背景：前期研究发现，补肾调肝方能明显改善高龄骨质疏松症患者的疼痛和中医临床症状，疗效肯定，但缺乏相关的实验依据。

目的：探讨补肾调肝方的安全性及其对泼尼松龙诱导骨质疏松的治疗作用。

方法：选取受精后5 h的鱼卵分到24孔板中，每孔20枚，将不同浓度的补肾调肝方(生药质量浓度10，50，100，250，500 mg/L)分别加入到相应的孔，分别于受精后24，48，72，96 h观察胚胎的发育情况，并统计胚胎累计存活率及孵化率以评价补肾调肝方的安全性；将受精后3 d的斑马鱼卵分为对照组(0.5%二甲基亚砜)、泼尼松龙模型组(25 μmol/L)、阿仑膦酸钠组(阳性药物)、补肾调肝方(生药质量浓度 10，50，100，250，500 mg/L)组，每组40条，每天换液至第8天后处死，用茜素红对各组斑马鱼幼鱼骨骼染色，进行显微检测、数码成像，并用图像软件定量分析骨骼染色区域来评价补肾调肝方抗骨质疏松的作用。

结果与结论：①低浓度补肾调肝方对胚胎存活率及孵化率无影响，而高浓度补肾调肝方可降低胚胎存活率及孵化率；②25 μmol/L泼尼松龙能成功诱导斑马鱼骨量显著丢失；③与模型组比较，50，100，250 mg/L的补肾调肝方可对抗泼尼松龙诱导的斑马鱼骨质疏松，并呈浓度依赖性；低质量浓度无明显的抗骨质疏松作用，高质量浓度抗骨质疏松作用呈下降趋势；④结果表明，25 μmol/L泼尼松龙能成功诱导斑马鱼骨质疏松模型的建立；成功评价了补肾调肝方的安全性，补肾调肝方在50 mg/L开始出现抗骨质疏松作用，在250 mg/L具有最为显著的抗骨质疏松作用。

BACKGROUND:Our previous studies have found thatBushen Tiaoganprescription can significantly reduce pain and improve the clinical symptoms of traditional Chinese medicine in elderly patients with clinical effect is positive, but there is no relevant experimental evidence.

OBJECTIVE:To explore the safety ofBushen Tiaoganprescription and its therapeutic effect on prednisolone-induced osteoporosis.

METHODS:The zebrafish juveniles 5 hours after fertilization were evenly distributed into 24-well plates at a density of 20 per concentrations ofBushen Tiaoganprescription (crude drug concentrations 10, 50, 100, 250, 500 mg/L) were added to the corresponding development of the embryos was observed at 24, 48, 72, 96 hours after fertilization, and the cumulative survival rate and hatching rate were used to evaluate the safety ofBushen zebrafish juveniles 3 days after fertilization were randomly divided into blank control group (0.5% dimethyl sulfoxide), 25 μmol/L prednisolone model group, alendronate sodium positive drug group andBushen Tiaoganprescription (10, 50, 100, 250, 500 mg/L) groups, with 40 in each culture fluid was changed every day and executed at the juveniles were dyed using alizarin red for microscopic detection and digital imaging and the bone staining area was quantitatively analyzed by image software to evaluate the anti-osteoporosis activity ofBushen Tiaoganprescription.

RESULTS AND CONCLUSION:The low-concentrationBushen Tiaoganprescription had no effect on embryo survival rate and hatching rate,while the high-concentrationBushen Tiaoganprescription could reduce embryo survival rate and hatching , 25 μmol/L, was able to successfully induce significant loss of bone mass in with the model group, 50, 100 and 250 mg/LBushen Tiaoganprescription could resist prednisolone-induced zebrafish osteoporosis in a concentration-dependent low concentrations,there was no significant anti-osteoporosis effect, and at high concentrations, anti-osteoporosis effect showed a downward findings indicate that 25 μmol/L prednisolone successfully induces the establishment of zebrafish osteoporosis model, and successfully evaluates the initial safety ofBushen Tiaoganprescription begins to show an anti-osteoporosis effect at 50 mg/L,and has the most significant anti-osteoporosis effect at 250 mg/L.

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文题释义：

斑马鱼骨质疏松模型：斑马鱼具有体型小、易养殖、成本低、发育迅速、产卵量大、对外界环境反应迅速、基因组与人类同源性高等特点而被广泛应用于科研领域。泼尼松龙诱导的斑马鱼骨质疏松模型具有简单、高效、高灵敏及低成本的优势，可克服哺乳动物实验强度大、耗时长、低灵敏、用样量大，以及细胞模型条件要求苛刻、作用环节单一等缺陷。该模型有望突破中药体内抗骨质疏松活性高效评价的制约，为早期、快速评价中药及其复方的抗骨质疏松活性提供思路与方法。

补肾调肝方：为自拟方，临床上应用多年，能明显改善高龄骨质疏松症患者疼痛症状和中医临床症状，方中包括骨碎补、狗脊、白芍、柴胡、郁金、当归、玫瑰花、川楝子、川芎、白术、合欢皮、菊花、石菖蒲、甘草，全方共奏补肾壮骨、疏肝解郁、理气止痛之功。

文章来源：《云南中医中药杂志》 网址: http://www.ynzyzyzz.cn/qikandaodu/2020/0722/383.html