y,Paris M,et al.Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene.Gene.2006;372(1-2):62-70.
[32]Komori T.Roles of Runx2 in skeletal development.Adv Exp Med Biol.2017;962:83-93.
[33]Rashid H,Ma C,Chen H,et al.Sp7 and Runx2 molecular complex synergistically regulate expression of target genes.Connect Tissue Res.2014;55 Suppl 1(Supp 1):83-87.
[34]Komori T.Regulation of osteoblast differentiation by transcription factors.J Cell Biochem.2010;99(5):1233-1239.