补肾调肝方逆转泼尼松龙诱导的斑马鱼骨质疏松

Bushen Tiaogan prescription reverses prednisone-induced osteoporosis in zebrafish

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(25μmol/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;②25μmol/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,25μmol/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 that Bushen Tiaogan prescription can significantly reduce pain and improve the clinical symptoms of traditional Chinese medicine in elderly patients with osteoporosis.The clinical effect is positive,but there is no relevant experimental evidence.OBJECTIVE:To explore the safety of Bushen Tiaogan prescription 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 well.Different concentrations of Bushen Tiaogan prescription(crude drug concentrations 10,50,100,250,500 mg/L)were added to the corresponding wells.The 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 of Bushen Tiaogan prescription.The zebrafish juveniles 3 days after fertilization were randomly divided into blank control group(0.5%dimethyl sulfoxide),25μmol/L prednisolone model group,alendronate sodium positive drug group and Bushen Tiaogan prescription(10,50,100,250,500 mg/L)groups,with 40 in each group.The culture fluid was changed every day and executed at the 8th day.Zebrafish 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 of Bushen Tiaogan prescription.RESULTS AND CONCLUSION:The low-concentration Bushen Tiaogan prescription had no effect on embryo survival rate and hatching rate,while the high-concentration Bushen Tiaogan prescription could reduce embryo survival rate and hatching rate.Prednisolone,25μmol/L,was able to successfully induce significant loss of bone mass in zebrafish.Compared with the model group,50,100 and 250 mg/L Bushen Tiaogan prescription could resist prednisolone-induced zebrafish osteoporosis in a concentration-dependent manner.At low concentrations,there was no significant anti-osteoporosis effect,and at high concentrations,anti-osteoporosis effect showed a downward trend.These findings indicate that 25μmol/L prednisolone successfully induces the establishment of zebrafish osteoporosis model,and successfully evaluates the initial safety of Bushen Tiaogan prescription.Bushen Tiaogan prescription begins to show an anti-osteoporosis effect at 50 mg/L,and has the most significant anti-osteoporosis effect at 250 mg/L.