基于斑马鱼毒/效联合评价的强骨胶囊抗骨质疏松活性与安全性研究

Anti-osteoporotic activity and safety of Strong Bone Capsules based on joint evaluation of toxicity and effect on zebrafish

目的用斑马鱼毒/效联合模型评价强骨胶囊(骨碎补总黄酮)抗骨质疏松活性与初步安全性。方法将受精后3 d的斑马鱼幼鱼暴露在对照组(0.4%DMSO胚胎培养用水),泼尼松龙组,依替膦酸二钠组和强骨胶囊组溶液中培养至9 d。采用茜素红对斑马鱼幼鱼头部骨骼染色,定量分析骨骼染色区域;将受精后24 h的健康斑马鱼胚胎置于不同浓度药物组中,观察记录给药后1至9 d的死亡数和3 d的斑马鱼幼鱼形态。结果 25μmol/L泼尼松龙能够成功诱导斑马鱼骨量显著丢失。与模型组比较,25 mg/L的强骨胶囊及30 mg/L依替膦酸二钠阳性药均能显著抑制泼尼松龙诱导的斑马鱼头部骨骼染色面积和累计光密度值的下降;强骨胶囊对于9 d斑马鱼的半数致死质量浓度LC50为529.87 mg/L,400 mg/L强骨胶囊溶液处理48 h斑马鱼未见明显脏器中毒,当质量浓度大于500 mg/L强骨胶囊均导致斑马鱼心脏中毒,出现心膜出血、血细胞在心区堆积及囊肿等现象。其最低中毒浓度为有效剂量浓度的20倍。结论斑马鱼毒/效联合模型成功评价了强骨胶囊的抗骨质疏松活性及其初步安全性。

AIM To use zebrafish to evaluate the preliminary safety and anti-osteoporosis activity of Strong Bone Capsules（total flavones from Drynaria fortunei（Kunze） J. Sm）. METHODS Zebrafish larvae were coexposed with the vehicle control group（0. 4% DMSO）,prednisolone group,etidronate disodium group,and different concentrations of Strong Bone Capsules with 25 μmol/L prednisolone from 3 to 9 days post fertilization（dpf）. Zebrafish skeletons at 9 dpf were fixed for staining with alizarin red. Quantitative analysis of the stained area was performed. Zebrafish embryos 24 hours post fertilization（hpf） were exposed with various concentrations of Strong Bone Capsules; embryonic morphology of zebrafish（3 dpf） was examined and the death number of the embryos was counted from 2 to 9 dpf. RESULTS The results indicated that 25 μmol/L prednisolone could make zebrafish bone loose significantly,and when compared with the model group,25 mg/L Strong Bone Capsules and etidronate disodium could restrain the decline of the mineralized area and integrated optical density（IOD） in the head bone. The lethal concentration of 50%（LC50） of Strong Bone Capusles to zebrafish（9 dpf） was 529. 87 mg/L; embryos（48 hpf） exposure to 400 mg/L Strong Bone Capsules did not show obvious organ toxicity,while cardiac toxicity of the zebrafish embryo appeared when the concentration was higher than 500 mg/L,such as endocardium bleeding,aggregation of blood cells in the chambers as well as swelling of the heart. The minimum toxic concentration was 20 times of the effective dose. CONCLUSION The zebrafish model is successfully used in evaluating anti-osteoporosis activity and preliminary safety of Strong Bone Capsules.