山茱萸总甙干预骨质疏松模型大鼠骨代谢:TRPV6、TRPV5通路的变化
|
摘要
背景:钙离子通道的调控直接影响骨的代谢,但骨代谢与TRPV6、TRPV5通路的联系及相互作用目前尚未明确。研究认为山茱萸具有防治骨质疏松作用。目的:探讨山茱萸总甙调节TRPV6、TRPV5通路表达防治骨质疏松症的机制。方法:健康6月龄雌性SD大鼠50只,体质量(350±15) g,由广州中医药大学实验动物中心提供。随机将大鼠分为5组,即正常对照组,模型组,山茱萸总甙干预低剂量组、中剂量组及高剂量组。除正常对照组外,其余各组大鼠行双侧卵巢切除术建立骨质疏松模型。造模后山茱萸总甙干预各组依剂量每天予药物灌胃,正常对照组及模型组予等量生理盐水灌胃,共12周。实验结束时,双能X射线骨密度仪扫描各组大鼠获取骨密度,取右侧股骨组织RT-PCR检测TRPV6、TRPV5 RNA基因表达情况。结果与结论:(1)山茱萸干预各组大鼠骨密度均较模型组提高(P<0.05);(2)各组大鼠骨组织均有TRPV6、TRPV5表达,其中山茱萸总甙干预各组TRPV6、TRPV5表达均较正常对照组及模型组增加,以中剂量组表达升高最明显,但高剂量组TRPV6/TRPV5倍比关系为最高,成骨效能最好;(3)结果说明,山茱萸总甙可调节骨质疏松大鼠骨组织TRPV6、TRPV5通道蛋白表达,改变TRPV6/TRPV5倍比关系,影响成骨细胞和破骨细胞功能,促进成骨方向的骨代谢转变及提高骨密度。
BACKGROUND: Regulation of calcium channel directly affects bone metabolism, but the correlation of bone metabolism with TRPV6/TRPV5 pathways remains unclear. Cornus officinalis has been shown to treat and prevent osteoporosis. OBJECTIVE: To explore the mechanism of Cornus officinalis glycosides underlying intervening the expression of TRPV6 and TRPV5 for preventing and treating osteoporosis. METHODS: Fifty healthy 6-month-old male Sprague-Dawley rats(provide by Lanoratory Animal Center of Guangzhou University of Chiense Medicine) were selected, weighting(350±15) g. The rats were randomly divided into control(normal saline treatment), model(normal saline treatment), low-, medium, and high-dose Cornus officinalis glycosides groups. The model of osteoporosis was established by bilateral ovariectomy in the latter four groups. The administration was given gastricly, and for 12 weeks. At the end of the experiment, the bone mineral density was detected by dual energy X-ray absorptiometry. The expression levels of TRPV6 and TRPV5 in the right femur were detected by RT-PCR. RESULTS AND CONCLUSION: The bone mineral density in the Cornus officinalis glycosides groups was significantly higher than that in the model group(P < 0.05). TRPV6 and TRPV5 were expressed in bone tissues of rats in each group. The expression levels of TRPV6 and TRPV5 in all the Cornus officinalis glycosides groups were higher than those in the control and model groups. The medium dose group had the highest expression levels of TRPV6 and TRPV5. The ratio of TRPV6 to TRPV5 and osteogenesis performance in the high-dose group was highest. These results imply that Cornus officinalis glycosides can promote the expression of TRPV6 and TRPV5 in bone tissue of osteoporotic rats, change the ratio of TRPV6 to TRPV5, affect the function of osteoblasts and osteoclasts, promote osteogenesis and improve bone mineral density.
BACKGROUND: Regulation of calcium channel directly affects bone metabolism, but the correlation of bone metabolism with TRPV6/TRPV5 pathways remains unclear. Cornus officinalis has been shown to treat and prevent osteoporosis. OBJECTIVE: To explore the mechanism of Cornus officinalis glycosides underlying intervening the expression of TRPV6 and TRPV5 for preventing and treating osteoporosis. METHODS: Fifty healthy 6-month-old male Sprague-Dawley rats(provide by Lanoratory Animal Center of Guangzhou University of Chiense Medicine) were selected, weighting(350±15) g. The rats were randomly divided into control(normal saline treatment), model(normal saline treatment), low-, medium, and high-dose Cornus officinalis glycosides groups. The model of osteoporosis was established by bilateral ovariectomy in the latter four groups. The administration was given gastricly, and for 12 weeks. At the end of the experiment, the bone mineral density was detected by dual energy X-ray absorptiometry. The expression levels of TRPV6 and TRPV5 in the right femur were detected by RT-PCR. RESULTS AND CONCLUSION: The bone mineral density in the Cornus officinalis glycosides groups was significantly higher than that in the model group(P < 0.05). TRPV6 and TRPV5 were expressed in bone tissues of rats in each group. The expression levels of TRPV6 and TRPV5 in all the Cornus officinalis glycosides groups were higher than those in the control and model groups. The medium dose group had the highest expression levels of TRPV6 and TRPV5. The ratio of TRPV6 to TRPV5 and osteogenesis performance in the high-dose group was highest. These results imply that Cornus officinalis glycosides can promote the expression of TRPV6 and TRPV5 in bone tissue of osteoporotic rats, change the ratio of TRPV6 to TRPV5, affect the function of osteoblasts and osteoclasts, promote osteogenesis and improve bone mineral density.
引文
[1]原发性骨质疏松症诊疗指南(2017)[J].中国实用内科杂志,2018,38(2):127-150.
[2]CLAPHAM DE.TRP channels as cellular sensors.Nature.2003;426(6966):517-524.
[3]Montell C,Birnbaumer L,Flockerzi V,et al.A unified nomenclature for the superfamily of TRP cation channels.Molecular Cell.2002;9(2):229-231.
[4]Shi DJ,Wang KW.The progress of TRP channels in structural studies.Sheng Li Ke Xue Jin Zhan.2014;45(6):401-409.
[5]Hoenderop JG,Nilius B,Bindels RJ.Molecular mechanism of active Ca2+reabsorption in the distal nephron.Annu Rev Physiol.2002;64:529-549.
[6]Hoenderop JG,Vennekens R,Müller D,et al.Function and expression of the epithelial Ca(2+)channel family:comparison of mammalian ECaC1 and 2.J Physiol.2001;537(Pt 3):747-761.
[7]Müller D,Hoenderop JG,Meij IC,et al.Molecular cloning,tissue distribution,and chromosomal mapping of the human epithelial Ca2+channel(ECAC1).Genomics.2000;67(1):48-53.
[8]Peng JB,Chen XZ,Berger UV,et al.Human calcium transport protein CaT1.Biochem Biophys Res Commun.2000;278(2):326-332.
[9]van de Graaf SF,Boullart I,Hoenderop JG,et al.Regulation of the epithelial Ca2+channels TRPV5 and TRPV6 by 1alpha,25-dihydroxy Vitamin D3 and dietary Ca2+J Steroid Biochem Mol Biol.2004;89-90(1-5):303-308.
[10]KIM M H,LEE G S,JUNG E M,et al.The negative effect of dexamethasone on calcium-processing gene expressions is associated with a glucocorticoid-induced calcium-absorbing disorder.Life Sci.2009;85(3-4):146-152.
[11]Van Cromphaut SJ,Stockmans I,Torrekens S,et al.Duodenal calcium absorption in dexamethasone-treated mice:functional and molecular aspects.Arch Biochem Biophys.2007;460(2):300-305.
[12]Weber K,Erben RG,Rump A,et al.Gene structure and regulation of the murine epithelial calcium channels ECaC1 and 2.Biochem Biophys Res Commun.2001;289(5):1287-1294..
[13]郭清河,叶添文.TRPV5/TRPV6调节骨代谢作用的研究进展[J].中国矫形外科杂志,2011,19(23):1976-1978.
[14]周京华,李春生,李电东.山茱萸有效化学成分的研究进展[J].中国新药杂志,2001,10(11):808-812.
[15]李晶.针灸及山茱萸总甙对去势大鼠骨代谢影响的生化研究[D].天津:天津中医学院,2004.
[16]Diederichs S,Freiberger F,van Griensven M.Effects of repetitive and short time strain in human bone marrow stromal cells.Journal of biomedical materials research Part A.2009;88(4):907-915.
[17]Hoenderop JG,van Leeuwen JP,van der Eerden BC,et al.Renal Ca2+wasting,hyperabsorption,and reduced bone thickness in mice lacking TRPV5.J Clin Invest.2003;112(12):1906-1914.
[18]van der Eerden BC,Hoenderop JG,de Vries TJ,et al.The epithelial Ca2+channel TRPV5 is essential for proper osteoclastic bone resorption.Proc Natl Acad Sci U S A.2005;102(48):17507-17512.
[19]车明学,谷贵山,李福春.新型钙离子通道TRPV5在骨质疏松症发病机制中的作用[J].中国老年学杂志,2007,27(17):1732-1734.
[20]Walters JR,Balesaria S,Chavele KM,et al.Calcium channel TRPV6 expression in human duodenum:different relationships to the vitamin D system and aging in men and women.J Bone Miner Res.2006;21(11):1770-1777.
[21]Barthel TK,Mathern DR,Whitfield GK,et al.1,25-Dihydroxyvitamin D3/VDR-mediated induction of FGF23 as well as transcriptional control of other bone anabolic and catabolic genes that orchestrate the regulation of phosphate and calcium mineral metabolism.J Steroid Biochem Mol Biol.2007;103(3-5):381-388.
[22]李福春.TRPV5/TRPV6对大鼠骨髓间充质干细胞成骨分化及成骨细胞信号传递的影响[D].长春:吉林大学,2008.
[23]那键.雌激素、维生素D_3对雌性大鼠成骨细胞增殖及钙通道TRPV5/TRPV6表达的影响[D].长春:吉林大学,2009.
[24]叶贤胜.中药山茱萸的化学成分和生物活性研究[D].北京:北京中医药大学,2017.
[25]郭富礼,张振凌,王洪波.不同山茱萸炮制品中熊果酸含量的比较[J].时珍国医国药,2002,13(7):404-405.
[26]刘洪,许惠琴.山茱萸及其主要成分的药理学研究进展[J].南京中医药大学学报(自然科学版),2003,19(4):254-256.
[27]张兰桐,任雷鸣,温进坤.山茱萸提取液抗心律失常有效部位的研究[J].中草药,2001,32(11):47-50.
[28]刘倩,范颖,姜开运,等山茱萸潜在功能的发掘与利用[J].时珍国医国药,2015,26(11):2764-5.
[29]陈涛.山茱萸水提液对骨质疏松模型小鼠骨形态学影响[J].天津药学,2003,15(4):5-6+32.
[30]李平,李晶,王惠明.山茱萸总苷对去势大鼠骨代谢和骨密度影响的实验研究[J].天津中医药,2007,24(4):315-317.
[31]李晶,王惠明,李平.山茱萸总苷对去势大鼠骨代谢影响的生化研究[J].辽宁中医杂志,2007,34(10):1480-1482.
[32]李晶,武峻艳,王慧明,等.山茱萸总苷对去势大鼠骨组织形态学影响的实验研究[J].上海中医药杂志,2010,44(1):69-72.
[33]王艳,宋春静,孙秀岩,等.山茱萸总苷及其含药血清对MSCs增殖的影响[J].天津中医药大学学报,2013,32(2):101-104.
[34]章晓云,夏天,陈跃平,等.Zip1转染骨髓间充质干细胞对Wnt1/β-catenin信号成骨通路的影响[J].中国骨质疏松杂志,2018,24(5):594-599.
[35]李庆帆,王佐林.ALP、Runx2及OCN在大鼠拔牙后牙槽骨来源BMSCs中的表达[J].口腔颌面外科杂志,2017,27(2):77-82.
[36]Saville PD,Lieber CS.Effect of alcohol on growth,bone density and muscle magnesium in the rat.J Nutr.1965;87(4):477-484.
[37]翁世芳,黄克,王洪复.大鼠骨密度测定在骨质疏松模型研制中的应用[C].西安:proceedings of the第三届全国骨质疏松研讨会,1994.
[38]周丽,邓伟民.去势雌性大鼠动物模型在绝经后骨质疏松症中的应用[J].中国组织工程研究与临床康复,2008,12(7):1323-1326.
[39]Rodgers JB,Monier-Faugere MC,Malluche H.Animal models for the study of bone loss after cessation of ovarian function.Bone.1993;14(3):369-377.
[40]Hel wig N,Albrecht N,Harteneck C,et al.Homo-and heteromeric assembly of TRPV channel subunits.J Cell Sci.2005;118(Pt 5):917-928.
[41]Nilius B,Prenen J,Hoenderop JG,et al.Fast and slow inactivation kinetics of the Ca2+channels ECaC1 and ECaC2(TRPV5 and TRPV6).Role of the intracellular loop located between transmembrane segments 2 and 3.J Biol Chem.2002;277(34):30852-30858.
[42]Vennekens R,Hoenderop JG,Prenen J,et al.Permeation and gating properties of the novel epithelial Ca(2+)channel.J Biol Chem.2000;275(6):3963-3969.
[43]Sternfeld L,Anderie I,Schmid A,et al.Identification of tyrosines in the putative regulatory site of the Ca2+channel TRPV6.Cell calcium.2007;42(1):91-102.
[44]Zheng W,Hu R,Cai R,et al.Identification and characterization of hydrophobic gate residues in TRP channels.FASEB J.2018;32(2):639-653.
[45]Xie H,Cui Z,Wang L,et al.PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis.Nat Med.2014;20(11):1270-1278.
[2]CLAPHAM DE.TRP channels as cellular sensors.Nature.2003;426(6966):517-524.
[3]Montell C,Birnbaumer L,Flockerzi V,et al.A unified nomenclature for the superfamily of TRP cation channels.Molecular Cell.2002;9(2):229-231.
[4]Shi DJ,Wang KW.The progress of TRP channels in structural studies.Sheng Li Ke Xue Jin Zhan.2014;45(6):401-409.
[5]Hoenderop JG,Nilius B,Bindels RJ.Molecular mechanism of active Ca2+reabsorption in the distal nephron.Annu Rev Physiol.2002;64:529-549.
[6]Hoenderop JG,Vennekens R,Müller D,et al.Function and expression of the epithelial Ca(2+)channel family:comparison of mammalian ECaC1 and 2.J Physiol.2001;537(Pt 3):747-761.
[7]Müller D,Hoenderop JG,Meij IC,et al.Molecular cloning,tissue distribution,and chromosomal mapping of the human epithelial Ca2+channel(ECAC1).Genomics.2000;67(1):48-53.
[8]Peng JB,Chen XZ,Berger UV,et al.Human calcium transport protein CaT1.Biochem Biophys Res Commun.2000;278(2):326-332.
[9]van de Graaf SF,Boullart I,Hoenderop JG,et al.Regulation of the epithelial Ca2+channels TRPV5 and TRPV6 by 1alpha,25-dihydroxy Vitamin D3 and dietary Ca2+J Steroid Biochem Mol Biol.2004;89-90(1-5):303-308.
[10]KIM M H,LEE G S,JUNG E M,et al.The negative effect of dexamethasone on calcium-processing gene expressions is associated with a glucocorticoid-induced calcium-absorbing disorder.Life Sci.2009;85(3-4):146-152.
[11]Van Cromphaut SJ,Stockmans I,Torrekens S,et al.Duodenal calcium absorption in dexamethasone-treated mice:functional and molecular aspects.Arch Biochem Biophys.2007;460(2):300-305.
[12]Weber K,Erben RG,Rump A,et al.Gene structure and regulation of the murine epithelial calcium channels ECaC1 and 2.Biochem Biophys Res Commun.2001;289(5):1287-1294..
[13]郭清河,叶添文.TRPV5/TRPV6调节骨代谢作用的研究进展[J].中国矫形外科杂志,2011,19(23):1976-1978.
[14]周京华,李春生,李电东.山茱萸有效化学成分的研究进展[J].中国新药杂志,2001,10(11):808-812.
[15]李晶.针灸及山茱萸总甙对去势大鼠骨代谢影响的生化研究[D].天津:天津中医学院,2004.
[16]Diederichs S,Freiberger F,van Griensven M.Effects of repetitive and short time strain in human bone marrow stromal cells.Journal of biomedical materials research Part A.2009;88(4):907-915.
[17]Hoenderop JG,van Leeuwen JP,van der Eerden BC,et al.Renal Ca2+wasting,hyperabsorption,and reduced bone thickness in mice lacking TRPV5.J Clin Invest.2003;112(12):1906-1914.
[18]van der Eerden BC,Hoenderop JG,de Vries TJ,et al.The epithelial Ca2+channel TRPV5 is essential for proper osteoclastic bone resorption.Proc Natl Acad Sci U S A.2005;102(48):17507-17512.
[19]车明学,谷贵山,李福春.新型钙离子通道TRPV5在骨质疏松症发病机制中的作用[J].中国老年学杂志,2007,27(17):1732-1734.
[20]Walters JR,Balesaria S,Chavele KM,et al.Calcium channel TRPV6 expression in human duodenum:different relationships to the vitamin D system and aging in men and women.J Bone Miner Res.2006;21(11):1770-1777.
[21]Barthel TK,Mathern DR,Whitfield GK,et al.1,25-Dihydroxyvitamin D3/VDR-mediated induction of FGF23 as well as transcriptional control of other bone anabolic and catabolic genes that orchestrate the regulation of phosphate and calcium mineral metabolism.J Steroid Biochem Mol Biol.2007;103(3-5):381-388.
[22]李福春.TRPV5/TRPV6对大鼠骨髓间充质干细胞成骨分化及成骨细胞信号传递的影响[D].长春:吉林大学,2008.
[23]那键.雌激素、维生素D_3对雌性大鼠成骨细胞增殖及钙通道TRPV5/TRPV6表达的影响[D].长春:吉林大学,2009.
[24]叶贤胜.中药山茱萸的化学成分和生物活性研究[D].北京:北京中医药大学,2017.
[25]郭富礼,张振凌,王洪波.不同山茱萸炮制品中熊果酸含量的比较[J].时珍国医国药,2002,13(7):404-405.
[26]刘洪,许惠琴.山茱萸及其主要成分的药理学研究进展[J].南京中医药大学学报(自然科学版),2003,19(4):254-256.
[27]张兰桐,任雷鸣,温进坤.山茱萸提取液抗心律失常有效部位的研究[J].中草药,2001,32(11):47-50.
[28]刘倩,范颖,姜开运,等山茱萸潜在功能的发掘与利用[J].时珍国医国药,2015,26(11):2764-5.
[29]陈涛.山茱萸水提液对骨质疏松模型小鼠骨形态学影响[J].天津药学,2003,15(4):5-6+32.
[30]李平,李晶,王惠明.山茱萸总苷对去势大鼠骨代谢和骨密度影响的实验研究[J].天津中医药,2007,24(4):315-317.
[31]李晶,王惠明,李平.山茱萸总苷对去势大鼠骨代谢影响的生化研究[J].辽宁中医杂志,2007,34(10):1480-1482.
[32]李晶,武峻艳,王慧明,等.山茱萸总苷对去势大鼠骨组织形态学影响的实验研究[J].上海中医药杂志,2010,44(1):69-72.
[33]王艳,宋春静,孙秀岩,等.山茱萸总苷及其含药血清对MSCs增殖的影响[J].天津中医药大学学报,2013,32(2):101-104.
[34]章晓云,夏天,陈跃平,等.Zip1转染骨髓间充质干细胞对Wnt1/β-catenin信号成骨通路的影响[J].中国骨质疏松杂志,2018,24(5):594-599.
[35]李庆帆,王佐林.ALP、Runx2及OCN在大鼠拔牙后牙槽骨来源BMSCs中的表达[J].口腔颌面外科杂志,2017,27(2):77-82.
[36]Saville PD,Lieber CS.Effect of alcohol on growth,bone density and muscle magnesium in the rat.J Nutr.1965;87(4):477-484.
[37]翁世芳,黄克,王洪复.大鼠骨密度测定在骨质疏松模型研制中的应用[C].西安:proceedings of the第三届全国骨质疏松研讨会,1994.
[38]周丽,邓伟民.去势雌性大鼠动物模型在绝经后骨质疏松症中的应用[J].中国组织工程研究与临床康复,2008,12(7):1323-1326.
[39]Rodgers JB,Monier-Faugere MC,Malluche H.Animal models for the study of bone loss after cessation of ovarian function.Bone.1993;14(3):369-377.
[40]Hel wig N,Albrecht N,Harteneck C,et al.Homo-and heteromeric assembly of TRPV channel subunits.J Cell Sci.2005;118(Pt 5):917-928.
[41]Nilius B,Prenen J,Hoenderop JG,et al.Fast and slow inactivation kinetics of the Ca2+channels ECaC1 and ECaC2(TRPV5 and TRPV6).Role of the intracellular loop located between transmembrane segments 2 and 3.J Biol Chem.2002;277(34):30852-30858.
[42]Vennekens R,Hoenderop JG,Prenen J,et al.Permeation and gating properties of the novel epithelial Ca(2+)channel.J Biol Chem.2000;275(6):3963-3969.
[43]Sternfeld L,Anderie I,Schmid A,et al.Identification of tyrosines in the putative regulatory site of the Ca2+channel TRPV6.Cell calcium.2007;42(1):91-102.
[44]Zheng W,Hu R,Cai R,et al.Identification and characterization of hydrophobic gate residues in TRP channels.FASEB J.2018;32(2):639-653.
[45]Xie H,Cui Z,Wang L,et al.PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis.Nat Med.2014;20(11):1270-1278.