Zhai Yuan-kun, Li Zhi-zhong, Chen Ke-ming, Zhang Na, Cheng Guo-zheng, Zhu Rui-qing
College of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China.
Zhong Yao Cai. 2011 Jun;34(6):917-22.
To investigate the effects of icariin on the proliferation, differentiation and maturation 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. Icariin was added into the culture at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol/L respectively. MTT method was adopted in proliferation analysis. The activity of ALP was assayed after 9 days' induced. Optimal concentration icariin was added into the medium, then the osteogenic differentiation markers including mineralized bone nodules, CFU-F(ALP) were compared between the icariin-added group and the control. Total RNA was isolated and the gene expressions of Runx-2 and Osterix were investigated by Real Time RT-PCR. Total protein was also isolated and the secretion of collagen I was examined by Western-blot.
The ROB proliferation was inhibited by icariin in a dose-dependent manner. But it evidently led to osteogenic process and maturation. 1 x 10(-5) mol/L was the best concentration. Icariin improved the secretion of collagen I, CFU-F(ALP) amounts and mineralized nodules significantly. It also enhanced the mRNA level of Runx-2 and Osterix.
The icariin with final concentration of 1 x 10(-5) mol/L can enhance the osteogenic differentiation and maturation of ROB significantly, suggesting that icariin has the activity of inducing bone formation, it has the potential to be developed into a new drug of anti-osteoporosis.
研究淫羊藿苷对大鼠颅骨成骨细胞（ROB）增殖、分化及成熟的影响。
分离新生SD大鼠颅骨，酶消化法获取ROB。每3天更换一次培养基。当细胞铺满培养皿80%时进行传代培养。分别以1×10⁻⁴、1×10⁻⁵、1×10⁻⁶、1×10⁻⁷mol/L的浓度向培养物中加入淫羊藿苷。采用MTT法进行增殖分析。诱导9天后检测碱性磷酸酶（ALP）活性。向培养基中加入最佳浓度的淫羊藿苷，然后比较添加淫羊藿苷组与对照组之间包括矿化骨结节、CFU-F（ALP）在内的成骨分化标志物。提取总RNA，通过实时荧光定量逆转录聚合酶链反应（Real Time RT-PCR）检测Runx-2和Osterix的基因表达。提取总蛋白，通过蛋白质免疫印迹法检测Ⅰ型胶原蛋白的分泌情况。
淫羊藿苷以剂量依赖性方式抑制ROB增殖。但它明显促进成骨过程和成熟。1×10⁻⁵mol/L为最佳浓度。淫羊藿苷显著提高了Ⅰ型胶原蛋白的分泌、CFU-F（ALP）数量和矿化结节。它还提高了Runx-2和Osterix的mRNA水平。
终浓度为1×10⁻⁵mol/L的淫羊藿苷可显著增强ROB的成骨分化和成熟，表明淫羊藿苷具有诱导骨形成的活性，有开发成为抗骨质疏松新药的潜力。
