葛根素影响雌激素受体α启动子甲基化调控成骨细胞增殖分化的研究

Puerarin reduces methylation of estrogen receptor a promoter in osteoblasts and regulates its proliferation and osteoblastic differentiation

目的探究葛根素对大鼠成骨细胞代谢调控的途径,为绝经后骨质疏松治疗提供理论依据。方法取新生SD大鼠的颅骨,胶原酶法培养成骨细胞,分别加入不同浓度(5、10、25、50、100μmol/L)葛根素分组培养,应用巢式甲基敏感性聚合酶链反应方法观察雌激素受体α(estrogen receptorα,ERα)启动子A区甲基化,反转录—聚合酶链反应方法观察ERαmRNA表达,MTT法和对硝基苯磷酸酯方法观察体外培养成骨细胞的增殖及碱性磷酸酶活性。结果随着葛根素浓度增加,ERα基因启动子甲基化程度逐渐减弱;同时发现细胞中ERαmRNA表达逐渐增强,25μmol/L葛根素浓度时表达最强(P<0.01)。葛根素能刺激成骨细胞增殖,提高碱性磷酸酶活性(P<0.01)。结论葛根素抑制ERα启动子甲基化及增加ERαmRNA表达,可能通过此途径促进成骨细胞增殖分化。

Objective The study shows that puerarin regulates pathway of metabolism in osteoblasts and provides the theoretical foundation for treating methods of postmenopausal osteoporosis.Methods Culture of primary calvarial osteoblasts in rats were digested,and used in experiments.Then,the cells were treated with different concentrations of puerarin（5,10,25,50, 100 μmol/L）in different groups,respectively.The methylation status of CpG islands in estrogen receptor α（ERα）gene promoter region of genome DNA was analyzed by nested-methylation-specific PCR;ER-α gene mRNA expression was examined by semiquantitative reverse transcription PCR analysis.The proliferation and viability of osteoblasts were assayed by MTT assay using the spectrophotometric method.Furthermore,the differentiation of osteoblast-like cells by p-nitrophenyl phosphate were identified.Results The cultured osteoblasts treated by puerarin with different concentrations reduced in de novo methylation in promoter region of ERαgene with a concentration-treating manner,and a dose-dependent raising effect on osteoblasts proliferation and differentiation （P 〈 0.01 ）.Meanwhile,ER-α gene mRNA expressions in osteoblasts treated by puerarin with different concentrations became stronger and stronger,until in the 25 μmol/L treating group,their expressions were the stongest（P 〈 0.01 ）.Conclusion Puerarin reduces DNA methylation in promoter region of ERα in osteoblasts and enhences ERαgene mRNA expressions in osteoblasts.Above results possible influence its proliferation and＆nbsp;osteoblastic differentiation.