罗汉果皂苷V刺激成骨细胞增殖与分化
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摘要
背景:骨在一生中不断地进行重塑,骨稳态是成骨细胞的骨形成和破骨细胞的骨吸收之间的动态平衡。在细胞水平严格控制骨重建,对于维持骨稳态以响应结构和代谢需求是至关重要的。目的:探讨罗汉果皂苷V对成骨细胞增殖、分化的影响。方法:采用两步酶消化法提取大鼠成骨细胞,通过MTT实验检测罗汉果皂苷V的细胞毒性;不同质量浓度(0,6.25×10~(-3),1.25×10~(-2),2.5×10~(-2)g/L)罗汉果皂苷V对成骨细胞干预2,4,6d后通过FDA/PI荧光染色观察成骨细胞活性,干预6d后通过碱性磷酸酶染色、茜素红染色及q RT-PCR检测观察罗汉果皂苷V对成骨细胞分化的影响。结果与结论:①MTT实验表明罗汉果皂苷V在6.25×10~(-3)-2.5×10~(-2) g/L时显著促进成骨细胞增殖;②FDA/PI荧光染色显示6.25×10~(-3),1.25×10~(-2),2.5×10~(-2) g/L罗汉果皂苷V可提高成骨细胞活力、促进细胞增殖,尤其以1.25×10~(-2) g/L效果显著;③罗汉果皂苷V干预6 d后,碱性磷酸酶染色(定量)及茜素红染色实验均显示罗汉果皂苷V促进成骨细胞成骨矿化,特别是在1.25×10~(-2)g/L时最为明显;④q RT-PCR结果显示罗汉果皂苷V组的成骨分化相关基因RUNX2、BSP、OCN和COL1A1表达高于对照组,特别是罗汉果皂苷V在1.25×10~(-2)g/L时表达最高;⑤结果表明,罗汉果皂苷V可刺激成骨细胞增殖、分化,在1.25×10~(-2)g/L时作用最为明显。
BACKGROUND: Bone remodeling is going on throughout the lifetime. Bone homeostasis is a dynamic balance between bone formation of osteoblasts and bone resorption of osteoclasts. Controlling of bone remodeling at the cellular level is critical to maintaining bone homeostasis in response to structural and metabolic demands.OBJECTIVE: To explore the effect of mogroside V on the proliferation and differentiation of osteoblasts.METHODS: Rat osteoblasts were extracted by two-step enzymatic digestion, followed by MTT assay to detect the toxicity of mogroside V.The proliferation of osteoblasts was observed by FDA/PI fluorescence staining at 2, 4 and 6 days after the treatment with mogroside V(0,6.25× 10~(-3), 1.25× 10~(-2), 2.5× 10~(-2) g/L). After 6 days of treatment on osteoblasts, the effect of mogroside V on osteoblast differentiation was studied by alkaline phosphatase staining, alizarin red staining and qRT-PCR.RESULTS AND CONCLUSION: MTT assay showed that the mogroside V at 6.25× 10~(-3) to 2.5× 10~(-2) g/L promoted osteoblast proliferation.FDA/PI fluorescence staining showed that mogroside V(6.25× 10~(-3), 1.25× 10~(-2), 2.5× 10~(-2) g/L) could improve osteoblast activity and promote cell proliferation, especially when the concentration of mogroside V was 1.25× 10~(-2) g/L. After 6 days of treatment, alkaline phosphatase staining and alizarin red staining showed that the mogroside V, especially at 1.25× 10~(-2) g/L, promoted osteogenesis and mineralization. qRT-PCR results showed that the expressions of runt related transcription factor 2, bone sialoprotein, osteocalcin and alpha-1 type I collagen genes were higher in the mogroside V group than the blank group, especially when the concentration of mogroside V was 1.25× 10~(-2) g/L. Therefore,mogroside V, especially at 1.25× 10~(-2) g/L, promotes bone formation by stimulating osteoblast proliferation and differentiation.
BACKGROUND: Bone remodeling is going on throughout the lifetime. Bone homeostasis is a dynamic balance between bone formation of osteoblasts and bone resorption of osteoclasts. Controlling of bone remodeling at the cellular level is critical to maintaining bone homeostasis in response to structural and metabolic demands.OBJECTIVE: To explore the effect of mogroside V on the proliferation and differentiation of osteoblasts.METHODS: Rat osteoblasts were extracted by two-step enzymatic digestion, followed by MTT assay to detect the toxicity of mogroside V.The proliferation of osteoblasts was observed by FDA/PI fluorescence staining at 2, 4 and 6 days after the treatment with mogroside V(0,6.25× 10~(-3), 1.25× 10~(-2), 2.5× 10~(-2) g/L). After 6 days of treatment on osteoblasts, the effect of mogroside V on osteoblast differentiation was studied by alkaline phosphatase staining, alizarin red staining and qRT-PCR.RESULTS AND CONCLUSION: MTT assay showed that the mogroside V at 6.25× 10~(-3) to 2.5× 10~(-2) g/L promoted osteoblast proliferation.FDA/PI fluorescence staining showed that mogroside V(6.25× 10~(-3), 1.25× 10~(-2), 2.5× 10~(-2) g/L) could improve osteoblast activity and promote cell proliferation, especially when the concentration of mogroside V was 1.25× 10~(-2) g/L. After 6 days of treatment, alkaline phosphatase staining and alizarin red staining showed that the mogroside V, especially at 1.25× 10~(-2) g/L, promoted osteogenesis and mineralization. qRT-PCR results showed that the expressions of runt related transcription factor 2, bone sialoprotein, osteocalcin and alpha-1 type I collagen genes were higher in the mogroside V group than the blank group, especially when the concentration of mogroside V was 1.25× 10~(-2) g/L. Therefore,mogroside V, especially at 1.25× 10~(-2) g/L, promotes bone formation by stimulating osteoblast proliferation and differentiation.
引文
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[5]Siris ES,Adler R,Bilezikian J,et al.The clinical diagnosis of osteoporosis:a position statement from the National Bone Health Alliance Working Group.Osteoporos Int.2014;25(5):1439-1443.
[6]Burge R,Dawson-Hughes B,Solomon DH,et al.Incidence and economic burden of osteoporosis-related fractures in the United States,2005-2025.J Bone Miner Res.2007;22(3):465-475.
[7]中华医学会骨质疏松和骨矿盐疾病分会.原发性骨质疏松症诊疗指南(2017)[J].中国全科医学,2017,20(32):3963-3982.
[8]Marie PJ,Kassem M.Osteoblasts in osteoporosis:past,emerging,and future anabolic targets.Eur J Endocrinol.2011;165(1):1-10.
[9]曹建平,汤杰,刘合生,等.罗汉果皂苷生物活性研究进展[J].食品工业科技,2014,35(24):384-388,395.
[10]Zhang M,Yang H,Zhang H,et al.Development of a process for separation of mogroside V from Siraitia grosvenorii by macroporous resins.Molecules.2011;16(9):7288-7301.
[11]Takasaki M,Konoshima T,Murata Y,et al.Anticarcinogenic activity of natural sweeteners,cucurbitane glycosides,from Momordica grosvenori.Cancer Lett.2003;198(1):37-42.
[12]Chen WJ,Wang J,Qi XY,et al.The antioxidant activities of natural sweeteners,mogrosides,from fruits of Siraitia grosvenori.Int J Food Sci Nutr.2007;58(7):548-556.
[13]Liu H,Qi X,Yu K,et al.AMPK activation is involved in hypoglycemic and hypolipidemic activities of mogroside-rich extract from Siraitia grosvenorii(Swingle)fruits on high-fat diet/streptozotocin-induced diabetic mice.Food Funct.2019;10(1):151-162.
[14]Callaway DA,Jiang JX.Reactive oxygen species and oxidative stress in osteoclastogenesis,skeletal aging and bone diseases.J Bone Miner Metab.2015;33(4):359-370.
[15]Lin J,Zhu J,Wang Y,et al.Chinese single herbs and active ingredients for postmenopausal osteoporosis:From preclinical evidence to action mechanism.Biosci Trends.2017;11(5):496-506.
[16]Xiao W,Wang Y,Pacios S,et al.Cellular and Molecular Aspects of Bone Remodeling.Front Oral Biol.2016;18:9-16.
[17]Du L,Nong MN,Zhao JM,et al.Polygonatum sibiricum polysaccharide inhibits osteoporosis by promoting osteoblast formation and blocking osteoclastogenesis through Wnt/β-catenin signalling pathway.Sci Rep.2016;6:32261.
[18]Liang D,Ren H,Qiu T,et al.Extracts from plastrum testudinis reverse glucocorticoid-induced spinal osteoporosis of rats via targeting osteoblastic and osteoclastic markers.Biomed Pharmacother.2016;82:151-160.
[19]Pellegrini GG,Morales CC,Wallace TC,et al.Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner.Nutrients.2016;8(7):E423.
[20]Kalyanaraman H,Ramdani G,Joshua J,et al.A Novel,Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice.J Bone Miner Res.2017;32(1):46-59.
[21]Kassem A,Lindholm C,Lerner UH.Toll-Like Receptor 2Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL.PLoS One.2016;11(6):e0156708.
[22]Zhang D,Fong C,Jia Z,et al.Icariin Stimulates Differentiation and Suppresses Adipocytic Transdifferentiation of Primary Osteoblasts Through Estrogen Receptor-Mediated Pathway.Calcif Tissue Int.2016;99(2):187-198.
[23]Choi BY,Park CH,Na YH,et al.Inhibition of RANKL-induced osteoclast differentiation through the downregulation of c-Fos and NFATc1 by Eremochloa ophiuroides(centipedegrass)extract.Mol Med Rep.2016;13(5):4014-4022.
[24]Baek JM,Kim JY,Yoon KH,et al.Ebselen Is a Potential Anti-Osteoporosis Agent by Suppressing Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclast Differentiation In vitro and Lipopolysaccharide-Induced Inflammatory Bone Destruction In vivo.Int J Biol Sci.2016;12(5):478-488.
[25]Sims NA,Martin TJ.Coupling Signals between the Osteoclast and Osteoblast:How are Messages Transmitted between These Temporary Visitors to the Bone Surface.Front Endocrinol(Lausanne).2015;6:41.
[26]Lassen NE,Andersen TL,Pl?en GG,et al.Coupling of Bone Resorption and Formation in Real Time:New Knowledge Gained From Human Haversian BMUs.J Bone Miner Res.2017;32(7):1395-1405.
[27]Long F.Building strong bones:molecular regulation of the osteoblast lineage.Nat Rev Mol Cell Biol.2011;13(1):27-38.
[28]Liu Y,Lin Z,Guo J,et al.Notoginsenoside R1 significantly promotes in vitro osteoblastogenesis.Int J Mol Med.2016;38(2):537-544.
[29]Huang L,Jin P,Lin X,et al.Beneficial effects of sulfonamide?based gallates on osteoblasts in vitro.Mol Med Rep.2017;15(3):1149-1156.
[30]闫海锋,李林轩,覃金萍,等.罗汉果研究综述[J].南方农业学报,2011,42(11):1387-1391.
[31]高媛,李慧敏,赵彤彤,等.罗汉果饮料的研制研究[J].食品安全质量检测学报,2018,9(14):3760-3764.
[32]万艳娟,吴军林,吴清平.功能性甜味剂罗汉果甜苷的生理功能及食品应用研究进展[J].食品与发酵科技,2015,51(5):51-56.
[33]Golub EE,Boesze-Battaglia K.The role of alkaline phosphatase in mineralization.Current Opinion in Orthopaedics.2007;18(5):444-448.
[34]Koromila T,Baniwal SK,Song YS,et al.Glucocorticoids antagonize RUNX2 during osteoblast differentiation in cultures of ST2 pluripotent mesenchymal cells.J Cell Biochem.2014;115(1):27-33.
[35]张弘,姚宇,孙佳栋,等.NELL-1调控RUNX2的P1启动子诱导成骨分化[J].中华口腔医学研究杂志(电子版),2017,11(4):197-203.
[36]陶周善,周皖舒,江云云,等.骨形成蛋白联合雷奈酸锶对成骨细胞增殖和分化的影响[J].中国骨质疏松杂志,2018,24(2):165-169.
[37]Foster BL,Ao M,Salmon CR,et al.Osteopontin regulates dentin and alveolar bone development and mineralization.Bone.2018;107:196-207.
[38]Hosseini S,Naderi-Manesh H,Vali H,et al.Contribution of osteocalcin-mimetic peptide enhances osteogenic activity and extracellular matrix mineralization of human osteoblast-like cells.Colloids Surf B Biointerfaces.2019;173:662-671.
[39]Zhang W,Chen J,Tao J,et al.The use of type 1 collagen scaffold containing stromal cell-derived factor-1 to create a matrix environment conducive to partial-thickness cartilage defects repair.Biomaterials.2013;34(3):713-723.
[2]Bliuc D,Alarkawi D,Nguyen TV,et al.Risk of subsequent fractures and mortality in elderly women and men with fragility fractures with and without osteoporotic bone density:the Dubbo Osteoporosis Epidemiology Study.J Bone Miner Res.2015;30(4):637-646.
[3]Compston JE,McClung MR,Leslie WD.Osteoporosis.Lancet.2019;393(10169):364-376.
[4]Abrahamsen B,Osmond C,Cooper C.Life Expectancy in Patients Treated for Osteoporosis:Observational Cohort Study Using National Danish Prescription Data.J Bone Miner Res.2015;30(9):1553-1559.
[5]Siris ES,Adler R,Bilezikian J,et al.The clinical diagnosis of osteoporosis:a position statement from the National Bone Health Alliance Working Group.Osteoporos Int.2014;25(5):1439-1443.
[6]Burge R,Dawson-Hughes B,Solomon DH,et al.Incidence and economic burden of osteoporosis-related fractures in the United States,2005-2025.J Bone Miner Res.2007;22(3):465-475.
[7]中华医学会骨质疏松和骨矿盐疾病分会.原发性骨质疏松症诊疗指南(2017)[J].中国全科医学,2017,20(32):3963-3982.
[8]Marie PJ,Kassem M.Osteoblasts in osteoporosis:past,emerging,and future anabolic targets.Eur J Endocrinol.2011;165(1):1-10.
[9]曹建平,汤杰,刘合生,等.罗汉果皂苷生物活性研究进展[J].食品工业科技,2014,35(24):384-388,395.
[10]Zhang M,Yang H,Zhang H,et al.Development of a process for separation of mogroside V from Siraitia grosvenorii by macroporous resins.Molecules.2011;16(9):7288-7301.
[11]Takasaki M,Konoshima T,Murata Y,et al.Anticarcinogenic activity of natural sweeteners,cucurbitane glycosides,from Momordica grosvenori.Cancer Lett.2003;198(1):37-42.
[12]Chen WJ,Wang J,Qi XY,et al.The antioxidant activities of natural sweeteners,mogrosides,from fruits of Siraitia grosvenori.Int J Food Sci Nutr.2007;58(7):548-556.
[13]Liu H,Qi X,Yu K,et al.AMPK activation is involved in hypoglycemic and hypolipidemic activities of mogroside-rich extract from Siraitia grosvenorii(Swingle)fruits on high-fat diet/streptozotocin-induced diabetic mice.Food Funct.2019;10(1):151-162.
[14]Callaway DA,Jiang JX.Reactive oxygen species and oxidative stress in osteoclastogenesis,skeletal aging and bone diseases.J Bone Miner Metab.2015;33(4):359-370.
[15]Lin J,Zhu J,Wang Y,et al.Chinese single herbs and active ingredients for postmenopausal osteoporosis:From preclinical evidence to action mechanism.Biosci Trends.2017;11(5):496-506.
[16]Xiao W,Wang Y,Pacios S,et al.Cellular and Molecular Aspects of Bone Remodeling.Front Oral Biol.2016;18:9-16.
[17]Du L,Nong MN,Zhao JM,et al.Polygonatum sibiricum polysaccharide inhibits osteoporosis by promoting osteoblast formation and blocking osteoclastogenesis through Wnt/β-catenin signalling pathway.Sci Rep.2016;6:32261.
[18]Liang D,Ren H,Qiu T,et al.Extracts from plastrum testudinis reverse glucocorticoid-induced spinal osteoporosis of rats via targeting osteoblastic and osteoclastic markers.Biomed Pharmacother.2016;82:151-160.
[19]Pellegrini GG,Morales CC,Wallace TC,et al.Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner.Nutrients.2016;8(7):E423.
[20]Kalyanaraman H,Ramdani G,Joshua J,et al.A Novel,Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice.J Bone Miner Res.2017;32(1):46-59.
[21]Kassem A,Lindholm C,Lerner UH.Toll-Like Receptor 2Stimulation of Osteoblasts Mediates Staphylococcus Aureus Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL.PLoS One.2016;11(6):e0156708.
[22]Zhang D,Fong C,Jia Z,et al.Icariin Stimulates Differentiation and Suppresses Adipocytic Transdifferentiation of Primary Osteoblasts Through Estrogen Receptor-Mediated Pathway.Calcif Tissue Int.2016;99(2):187-198.
[23]Choi BY,Park CH,Na YH,et al.Inhibition of RANKL-induced osteoclast differentiation through the downregulation of c-Fos and NFATc1 by Eremochloa ophiuroides(centipedegrass)extract.Mol Med Rep.2016;13(5):4014-4022.
[24]Baek JM,Kim JY,Yoon KH,et al.Ebselen Is a Potential Anti-Osteoporosis Agent by Suppressing Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclast Differentiation In vitro and Lipopolysaccharide-Induced Inflammatory Bone Destruction In vivo.Int J Biol Sci.2016;12(5):478-488.
[25]Sims NA,Martin TJ.Coupling Signals between the Osteoclast and Osteoblast:How are Messages Transmitted between These Temporary Visitors to the Bone Surface.Front Endocrinol(Lausanne).2015;6:41.
[26]Lassen NE,Andersen TL,Pl?en GG,et al.Coupling of Bone Resorption and Formation in Real Time:New Knowledge Gained From Human Haversian BMUs.J Bone Miner Res.2017;32(7):1395-1405.
[27]Long F.Building strong bones:molecular regulation of the osteoblast lineage.Nat Rev Mol Cell Biol.2011;13(1):27-38.
[28]Liu Y,Lin Z,Guo J,et al.Notoginsenoside R1 significantly promotes in vitro osteoblastogenesis.Int J Mol Med.2016;38(2):537-544.
[29]Huang L,Jin P,Lin X,et al.Beneficial effects of sulfonamide?based gallates on osteoblasts in vitro.Mol Med Rep.2017;15(3):1149-1156.
[30]闫海锋,李林轩,覃金萍,等.罗汉果研究综述[J].南方农业学报,2011,42(11):1387-1391.
[31]高媛,李慧敏,赵彤彤,等.罗汉果饮料的研制研究[J].食品安全质量检测学报,2018,9(14):3760-3764.
[32]万艳娟,吴军林,吴清平.功能性甜味剂罗汉果甜苷的生理功能及食品应用研究进展[J].食品与发酵科技,2015,51(5):51-56.
[33]Golub EE,Boesze-Battaglia K.The role of alkaline phosphatase in mineralization.Current Opinion in Orthopaedics.2007;18(5):444-448.
[34]Koromila T,Baniwal SK,Song YS,et al.Glucocorticoids antagonize RUNX2 during osteoblast differentiation in cultures of ST2 pluripotent mesenchymal cells.J Cell Biochem.2014;115(1):27-33.
[35]张弘,姚宇,孙佳栋,等.NELL-1调控RUNX2的P1启动子诱导成骨分化[J].中华口腔医学研究杂志(电子版),2017,11(4):197-203.
[36]陶周善,周皖舒,江云云,等.骨形成蛋白联合雷奈酸锶对成骨细胞增殖和分化的影响[J].中国骨质疏松杂志,2018,24(2):165-169.
[37]Foster BL,Ao M,Salmon CR,et al.Osteopontin regulates dentin and alveolar bone development and mineralization.Bone.2018;107:196-207.
[38]Hosseini S,Naderi-Manesh H,Vali H,et al.Contribution of osteocalcin-mimetic peptide enhances osteogenic activity and extracellular matrix mineralization of human osteoblast-like cells.Colloids Surf B Biointerfaces.2019;173:662-671.
[39]Zhang W,Chen J,Tao J,et al.The use of type 1 collagen scaffold containing stromal cell-derived factor-1 to create a matrix environment conducive to partial-thickness cartilage defects repair.Biomaterials.2013;34(3):713-723.