基于斑马鱼整合评价补骨脂粗多糖对补骨脂中香豆素组分的代谢及毒效关系的影响

Effects of Psoraleae Crude Plysaccharide on Metabolism and Toxicity/Efficacy Relationship of Coumarin Components from Psoraleae Fructus Based on Zebrafish Integrated Evaluation

关键词:  补骨脂  斑马鱼  粗多糖  香豆素  毒性  代谢  骨质疏松

Key words:  Psoraleae Fructus  zebrafish  crude polysaccharide  coumarin  toxicity  metabolism  osteoporosis

摘要:

目的 用斑马鱼整合评价探讨补骨脂粗多糖(psoraleae crude polysaccharide，PPS)对补骨脂中香豆素组分(coumarin components of Psoraleae Fructus，CCPF)的代谢及毒效关系影响。方法 用受精后天数(days post fertilization，dpf)1~6 dpf的斑马鱼评价CCPF、PPS及二者组合物的安全性，观察斑马鱼脏器形态，记录死亡数并计算斑马鱼半数死亡浓度(LC₅₀)；用1~6 dpf斑马鱼暴露于CCPF及其与PPS组合物，分析补骨脂苷和异补骨脂苷及其代谢物补骨脂素和异补骨脂素的动态变化；用25 μmol·L^-1泼尼松龙诱导斑马鱼骨质疏松模型，采用茜素红对培养至8 dpf的各给药组斑马鱼幼鱼骨骼染色，进行显微观察、数码成像，并用图像软件定量分析骨骼染色区域来评价上述样品的抗骨质疏松活性。结果 CCPF与PPS组合物对斑马鱼的毒性大于二者单独给药，且组合物PPS的比例增加，毒性增大：PPS能够降低斑马鱼中毒浓度，致斑马鱼脏器形态畸变严重，缩短斑马鱼死亡时间，增加斑马鱼死亡率；PPS能明显加速CCPF中补骨脂苷和异补骨脂苷脱糖基转化为潜在肝损伤代谢产物补骨脂素和异补骨脂素；CCPF及其与PPS组合物可增加斑马鱼头骨累积光密度值，且组合物具有一定增效作用。结论 用斑马鱼整合评价高效揭示了PPS对CCPF的代谢与毒效关系的影响，为研究PPS对其他结构类型成分的毒效关系提供高效的方法与思路。

Abstract:

OBJECTIVE To evaluate the effects of psoraleae crude polysaccharide(PPS) on metabolism and toxicity/efficacy relationship of coumarin components of Psoraleae Fructus(CCPF) by zebrafish integrated evaluation. METHODS Zebrafish(1-6 days post fertilization, dpf) was used to evaluate the safety of CCPF, PPS and their combination; the morphologies of zebrafish organs was observed and the number of deaths was recorded and the half death concentration of zebrafish(LC₅₀) was calculated. Zebrafish(1-6 dpf) were exposed to CCPF and its combination with PPS; the dynamic changes of psoralenoside and isopsoralenoside and their metabolites psoralen and isopsoralen were analyzed. The zebrafish osteoporosis model was induced with 25 μmol·L^-1 prednisolone; microscopic observation and digital imaging of zebrafish larvae of each treatment group cultured to 8 dpf were performed using alizarin red, and the bone staining area was quantitatively analyzed by image software to evaluate the anti-osteoporosis activity of above samples. RESULTS Evaluation of the safety of CCPF, PPS and their combination by interaction with zebrafish juveniles. The toxicity of the combination of CCPF and PPS to zebrafish was greater than that of CCPF or PPS alone, and the toxicity increased with the increase of the proportion of PPS: PPS reduced the poisoning concentration of zebrafish, causing serious morphological distortion of zebrafish organs, shortening the death time of zebrafish and increasing the death rate of zebrafish. PPS obviously accelerated the deglycosylation of psoralenoside and isopsoralenoside in CCPF into psoralen and isopsoralen, which were potential metabolites of liver injury. CCPF and its combination with PPS increased the mineralized area and cumulative optical density of zebrafish skull and the combination had a certain synergistic effect, which suggested that PPS increased the anti-osteoporosis activity of CCPF to some extent. CONCLUSION Based on the integrated evaluation of zebrafish, the effects of PPS on the metabolism and toxicity/efficacy relationship of CCPF are revealed, which provides an efficient method and idea for revealing the toxicity/efficacy relationship action of PPS on other structural components.