葛根素对MC3T3-E1细胞增殖、分化和矿化及TRPM3 mRNA表达的影响
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摘要
目的观察葛根素对小鼠成骨细胞MC3T3-E1细胞增殖、分化和矿化及TRPM3 mRNA表达的影响。方法分别采用CCK-8法、碱性磷酸酶(ALP)活性、茜素红染色测定葛根素对MC3T3-E1细胞增殖、成骨分化、矿化作用的影响。流式细胞术检测葛根素对MC3T3-E1细胞周期及细胞内钙离子浓度的影响。RT-PCR检测葛根素对TRPM3基因m RNA表达的影响。结果 0.1、1、10μmol·L~(-1)葛根素能明显促进MC3T3-E1细胞增殖,使G1期细胞比例减少,G_2+S期细胞比例增加,其中0.1μmol·L~(-1)浓度效果最为明显;与正常对照组相比较,0.1μmol·L~(-1)葛根素组细胞的ALP活性和矿化结节面积均明显提高;0.1μmol·L~(-1)葛根素组细胞的TRPM3 m RNA表达水平和细胞内钙离子浓度明显降低。结论葛根素可促进MC3T3-E1细胞的增殖、分化和矿化,可降低TRPM3 mRNA表达水平和细胞内钙离子浓度。
Aim To investigate the effect of puerarin on proliferation,differentiation,and mineralization of MC3T3-E1 cells and the expression of TRPM3 mR NA.Methods Proliferation,differentiation,and mineralization of puerarin in MC3T3-E1 cells were determined using CCK-8 assay,alkaline phosphatase(ALP) activity assay,and Alizarin Red Staining,respectively.Effect of puerarin on cell cycle and intracellular calcium concentration of MC3T3-E1 cells was detected by flow cytometry.RT-PCR was used to detect the effect of puerarin on TRPM3 mR NA expression.Resuls0.1,1,10 μmol·L~(-1)puerarin significantly promoted the proliferation of MC3T3-E1 cells,reduced the proportion of cells in G1 phase,increased the proportion of G_2 and S phase,of which 0.1 μmol · L~(-1)concentration effect was the most significant.Compared with control group,the ALP activity and mineralized nodule area of 0.1 μmol · L~(-1)puerarin group were significantly increased.The expression of TRPM3 mR NA and the intracellular calcium concentration were significantly decreased in 0.1 μmol · L~(-1)puerarin group.Conclusion Puerarin can promote the proliferation,differentiation,and mineralization of MC3T3-E1 cells,and reduce the expression level of TRPM3 mR NA and intracellular calcium concentration.
Aim To investigate the effect of puerarin on proliferation,differentiation,and mineralization of MC3T3-E1 cells and the expression of TRPM3 mR NA.Methods Proliferation,differentiation,and mineralization of puerarin in MC3T3-E1 cells were determined using CCK-8 assay,alkaline phosphatase(ALP) activity assay,and Alizarin Red Staining,respectively.Effect of puerarin on cell cycle and intracellular calcium concentration of MC3T3-E1 cells was detected by flow cytometry.RT-PCR was used to detect the effect of puerarin on TRPM3 mR NA expression.Resuls0.1,1,10 μmol·L~(-1)puerarin significantly promoted the proliferation of MC3T3-E1 cells,reduced the proportion of cells in G1 phase,increased the proportion of G_2 and S phase,of which 0.1 μmol · L~(-1)concentration effect was the most significant.Compared with control group,the ALP activity and mineralized nodule area of 0.1 μmol · L~(-1)puerarin group were significantly increased.The expression of TRPM3 mR NA and the intracellular calcium concentration were significantly decreased in 0.1 μmol · L~(-1)puerarin group.Conclusion Puerarin can promote the proliferation,differentiation,and mineralization of MC3T3-E1 cells,and reduce the expression level of TRPM3 mR NA and intracellular calcium concentration.
引文
[1]Marie P J,Kassem M.Osteoblasts in osteoporosis:past,emerging,and future anabolic targets[J].Eur J Endocrinol,2011,165(1):1-10.
[2]Shibata S,Murakami T,Nishkawa Y,et al.The constituents of Pueraria root[J].Chem Pharm Bull,1959,7(1):134-6.
[3]Wei S Y,Chen Y,Xu X Y.Progress on the pharmacological research of puerarin:a review[J].Chin J Nat Med,2014,12(6):407-14.
[4]Liu H,Li W,Ge X,et al.Coadministration of puerarin(low dose)and zinc attenuates bone loss and suppresses bone marrow adiposity in ovariectomized rats[J].Life Sci,2016,166:20-6.
[5]张莹莹,周建斌,曾祥伟,等.葛根素对成骨细胞增殖能力及靶向Runx2的mi RNA的影响[J].中国药理学通报,2016,32(10):1457-62.[5]Zhang Y Y,Zhou J B,Zeng X W,et al.Effects of puerarin on proliferation of osteoblasts and Runx2-targeting mi RNAs[J].Chin Pharmacol Bull,2016,32(10):1457-62.
[6]Cost N G,Czyzykkrzeska M F.Regulation of autophagy by two products of one gene:TRPM3 and mi R-204[J].Mol Cell Oncol,2015,2(4):e1002712.
[7]Abed E,Labelle D,Martineau C,et al.Expression of transient receptor potential(TRP)channels in human and murine osteoblast-like cells[J].Mol Membr Biol,2009,26(3):146-58.
[8]Addison W N,Azari F,Srensen E S,et al.Pyrophosphate Inhibits mineralization of osteoblast cultures by binding to mineral,up-regulating osteopontin,and inhibiting alkaline Phosphatase activity[J].J Biol Chem,2007,282(21):15872-83.
[9]Hoac B,Kiffer-Moreira T,Millán J L,et al.Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures[J].Bone,2013,53(2):478-86.
[10]Lee Y S,Choi E M.Costunolide stimulates the function of osteoblastic MC3T3-E1 cells[J].Int Immunopharmacol,2011,11(6):712-8.
[11]Abdi F,Alimoradi Z,Haqi P,et al.Effects of phytoestrogens on bone mineral density during the menopause transition:a systematic review of randomized,controlled trials[J].Climacteric,2016,19(6):535-45.
[12]Wang C,Meng M X,Tang X L,et al.The proliferation,differentiation,and mineralization effects of puerarin on osteoblasts in vitro[J].Chin J Nat Med,2014,12(6):436-42.
[13]Ding M,Lin B,Li T,et al.A dual yet opposite growth-regulating function of mi R-204 and its target XRN1 in prostate adenocarcinoma cells and neuroendocrine-like prostate cancer cells[J].Oncotarget,2015,6(10):7686-700.
[14]Ying Z,Li Y,Wu J,et al.Loss of mi R-204 expression enhances glioma migration and stem cell-like phenotype[J].Cancer Res,2013,73(2):990-9.
[15]Lee N,Chen J,Sun L,et al.Expression and characterization of human transient receptor potential melastatin 3(h TRPM3)[J].J Biol Chem,2003,278(23):20890-7.
[16]Zayzafoon M.Calcium/calmodulin signaling controls osteoblast growth and differentiation[J].J Cell Biochem,2006,97(1):56-70.
[17]徐晓虹,郑筱祥.葛根素对氧糖剥夺大鼠海马神经细胞Ca2+和NO的影响[J].中国药学杂志,2008,43(17):1308-12.[17]Xu X H,Zheng X X.Effects of puerarin on the levels of Calcium and Nitric Oxide in cultured hippocampal neurons during oxygenglucose deprivation[J].Chin Pharm J,2008,43(17):1308-12.
[18]黄绵庆,杨照新,姚茂忠,等.葛根素通过神经酰胺通路抑制UVB诱导的Ha Ca T细胞DNA损伤[J].药学学报,2012,47(5):609-13.[18]Huang M Q,Yang Z X,Yao M Z,et al.Puerarin inhibits DNA damage of Ha Ca T cells induced by UVB via ceramide pathway[J].Acta Pharm Sin,2012,47(5):609-13.
[19]肖桂凤,宁钢民,葛亚坤,等.葛根素对培养人脐静脉内皮细胞钙超载的影响[J].中国药理学通报,2005,21(11):1388-92.[19]Xiao G F,Ning G M,Ge Y K,et al.Effect of Puerarin on calcium overload in cultured human umbilical vascular endothelial cells[J].Chin Pharmacol Bull,2005,21(11):1388-92.
[20]杨冰,谭彤燕,梁笃,等.雌激素相关受体α特异性抑制剂XCT-790对成骨细胞株MG-63增殖分化的影响[J].广州中医药大学学报,2011,28(1):43-5.[20]Yang B,Tan T Y,Liang D,et al.Effect of estrogen-related receptor-alpha specific antagonist XCT-790 on proliferation and differentiation of osteoblast-like cell line MG-63[J].J Guangzhou Univer Tradit Chin Med,2011,28(1):43-5.
[2]Shibata S,Murakami T,Nishkawa Y,et al.The constituents of Pueraria root[J].Chem Pharm Bull,1959,7(1):134-6.
[3]Wei S Y,Chen Y,Xu X Y.Progress on the pharmacological research of puerarin:a review[J].Chin J Nat Med,2014,12(6):407-14.
[4]Liu H,Li W,Ge X,et al.Coadministration of puerarin(low dose)and zinc attenuates bone loss and suppresses bone marrow adiposity in ovariectomized rats[J].Life Sci,2016,166:20-6.
[5]张莹莹,周建斌,曾祥伟,等.葛根素对成骨细胞增殖能力及靶向Runx2的mi RNA的影响[J].中国药理学通报,2016,32(10):1457-62.[5]Zhang Y Y,Zhou J B,Zeng X W,et al.Effects of puerarin on proliferation of osteoblasts and Runx2-targeting mi RNAs[J].Chin Pharmacol Bull,2016,32(10):1457-62.
[6]Cost N G,Czyzykkrzeska M F.Regulation of autophagy by two products of one gene:TRPM3 and mi R-204[J].Mol Cell Oncol,2015,2(4):e1002712.
[7]Abed E,Labelle D,Martineau C,et al.Expression of transient receptor potential(TRP)channels in human and murine osteoblast-like cells[J].Mol Membr Biol,2009,26(3):146-58.
[8]Addison W N,Azari F,Srensen E S,et al.Pyrophosphate Inhibits mineralization of osteoblast cultures by binding to mineral,up-regulating osteopontin,and inhibiting alkaline Phosphatase activity[J].J Biol Chem,2007,282(21):15872-83.
[9]Hoac B,Kiffer-Moreira T,Millán J L,et al.Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures[J].Bone,2013,53(2):478-86.
[10]Lee Y S,Choi E M.Costunolide stimulates the function of osteoblastic MC3T3-E1 cells[J].Int Immunopharmacol,2011,11(6):712-8.
[11]Abdi F,Alimoradi Z,Haqi P,et al.Effects of phytoestrogens on bone mineral density during the menopause transition:a systematic review of randomized,controlled trials[J].Climacteric,2016,19(6):535-45.
[12]Wang C,Meng M X,Tang X L,et al.The proliferation,differentiation,and mineralization effects of puerarin on osteoblasts in vitro[J].Chin J Nat Med,2014,12(6):436-42.
[13]Ding M,Lin B,Li T,et al.A dual yet opposite growth-regulating function of mi R-204 and its target XRN1 in prostate adenocarcinoma cells and neuroendocrine-like prostate cancer cells[J].Oncotarget,2015,6(10):7686-700.
[14]Ying Z,Li Y,Wu J,et al.Loss of mi R-204 expression enhances glioma migration and stem cell-like phenotype[J].Cancer Res,2013,73(2):990-9.
[15]Lee N,Chen J,Sun L,et al.Expression and characterization of human transient receptor potential melastatin 3(h TRPM3)[J].J Biol Chem,2003,278(23):20890-7.
[16]Zayzafoon M.Calcium/calmodulin signaling controls osteoblast growth and differentiation[J].J Cell Biochem,2006,97(1):56-70.
[17]徐晓虹,郑筱祥.葛根素对氧糖剥夺大鼠海马神经细胞Ca2+和NO的影响[J].中国药学杂志,2008,43(17):1308-12.[17]Xu X H,Zheng X X.Effects of puerarin on the levels of Calcium and Nitric Oxide in cultured hippocampal neurons during oxygenglucose deprivation[J].Chin Pharm J,2008,43(17):1308-12.
[18]黄绵庆,杨照新,姚茂忠,等.葛根素通过神经酰胺通路抑制UVB诱导的Ha Ca T细胞DNA损伤[J].药学学报,2012,47(5):609-13.[18]Huang M Q,Yang Z X,Yao M Z,et al.Puerarin inhibits DNA damage of Ha Ca T cells induced by UVB via ceramide pathway[J].Acta Pharm Sin,2012,47(5):609-13.
[19]肖桂凤,宁钢民,葛亚坤,等.葛根素对培养人脐静脉内皮细胞钙超载的影响[J].中国药理学通报,2005,21(11):1388-92.[19]Xiao G F,Ning G M,Ge Y K,et al.Effect of Puerarin on calcium overload in cultured human umbilical vascular endothelial cells[J].Chin Pharmacol Bull,2005,21(11):1388-92.
[20]杨冰,谭彤燕,梁笃,等.雌激素相关受体α特异性抑制剂XCT-790对成骨细胞株MG-63增殖分化的影响[J].广州中医药大学学报,2011,28(1):43-5.[20]Yang B,Tan T Y,Liang D,et al.Effect of estrogen-related receptor-alpha specific antagonist XCT-790 on proliferation and differentiation of osteoblast-like cell line MG-63[J].J Guangzhou Univer Tradit Chin Med,2011,28(1):43-5.