基于斑马鱼毒/效同步评价的壮骨关节丸促骨骼发育作用与初步安全性研究

Research on Bone Development Promotion and Preliminary Safety of Zhuanggu Guanjie Pill based on Synchronous Evaluation of Toxicity and Efficacy Using Zebrafish

关键词:  斑马鱼  壮骨关节丸  毒性  骨质疏松

Key words:  zebrafish  Zhuanggu Guanjie pill  toxicity  osteoporosis

摘要:

目的 用斑马鱼毒/效同步法评价壮骨关节丸的促骨骼发育作用及初步安全性。方法 将受精后3 d（3 day post fertilization，3 dpf）的斑马鱼幼鱼暴露于25 μmol·L^-1泼尼松龙（prednisolone，PN），壮骨关节丸溶液及含PN的壮骨关节丸溶液（12.5，25，50，100，200，300和400 μg·mL^-1）中，0.5% DMSO胚胎培养基为空白对照，于28℃培养至8 dpf，隔天换一半药液。采用茜素红对8 dpf的斑马鱼幼鱼头部骨骼染色，定量分析骨骼染色区域；给药期间每天同步观察鱼死亡及脏器中毒情况。结果 在8 dpf时，100 μg·mL^-1及以上浓度的壮骨关节丸健康鱼组和含PN的壮骨关节丸模型鱼组斑马鱼死亡率>80%，未进行骨骼染色。壮骨关节丸健康鱼组（12.5，25，50 μg·mL^-1）的斑马鱼头骨矿化面积和累积光密度值IOD与PN组比较显著增加（P<0.01或<0.05），而含PN的壮骨关节丸模型鱼组（12.5，25，50 μg·mL^-1）斑马鱼的头骨矿化面积或IOD结果显示阻止PN致骨丢失的趋势，但均低于健康鱼组。给予壮骨关节丸后，PN模型鱼的死亡速度和死亡率较健康鱼高，鱼死亡率与给药浓度和时间呈依赖性；PN模型鱼的6 dpf和8 dpf半数致死浓度值（63.92，63.92 μg·mL^-1）均低于健康鱼（94.66，87.84 μg·mL^-1）。结论 斑马鱼毒/效同步法快速评价了壮骨关节丸对健康鱼和模型鱼的骨骼发育作用及初步安全性。壮骨关节丸对健康鱼和模型鱼的作用及毒性有差别。

Abstract:

OBJECTIVE To evaluate bone development promotion and preliminary safety of Zhuanggu Guanjie pill based on synchronous evaluation of toxicity and efficacy using zebrafish. METHODS Zebrafish embryos at 3 day post fertilization (dpf) were exposed to prednisolone (PN) 25 μmol·L^-1, different concentration of Zhuanggu Guanjie pill and its solution with PN(12.5, 25, 50, 100, 200, 300, 400 μg·mL^-1), and embryonic medium(0.5% DMSO) as a blank control, all groups were incubated at 28℃ until 8 dpf. The medium/solution was changed half every other day. Zebrafish skeleton at 8 dpf were stained with alizarin red, and the stained area of bone was quantitative analyzed, at the same time, the death number and organ toxicity of zebrafish were also observed. RESULTS At 8 dpf, more than 80% of zebrafish which exposed in Zhuanggu Guanjie pill (healthy fish group) and its solution with PN (model fish group) at 100 μg·mL^-1 and above concentration were died without bone staining. Compared with PN group, cumulative optical density value (IOD) and skull mineralization area of Zhuanggu Guanjie pill healthy fish group(12.5, 25, 50 μg·mL^-1) increased significantly(P<0.01 or < 0.05). However, the skull mineralization area or IOD of Zhuanggu Guanjie pill solution with PN(12.5, 25 and 50 μg·mL^-1) had tendency to prevent the bone loss induced by PN, while the effect on model fish were weaker than that of healthy fish. After dosed Zhuanggu Guanjie pill, mortality speed and mortality rate of PN-treated model zebrafish were higher than that of healthy fish, the mortality rate was correlation with both drug concentration and dosed time. The LC₅₀ values at 6 dpf and 8 dpf of PN-treated model zebrafish (63.92 and 63.92 μg·mL^-1) were lower than that of healthy fish(94.66 and 87.84 μg·mL^-1). CONCLUSION The synchronous evaluation method of toxicity and efficacy using zebrafish is successfully used in evaluating bone development activity and preliminary safety of Zhuanggu Guanjie pill. And the effect and toxicity of Zhuanggu Guanjie pill on healthy fish and model fish are different.