淫羊藿总黄酮对泼尼松龙诱导斑马鱼致骨质疏松的防治作用

Preventive effect of total flavonoids of Epimedii Folium on the zebrafish osteoporosis model induced by prednisolone

目的:利用泼尼松龙诱导的斑马鱼骨质疏松模型评价淫羊藿总黄酮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不同浓度泼尼松龙(1,10,25μmol·L-1)模型药溶液,得出泼尼松龙诱导斑马鱼骨质疏松的最佳剂量,以及含25μmol·L-1泼尼松龙的不同浓度淫羊藿总黄酮(0.12,0.6,3,6μg·ml-1)溶液,含25μmol·L-1泼尼松龙的15μg·ml-1依替膦酸二钠阳性药溶液,0.5%DMSO溶媒对照溶液和培养基阴性对照溶液中,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DMSO溶媒和培养基阴性对照组相比,25μmol·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,15μg·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,6μg·ml-1淫羊藿总黄酮能显著提高斑马鱼头部骨骼的染色面积和染色光密度值。结论:淫羊藿总黄酮能对抗泼尼松龙诱导的斑马鱼骨质疏松,同时表明该模型可成功用于评价淫羊藿总黄酮抗骨质疏松活性。

OBJECTIVE To explore the anti osteoporosis effect of total flavonoids of Epimedii Folium（EFs）by using prednis olone-induced zebrafish osteoporosis model. METHODS Zebrafish larvae were exposured to a series of prednisolone solutions with a range of concentration （1,111 and 25 μmol·L-1 ） at 4 days post fertilization （d. p. f. ）. The 25 μmol·L-1 prednisolone was selected as model group. Zebrafish larvae were co administered with 25 μmol·L-1 prednisolone and a series of EFs solu- tions with a range of concentration （0. 12,0. 6,3,6μg·ml-1） at 4 d. p. f. , 15 μg·ml-1 etidronate disodium with 25 μmol·L-1 prednisolone as positive group, 0. 5% DMSO was selected 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 d. p. f.. Zebrafish skeleton at 9 d. p. f. were stained with alizarin red. Quantitative analysis of the stained area was performed by microscopic inspection and digital imaging methods to reflect the amount of zebrafish head skeleton mineral area. RESULTS The head skeleton mineral area and integrated optical density（ IOD） of 25μmol·L-1 prednisolone model group were significantly decreased when compared with vehicle control group and negative control group and 15μg·ml-1 etidronate disodium could increase the mineralized matrix of zebrafish head skeleton when compared with model group, and prevented osteoporosis induced by prednisolone. The head skeleton mineral area and IOD of 6 μg·ml-1 EFs groups were significantly increased when compared with model group. CONCLUSION The results showed that EFs can prevent zebrafish osteoporosis induced by prednisolone, in addition, this model can be successfully used for the evaluation of anti osteoporosis activity of EFs.