苦味酸类成分蛇麻酮和葎草酮对大鼠成骨细胞和破骨细胞的干预作用
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摘要
目的研究苦味酸类成分蛇麻酮和葎草酮对大鼠成骨细胞及破骨细胞活性的干预作用。方法以新生24 h的Wistar大鼠所分离的成骨细胞和破骨细胞为研究对象,设置对照组,蛇麻酮处理低(10-15mol/L)、中(10-14 mol/L)、高(10-13 mol/L)剂量组,以及葎草酮处理低(10-15 mol/L)、中(10-14 mol/L)、高(10-13 mol/L)剂量组。药物处理后,分别用MTT法、碱性磷酸酶(ALP)活性检测以及茜素红染色法评价蛇麻酮和葎草酮对成骨细胞增殖、分化及骨矿化水平的影响。采用破骨细胞计数以及抗酒石酸酸性磷酸酶(TRAP)活性检测评价蛇麻酮和葎草酮对破骨细胞活性的影响。采用试剂盒法检测骨钙素(OCN)水平,采用蛋白质印迹法检测骨形成相关蛋白骨桥蛋白(OPN)、骨涎蛋白(BSP)、骨形成蛋白(BMP-2)以及骨吸收相关蛋白组织蛋白酶K(CK)、基质金属蛋白酶9(MMP-9)的表达,评价蛇麻酮和葎草酮在骨代谢调控方面的作用。结果在大鼠成骨细胞水平上,与对照组相比,蛇麻酮在10-15、10-14 mol/L浓度下可促进成骨细胞增殖(P<0.05),在10-14、10-13 mol/L浓度下可提高ALP活性以及骨矿化水平(P<0.05,P<0.01),在10-13 mol/L浓度下可促进OCN的表达(P<0.01),在10-14、10-13 mol/L浓度下可提高骨形成相关蛋白BSP和BMP-2的表达(P<0.05);葎草酮在10-15～10-13 mol/L浓度下可促进成骨细胞增殖、提高ALP活性以及骨矿化水平(P<0.01)、提高骨形成相关蛋白OCN、OPN的表达(P<0.05,P<0.01),在10-14、10-13 mol/L浓度下可促进BSP和BMP-2的表达(P<0.05)。在大鼠破骨细胞水平上,与对照组相比,蛇麻酮和葎草酮在10-15～10-13mol/L浓度下均可降低破骨细胞数目(P<0.01),抑制CK的表达(P<0.05,P<0.01);葎草酮在10-15～10-13 mol/L浓度下可抑制MMP-9的表达(P<0.05,P<0.01)。结论本研究在细胞水平上初步明确了蛇麻酮和葎草酮可通过促进成骨细胞骨形成、抑制破骨细胞骨吸收来防治骨丢失,为骨质疏松药物的开发提供了新资源。
Objective To explore the effects of lupulone(LUP) and humulone(HUM) in Humulus lupulus L. on osteoblasts and osteoclasts of rats. Methods Osteoblasts and osteoclasts isolated from 24-h-old Wistar rats were studied and divided into control group, LUP-treated low(10-15 mol/L)-, medium(10-14 mol/L)-and high(10-13 mol/L)-dose groups, and HUM-treated low(10-15 mol/L)-, medium(10-14 mol/L)-and high(10-13 mol/L)-dose groups. After drug treatment, the proliferation, differentiation and bone mineralization of osteoblasts were determined by MTT assay, alkaline phosphatase(ALP) activity assay and alizarin red staining, respectively. Osteoclasts were counted and tartrate-resistant acid phosphatase(TRAP) activity was measured to evaluate the effects of LUP and HUM on the activity of osteoclasts. Osteocalcin(OCN) levels were measured by kit assay, and the expression levels of bone formation related proteins osteopontin(OPN), bone sialoprotein(BSP), bone morphogenetic protein 2(BMP-2), bone resorption related proteins cathepsin K(CK) and matrix metalloproteinase 9(MMP-9) were measured by Western blotting analysis to evaluate the effects of LUP and HUM on bone metabolism. Results At the osteoblast level, LUP at dosages of 10-15 and 10-14 mol/L could significantly promote the cell proliferation(P<0.05). LUP at dosages of 10-14 and 10-13 mol/L could significantly improve ALP activity and bone mineralization(P<0.05, P<0.01). LUP at dosage of 10-13 mol/L could significantly induce the expression of OCN(P<0.01). Furthermore, LUP at dosages of 10-14 and 10-13 mol/L could significantly increase the expression of BSP and BMP-2(P<0.05). HUM at dosages of 10-15-10-13 mol/L could also significantly promote the osteoblastic proliferation, ALP activity and bone mineralization(P<0.01), and could significantly increase the expression of OCN and OPN(P<0.05, P<0.01). Additionally, HUM at dosages of 10-14 and 10-13 mol/L could significantly increase the expression of BSP and BMP-2(P<0.05). At the osteoclast level, both LUP and HUM at dosages of 10-15-10-13 mol/L could significantly reduce the number of osteoclasts(P<0.01) and could significantly inhibit the expression of CK(P<0.05, P<0.01). HUM at dosages of 10-15-10-13 mol/L could also significantly inhibit the expression of MMP-9(P<0.05, P<0.01). Conclusion This study preliminarily clarifies that LUP and HUM can prevent bone loss by promoting bone formation and inhibiting bone resorption, which provides a new reference for the development of osteoporosis drugs.
Objective To explore the effects of lupulone(LUP) and humulone(HUM) in Humulus lupulus L. on osteoblasts and osteoclasts of rats. Methods Osteoblasts and osteoclasts isolated from 24-h-old Wistar rats were studied and divided into control group, LUP-treated low(10-15 mol/L)-, medium(10-14 mol/L)-and high(10-13 mol/L)-dose groups, and HUM-treated low(10-15 mol/L)-, medium(10-14 mol/L)-and high(10-13 mol/L)-dose groups. After drug treatment, the proliferation, differentiation and bone mineralization of osteoblasts were determined by MTT assay, alkaline phosphatase(ALP) activity assay and alizarin red staining, respectively. Osteoclasts were counted and tartrate-resistant acid phosphatase(TRAP) activity was measured to evaluate the effects of LUP and HUM on the activity of osteoclasts. Osteocalcin(OCN) levels were measured by kit assay, and the expression levels of bone formation related proteins osteopontin(OPN), bone sialoprotein(BSP), bone morphogenetic protein 2(BMP-2), bone resorption related proteins cathepsin K(CK) and matrix metalloproteinase 9(MMP-9) were measured by Western blotting analysis to evaluate the effects of LUP and HUM on bone metabolism. Results At the osteoblast level, LUP at dosages of 10-15 and 10-14 mol/L could significantly promote the cell proliferation(P<0.05). LUP at dosages of 10-14 and 10-13 mol/L could significantly improve ALP activity and bone mineralization(P<0.05, P<0.01). LUP at dosage of 10-13 mol/L could significantly induce the expression of OCN(P<0.01). Furthermore, LUP at dosages of 10-14 and 10-13 mol/L could significantly increase the expression of BSP and BMP-2(P<0.05). HUM at dosages of 10-15-10-13 mol/L could also significantly promote the osteoblastic proliferation, ALP activity and bone mineralization(P<0.01), and could significantly increase the expression of OCN and OPN(P<0.05, P<0.01). Additionally, HUM at dosages of 10-14 and 10-13 mol/L could significantly increase the expression of BSP and BMP-2(P<0.05). At the osteoclast level, both LUP and HUM at dosages of 10-15-10-13 mol/L could significantly reduce the number of osteoclasts(P<0.01) and could significantly inhibit the expression of CK(P<0.05, P<0.01). HUM at dosages of 10-15-10-13 mol/L could also significantly inhibit the expression of MMP-9(P<0.05, P<0.01). Conclusion This study preliminarily clarifies that LUP and HUM can prevent bone loss by promoting bone formation and inhibiting bone resorption, which provides a new reference for the development of osteoporosis drugs.
引文
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[9]张嘉,李多伟,张小燕,董崇强,赵蓉,张媛媛.超声提取啤酒花中植物雌激素样物质工艺的研究[J].西北药学杂志,2007,22:14-15.
[10]翟远坤,牛银波,潘亚磊,李晨睿,武祥龙,梅其炳.柚皮苷对体外培养乳鼠颅骨成骨细胞增殖和分化成熟的影响[J].中国中药杂志,2013,38:105-111.
[11]张乃丹.基于分子对接策略的熟地黄防治糖尿病性骨质疏松症有效成分及其作用机制研究[D].上海:第二军医大学,2016.
[12]薛黎明.基于蛋白质组学淫羊藿苷防治骨质疏松作用机理及药对“淫羊藿仙茅”配伍机制研究[D].上海:第二军医大学,2012.
[13]张葆鑫,王兴国,郝廷.成骨细胞、破骨细胞与骨折愈合的相关性研究进展[J].中国现代医生,2017,55:161-164.
[14]MANOLAGAS S C.Birth and death of bone cells:basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis[J].Endocr Rev,2000,21:115-137.
[15]童安莉,陈璐璐,丁桂芝.成骨细胞骨形成机制研究进展[J].中国骨质疏松杂志,1999,5:60-64.
[16]ROOHANI-ESFAHANI S I,NO Y J,LU Z,NG P Y,CHEN Y,SHI J,et al.A bioceramic with enhanced osteogenic properties to regulate the function of osteoblastic and osteocalastic cells for bone tissue regeneration[J/OL].Biomed Mater,2016,11:35018.doi:10.1088/1748-6041/11/3/035018.
[17]ATANG E,AUWRT,WETTERWALD A,HOFSTETTER W.TNFαinhibits the development of osteoclasts through osteoblast-derived GM-CSF[J].Bone,2011,49:1090-1100.
[18]安新玲,韩金祥,王世立.骨形态发生蛋白的研究进展[J].食品与药品,2009,11:69-73.
[19]COSTA A G,CUSANO N E,SILVA B C,CREMERS S,BILEZIKIAN J P.Cathepsin K:its skeletal actions and role as a therapeutic target in osteoporosis[J/OL].Nat Rev Rheumatol,2011,7:447.doi:10.1038/nrrheum.2011.77.
[20]ZHAO Q,JIA Y,XIAO Y.Cathepsin K:a therapeutic target for bone diseases[J].Biochem Biophys Res Commun,2009,380:721-723.
[21]HE B,HU M,LI S D,YANG X T,LU Y Q,LIU J X,et al.Effects of geraniin on osteoclastic bone resorption and matrix metalloproteinase-9 expression[J].Bioorg Med Chem Lett,2013,23:630-634.
[2]Consensus development conference:diagnosis,prophylaxis and treatment of osteoporosis[J].Am J Med,1993,94:646-650.
[3]DI VIESTI V,CARNEVALE G,ZAVATTI M,BENELLIA,ZANOLI P.Increased sexual motivation in female rats treated with Humulus lupulus L.extract[J].JEthnopharmacol,2011,134:514-517.
[4]ZHANG B,CHU W,WEI P,LIU Y,WEI T.Xanthohumol induces generation of reactive oxygen species and triggers apoptosis through inhibition of mitochondrial electron transfer chain complexⅠ[J].Free Radical Biol Med,2015,89:486-497.
[5]GERH?USER C.Broad spectrum antiinfective potential of xanthohumol from hop(Humulus lupulus L.)in comparison with activities of other hop constituents and xanthohumol metabolites[J].Mol Nutr Food Res,2005,49:827-831.
[6]KAO T H,WU G Y.Simultaneous determination of prenylflavonoid and hop bitter acid in beer lee by HPLC-DAD-MS[J].Food Chem,2013,141:1218-1226.
[7]YAJIMA H,IKESHIMA E,SHIRAKI M,KANAYA T,FUJIWARA D,ODAI H,et al.Isohumulones,bitter acids derived from hops,activate both peroxisome proliferatoractivated receptor alpha and gamma and reduce insulin resistance[J].J Biol Chem,2004,279:33456-33462.
[8]HALL A J,BABISH J G,DARLAND G K,CARROLLB J,KONDA V R,LERMAN R H,et al.Safety,efficacy and anti-inflammatory activity of rho iso-alpha-acids from hops[J].Phytochemistry,2008,69:1534-1547.
[9]张嘉,李多伟,张小燕,董崇强,赵蓉,张媛媛.超声提取啤酒花中植物雌激素样物质工艺的研究[J].西北药学杂志,2007,22:14-15.
[10]翟远坤,牛银波,潘亚磊,李晨睿,武祥龙,梅其炳.柚皮苷对体外培养乳鼠颅骨成骨细胞增殖和分化成熟的影响[J].中国中药杂志,2013,38:105-111.
[11]张乃丹.基于分子对接策略的熟地黄防治糖尿病性骨质疏松症有效成分及其作用机制研究[D].上海:第二军医大学,2016.
[12]薛黎明.基于蛋白质组学淫羊藿苷防治骨质疏松作用机理及药对“淫羊藿仙茅”配伍机制研究[D].上海:第二军医大学,2012.
[13]张葆鑫,王兴国,郝廷.成骨细胞、破骨细胞与骨折愈合的相关性研究进展[J].中国现代医生,2017,55:161-164.
[14]MANOLAGAS S C.Birth and death of bone cells:basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis[J].Endocr Rev,2000,21:115-137.
[15]童安莉,陈璐璐,丁桂芝.成骨细胞骨形成机制研究进展[J].中国骨质疏松杂志,1999,5:60-64.
[16]ROOHANI-ESFAHANI S I,NO Y J,LU Z,NG P Y,CHEN Y,SHI J,et al.A bioceramic with enhanced osteogenic properties to regulate the function of osteoblastic and osteocalastic cells for bone tissue regeneration[J/OL].Biomed Mater,2016,11:35018.doi:10.1088/1748-6041/11/3/035018.
[17]ATANG E,AUWRT,WETTERWALD A,HOFSTETTER W.TNFαinhibits the development of osteoclasts through osteoblast-derived GM-CSF[J].Bone,2011,49:1090-1100.
[18]安新玲,韩金祥,王世立.骨形态发生蛋白的研究进展[J].食品与药品,2009,11:69-73.
[19]COSTA A G,CUSANO N E,SILVA B C,CREMERS S,BILEZIKIAN J P.Cathepsin K:its skeletal actions and role as a therapeutic target in osteoporosis[J/OL].Nat Rev Rheumatol,2011,7:447.doi:10.1038/nrrheum.2011.77.
[20]ZHAO Q,JIA Y,XIAO Y.Cathepsin K:a therapeutic target for bone diseases[J].Biochem Biophys Res Commun,2009,380:721-723.
[21]HE B,HU M,LI S D,YANG X T,LU Y Q,LIU J X,et al.Effects of geraniin on osteoclastic bone resorption and matrix metalloproteinase-9 expression[J].Bioorg Med Chem Lett,2013,23:630-634.