目的 本实验利用泼尼松龙诱导的斑马鱼骨质疏松模型评价微量淫羊藿苷和朝藿定B抗斑马鱼骨质疏松的作用。方法 将受精后4 d的斑马鱼幼鱼暴露在含25 μmol·L-1泼尼松龙的不同浓度淫羊藿苷(0.2、1、5和10 μmol·L-1)和朝藿定B(0.2、1、5和10 μmol·L-1), 同时设25 μmol·L-1泼尼松龙模型药物组, 含25 μmol·L-1泼尼松龙的15 μg·mL-1依替膦酸二钠阳性药物组, 0.5% 二甲基亚砜溶媒对照组和培养基阴性对照组。28.5 ℃下在24孔板中培养, 每天换液至第9天处死。采用茜素红对培养9 d的各组斑马鱼幼鱼骨骼染色, 并以显微检测、数码成像方法定量分析骨骼染色区域。结果 与二甲基亚砜溶媒和培养基阴性对照组相比, 25 μmol·L-1泼尼松龙模型组的斑马鱼头部骨骼染色面积和染色光密度值显著减少;与模型组比较, 15 μg·mL-1依替膦酸二钠能增加斑马鱼头骨矿化量并有效防止泼尼松龙诱导斑马鱼产生的骨质疏松;与模型组比较, 10和5 μmol·L-1的淫羊藿苷和1 μmol·L-1朝藿定B都能显著提高斑马鱼头部骨骼的染色面积和染色累积光密度值, 提示淫羊藿苷和朝藿定B能防止泼尼松龙诱导的斑马鱼骨质疏松。结论 斑马鱼骨质疏松模型具简单、高效、化合物用量少及可操作性强的优势, 成功用于评价微量淫羊藿苷和朝藿定B的抗骨质疏松活性。
Abstract
OBJECTIVE Prednisolone-induced osteoporosis model using zebrafish was used to evaluate the antiosteoporotic activity of micro amount icariin and epimedin B.METHODS Zebrafish larvae were co-exposed with 25 μmol·L-1 prednisolone and a series of icariin and epimedin B solutions with a range of concentration (0.2, 1, 5 and 10 μmol·L-1) from 4 to 9 days post fertilization (dpf), 25 μmol·L-1 prednisolone was selected as model group, 15 μg·mL-1 etidronate disodium with 25 μmol·L-1 prednisolone as positive group, 0.5% DMSO as the vehicle control group, the medium as a negative blank control group, all groups were incubated in 24-well plates (28.5 ℃) until 9 dpf. The medium/solution was changed half every day. Zebrafish skeleton at 9 dpf were anesthetized and fixed for staining with alizarin red. Quantitative analysis of the stained area was performed by microscopic inspection and digital imagingMETHODS to reflect the amount of zebrafish head skeleton mineralization. RESULTS The RESULTS indicated head skeleton mineral aera and integrated optical density (IOD) of 25 μmol·L-1 prednisolone model group were significantly decreased when compared with vehicle control group and negative control group; and 15 μg·mL-1 etidronate disodium can increase the mineralized matrix of zebrafish head skeleton when compared with model group and prevent osteoporosis induced by prednisolone; head skeleton mineral aera and IOD of 10 and 5 μmol·L-1 icariin groups and 1 μmol·L-1 epimedin B groups were significantly increased when compared with model group, which indicated that icariin and epimedin B can prevent zebrafish osteoporosis induced by prednisolone. CONCLUSION This novel osteoporosis model using zebrafish has advantages of simple, high efficiency, far less amount of compound needed and easy to perform, and it was successfully applied in quickly evaluating the antiosteoporosis activity of micro amount compounds such as icariin and epimedin B.
关键词
斑马鱼 /
骨质疏松 /
淫羊藿苷 /
朝藿定B /
泼尼松龙 /
依替膦酸二钠
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Key words
zebrafish /
osteoporosis /
icariin /
epimedin B /
prednisolone /
etidronate disodium
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中图分类号：
R965
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参考文献
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基金
国家自然科学基金资助项目(81274088;30973978);江苏省自然科学基金资助项目(BK2011866);江苏省“六大人才高峰”项目(YY006)
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