Zhai Yuan-Kun, Niu Yin-Bo, Pan Ya-Lei, Li Chen-Rui, Wu Xiang-Long, Mei Qi-Bing
Key Laboratory for Space Bioscience and Biotechnology, College of Life Science, Northwestern Polytechnical University, Xi'an 710072, China.
Zhongguo Zhong Yao Za Zhi. 2013 Jan;38(1):105-11.
To investigate the effects of naringin on the proliferation, differention and maturaion of rat calvarial osteoblasts (ROB).
Segregated neonatal SD rat skull, enzyme digestion to obtain ROB. The culture medium was replaced every three days. Serial subcultivation proceeded when cells covered with 80% culture dish. Naringin supplemented into the culture at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) respectively. MTT method was adopted in proliferation analysis and the activity of ALP was examined after induced 9 days. Search the best concentration and supplemented into the medium, then the osteogenic differentiation markers including the secretion amount of osteocalcin, osteopontin and bone morphogenetic protein-2 were compared between the naringin-supplemented group and the control. Total RNA was isolated and the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERa and ERbeta was investigated by Real time RT-PCR. Total protein also was isolated and the expression ERa, ERbeta and collagen I was examined by Western blot. After the addition of ICI 182.780, an inhibitor of the estrogen signal pathway, these index also was examined and the changes were compared.
The ROB proliferation was motivated by naringin dose-dependently. And it evidently leads to osteogenic process and maturation. 1 x 10(-5) mol x L(-1) is the best concentration. Naringin improved the secretion of osteocalcin, osteopontin, bone morphogenetic protein-2 and collagen I significantly. Besides, it can also enhanced the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERalpha and ERbeta. While all these effects can be restrained by ICI 182.780.
The naringin with final concentration of 1 x 10(-5) mol x L(-1) enhances the osteogenic differentiation and maturation of ROB significantly, while the promoting effects vanished after the addition of ICI 182.780. These results suggesting that naringin is one of the phytoestrogens and have the activity of bone formation may via estrogen signal pathway, it can be developed into a new drug for osteoporosis therapy.
研究柚皮苷对大鼠颅骨成骨细胞（ROB）增殖、分化和成熟的影响。
分离新生SD大鼠颅骨，酶消化法获取ROB。每3天更换一次培养液。细胞铺满培养皿80%时进行传代培养。分别以1×10⁻⁴、1×10⁻⁵、1×10⁻⁶、1×10⁻⁷mol·L⁻¹的浓度向培养液中添加柚皮苷。采用MTT法分析增殖情况，诱导9天后检测碱性磷酸酶（ALP）活性。寻找最佳浓度并添加到培养液中，然后比较添加柚皮苷组与对照组中成骨分化标志物骨钙素、骨桥蛋白和骨形态发生蛋白-2的分泌量。提取总RNA，通过实时逆转录聚合酶链反应（Real time RT-PCR）检测碱性成纤维细胞生长因子（bFGF）、胰岛素样生长因子-1（IGF-1）、核心结合因子α1（Runx-2）、osterix、雌激素受体α（ERα）和雌激素受体β（ERβ）的mRNA水平。提取总蛋白，通过蛋白质免疫印迹法检测ERα、ERβ和Ⅰ型胶原的表达。添加雌激素信号通路抑制剂ICI 182.780后，检测上述指标并比较变化情况。
柚皮苷能剂量依赖性地促进ROB增殖，明显促进其成骨过程和成熟。1×10⁻⁵mol·L⁻¹为最佳浓度。柚皮苷显著提高骨钙素、骨桥蛋白、骨形态发生蛋白-2和Ⅰ型胶原的分泌量。此外，还能提高bFGF、IGF-1、Runx-2、osterix、ERα和ERβ的mRNA水平。而ICI 182.780可抑制所有这些作用。
终浓度为1×10⁻⁵mol·L⁻¹的柚皮苷能显著增强ROB的成骨分化和成熟，添加ICI 182.780后促进作用消失。这些结果表明柚皮苷是一种植物雌激素，可能通过雌激素信号通路发挥促进骨形成的活性，有望开发成为治疗骨质疏松症的新药。
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