葛根素抑制人成骨样MG-63细胞凋亡的分子机制研究
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摘要
目的研究葛根素抑制人成骨样MG-63细胞凋亡的分子机制。方法建立顺铂诱导的成骨细胞凋亡模型,DAPI染色法检测葛根素对MG-63细胞凋亡的影响,RT-PCR和蛋白免疫印迹法分别检测凋亡相关因子Bcl-xL mRNA和蛋白的表达水平,应用ER阻断剂ICI 182,780和小RNA干扰技术探讨葛根素调节成骨细胞凋亡的作用机制。结果顺铂10μmol/L组MG-63细胞凋亡率高于对照组,葛根素+顺铂组低于顺铂10μmol/L组(P <0. 05)。不同浓度葛根素组MG-63细胞Bcl-xL mRNA和蛋白表达水平均高于对照组(P <0. 05)。葛根素+ICI+顺铂组成骨细胞凋亡率高于葛根素+顺铂组(P <0. 05)。葛根素+顺铂组MG-63/scrambled、MG-63/ERα和MG-63/ERβshRNA细胞凋亡低于顺铂组(P <0. 05)。结论葛根素抑制顺铂诱导的人成骨样MG-63细胞凋亡作用是经ER途径,由ERα和ERβ共同介导的。
Objective To study molecular mechanism of Puerarin inhibiting apoptosis of human osteoblastic MG-63 cells. Methods Apoptotic models of osteoblasts induced by Cisplatin were established. Effect of Puerarin on apoptosis of MG-63 cells was detected by DAPI staining. Expressions of Bcl-xL mRNA and protein were detected by reverse transcription polymerase chain reaction( RT-PCR) and Western blot methods respectively. Mechanism of Puerarin regulating apoptosis of osteoblastic cells was investigated by using estrogen receptor( ER) blocking agent ICI 182,780 and small RNA interference technology. Results Apoptotic rate of MG-63 cells in 10 μmol/L Cisplatin group was significantly higher than that in control group,and the rate in Puerarin + Cisplatin group was significantly lower than that in 10μmol/L Cisplatin group( P < 0. 05). Expressions of Bcl-xL in MG-63 cells in different concentrations Puerarin groups were significantly higher than that in control group( P < 0. 05). Apoptotic rate of osteoblasts in Puerarin + ICI + Cisplatin group was significantly higher than that in Puerarin + Cisplatin group( P < 0. 05). Apoptotic rates of MG-63/scrambled,MG-63/ERα and MG-63/ERβ shRNA in Puerarin + Cisplatin group were significantly lower than those in Cisplatin group( P < 0. 05). Conclusion Puerarin inhibiting Cisplatin-induced apoptosis in human osteoblastic MG-63 cells is through ER pathway and mediated by both ERα and ERβ.
Objective To study molecular mechanism of Puerarin inhibiting apoptosis of human osteoblastic MG-63 cells. Methods Apoptotic models of osteoblasts induced by Cisplatin were established. Effect of Puerarin on apoptosis of MG-63 cells was detected by DAPI staining. Expressions of Bcl-xL mRNA and protein were detected by reverse transcription polymerase chain reaction( RT-PCR) and Western blot methods respectively. Mechanism of Puerarin regulating apoptosis of osteoblastic cells was investigated by using estrogen receptor( ER) blocking agent ICI 182,780 and small RNA interference technology. Results Apoptotic rate of MG-63 cells in 10 μmol/L Cisplatin group was significantly higher than that in control group,and the rate in Puerarin + Cisplatin group was significantly lower than that in 10μmol/L Cisplatin group( P < 0. 05). Expressions of Bcl-xL in MG-63 cells in different concentrations Puerarin groups were significantly higher than that in control group( P < 0. 05). Apoptotic rate of osteoblasts in Puerarin + ICI + Cisplatin group was significantly higher than that in Puerarin + Cisplatin group( P < 0. 05). Apoptotic rates of MG-63/scrambled,MG-63/ERα and MG-63/ERβ shRNA in Puerarin + Cisplatin group were significantly lower than those in Cisplatin group( P < 0. 05). Conclusion Puerarin inhibiting Cisplatin-induced apoptosis in human osteoblastic MG-63 cells is through ER pathway and mediated by both ERα and ERβ.
引文
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[19]张立朋,李玲,金鑫,等.葛根素促人成骨样MG-63细胞增殖的分子机制研究[J].武警后勤学院学报:医学版,2016,25(1):10-14,封2.
[20]罗婷,李乐飞,郭小芹,等.葛根素抑制人成骨样MG-63细胞分泌IL-6的分子机制研究[J].中国药理学通报,2015,31(11):1524-1528,1529.
[21]Yuan S Y,Sheng T,Liu L Q,et al. Puerarin prevents bone loss in ovariectomized mice and inhibits osteoclast formation in vitro[J]. Chin J Nat Med,2016,14(4):265-269.
[22]卢宇,费小蔷,杨淑芳,等.葛根素抑制吡格列酮诱导的成骨细胞凋亡[J].中国骨质疏松杂志,2017,23(2):231-235.
[23]于冬冬,赵丹阳.葛根素通过p38MAPK通路抑制MC3T3-E1成骨细胞凋亡[J].中国现代医学杂志,2017,27(5):7-12.
[24]Kallio A,Guo T,Lamminen E,et al. Estrogen and the selective estrogen receptor modulator(SERM)protection against cell death in estrogen receptor alpha and beta expressing U2OS cells[J]. Mol Cell Endocrinol,2008,289(1-2):38-48.
[25]Zallone A. Direct and indirect estrogen actions on osteoblasts and osteoclasts[J]. Ann N Y Acad Sci,2006,1068:173-179.
[26]Ayaloglu-Butun F,Terzioglu-Kara E,Tokcaer-Keskin Z,et al. The effect of estrogen on bone marrow-derived rat mesenchymal stem cell maintenance:inhibiting apoptosis through the expression of Bcl-xL and Bcl-2[J]. Stem Cell Rev,2012,8(2):393-401.
[2]白璧辉,谢兴文,李鼎鹏,等.我国近5年来骨质疏松症流行病学研究现状[J].中国骨质疏松杂志,2018,24(2):253-258.
[3]崔莹,冯正平.抗骨质疏松药物的研究进展[J].中国骨质疏松杂志,2015,21(3):367-371.
[4]Bernabei R,Martone A M,Ortolani E,et al. Screening,diagnosis and treatment of osteoporosis:a brief review[J]. Clin Cases Miner Bone Metab,2014,11(3):201-207.
[5]张昌攀,陈凯,陈海鹏.中西医药物治疗原发性骨质疏松症研究进展[J].中医药临床杂志,2018,30(1):171-175.
[6]吴绍刚.葛根素的药理作用解析及临床合理应用[J].基层医学论坛,2017,21(25):3462.
[7]曾祥伟,冯倩,张莹莹,等.葛根素对炎症相关疾病研究进展[J].中国药理学通报,2018,34(1):8-11.
[8]张明.葛根素对骨质疏松症治疗作用及机制的实验研究[D].大理:大理大学,2016.
[9]李灵芝,刘启兵,张永亮,等.葛根素对UMR106细胞增殖及碱性磷酸酶活性的影响[J].中国新药杂志,2005,14(11):1291-1294.
[10]赵艳威,李宗旻,宋光明,等.葛根素促进人成骨样MG-63细胞分化的分子机制研究[J].中草药,2014,45(4):536-540.
[11]Wang Y,Li L Z,Zhang Y L,et al. LC,a novel estronerhein hybrid compound,concurrently stimulates osteoprotegerin and inhibits receptor activator of NF-κB ligand(RANKL)and interleukin-6 production by human osteoblastic cells[J]. Mol Cell Endocrinol,2011,337(1-2):43-51.
[12]Farr J N,Fraser D G,Wang H,et al. Identification of Senescent Cells in the Bone Microenvironment[J]. J Bone Miner Res,2016,31(11):1920-1929.
[13]Rutkovskiy A,Stenslkken K O,Vaage I J. Osteoblast Differentiation at a Glance[J]. Med Sci Monit Basic Res,2016,22:95-106.
[14]周立新,宋倩,薛鹏,等.雌激素对成骨细胞作用机制的研究进展[J].河北医科大学学报,2014,35(5):613-616.
[15]Khosla S,Oursler M J,Monroe D G. Estrogen and the skeleton[J]. Trends Endocrinol Metab,2012,23(11):576-581.
[16]Kousteni S,Bellido T,Plotkin L I,et al. Nongenotropic,sex-nonspecific signaling through the estrogen or androgen receptors:dissociation from transcriptional activity[J].Cell,2001,104(5):719-730.
[17]Kousteni S,Han L,Chen J R,et al. Kinase-mediated regulation of common transcription factors accounts for the bone-protective effects of sex steroids[J]. J Clin Invest,2003,111(11):1651-1664.
[18]徐霞,贡沁燕,鲁映青,等.大豆异黄酮对去卵巢大鼠骨密度和雌激素活性的影响(英文)[J].中国新药与临床杂志,2002,21(6):321-325.
[19]张立朋,李玲,金鑫,等.葛根素促人成骨样MG-63细胞增殖的分子机制研究[J].武警后勤学院学报:医学版,2016,25(1):10-14,封2.
[20]罗婷,李乐飞,郭小芹,等.葛根素抑制人成骨样MG-63细胞分泌IL-6的分子机制研究[J].中国药理学通报,2015,31(11):1524-1528,1529.
[21]Yuan S Y,Sheng T,Liu L Q,et al. Puerarin prevents bone loss in ovariectomized mice and inhibits osteoclast formation in vitro[J]. Chin J Nat Med,2016,14(4):265-269.
[22]卢宇,费小蔷,杨淑芳,等.葛根素抑制吡格列酮诱导的成骨细胞凋亡[J].中国骨质疏松杂志,2017,23(2):231-235.
[23]于冬冬,赵丹阳.葛根素通过p38MAPK通路抑制MC3T3-E1成骨细胞凋亡[J].中国现代医学杂志,2017,27(5):7-12.
[24]Kallio A,Guo T,Lamminen E,et al. Estrogen and the selective estrogen receptor modulator(SERM)protection against cell death in estrogen receptor alpha and beta expressing U2OS cells[J]. Mol Cell Endocrinol,2008,289(1-2):38-48.
[25]Zallone A. Direct and indirect estrogen actions on osteoblasts and osteoclasts[J]. Ann N Y Acad Sci,2006,1068:173-179.
[26]Ayaloglu-Butun F,Terzioglu-Kara E,Tokcaer-Keskin Z,et al. The effect of estrogen on bone marrow-derived rat mesenchymal stem cell maintenance:inhibiting apoptosis through the expression of Bcl-xL and Bcl-2[J]. Stem Cell Rev,2012,8(2):393-401.
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