补骨脂水煎液和醇提物对斑马鱼骨骼发育的影响及其毒性作用  

Effect of extract ethanol and water decoction of Psoralea corylifolia L.on bone development and their toxicities in zebrafish

作　　者：陈颖[1,2] 王茉[3] 宋捷[1,2] 詹扬[2] 景莉君 陈书芹 贾晓斌[1,2] 韦英杰[1,2] 

机构地区：[1]南京中医药大学第三临床医学院 [2]江苏省中医药研究院国家中医药管理局中药释药系统重点研究室 [3]中国人民解放军93038部队医院

出　　处：《中国药理学与毒理学杂志》2017年第6期661-669,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：国家科技重大专项(2017ZX09301-056);中医药行业科研专项(201507004-10);国家自然科学基金(81573833);江苏省自然科学基金(BK20141507);江苏省自然科学基金(BK2011866);江苏省六大人才高峰基金(2013-YY006);江苏省第四期"333工程"科研项目(BRA2014348)~~

摘　　要：目的研究补骨脂水煎液(WD)和醇提物(EE)促骨骼发育与安全性的差异。方法将受精后3 d(3 dpf)斑马鱼幼鱼暴露于泼尼松龙(PN)25μmol·L^(-1)和WD或EE 0.1,1,10和100 mg生药·L^(-1),以及依替膦酸二钠(ED)30 mg·L^(-1)溶液中,隔天换液至9 dpf处死。茜素红染色光学显微镜观察斑马鱼头骨骨矿化面积和骨密度,荧光定量PCR法检测9 dpf斑马鱼骨保护素(OPG)和NF-κB受体活化因子配体(RANKL)m RNA表达。另将1 dpf斑马鱼胚胎置于EE 10,20,30,35,40,50和60 mg生药·L^(-1)、WD 10,50,100,125,150,175,200和500 mg生药·L^(-1)、补骨脂素(PS)12.5,25,50,100,200和400μmol·L^(-1)及补骨脂酚(BK)1,5,10,25和50μmol·L^(-1)溶液中,光镜检测3 dpf斑马鱼胚胎形态,观察记录给药后2~9 dpf胚胎或幼鱼死亡数,SPSS软件计算4,6和9 dpf LC50;另用速率法检测6 dpf斑马幼鱼谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性。结果与模型组相比,EE 0.1 mg生药·L^(-1)和WD 1 mg生药·L^(-1)能显著增加斑马鱼头部骨骼染色面积和骨密度(P<0.01),提示增加骨矿化量;且WD和EE 1 mg生药·L^(-1)均上调OPG m RNA、下调RANKL m RNA表达水平(P<0.01),OPG/RANKL比值显著提高(P<0.01)。EE,WD,PS和BK可致斑马鱼心包、卵黄囊肿大,GOT活性降低(P<0.01),WD和PS的LC50值分别为EE和BK的4~7倍和4.3~20倍。结论 EE较WD促骨骼发育活性强,且毒性更大,提示脂溶性特征性成分可能是起效或致毒的关键成分。OBJECTIVE To investigate the bone development activity and differences in safety of ethanol extract（EE） and water decoction（WD） of Psoralea corylifolia L.efficiently. METHODS Zebrafish larvae were co-exposed to prednisolone 25 μmol·L-1and different concentrations of EE and WD（0.1,1.0, 10 and 100 mg crude drug·L-1）, and etidronate disodium（ED） 30 mg·L-1. All these groups were incubated at 28.5℃ until 9 dpf. The medium solution was changed every other day. Zebrafish skeleton at 9 dpf was stained with alizarin red and inspected under an optical microscope, in addition, the death toll and organ toxicity of zebrafish were also observed. The m RNA expression of osteoprotegrin（OPG）and receptor activator of NF-κB ligand（RANKL） in 9 dpf zebrafish were determined with fluorescence quantitative PCR. Zebrafish embryos（1 dpf） were exposed to various concentrations of EE（10, 20, 30,35, 40, 50 and 60 mg crude drug·L-1）, WD（10, 50, 100, 125, 150, 175, 200 and 500 mg crude drug·L-1）,psoralen（12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 μmol · L-1） and bakuchiol（1, 5, 10, 25 and50 μmol·L-1）. Embryonic morphology of zebrafish（3 dpf） was inspected with an optical microscope and the death toll of embryos or larvale was counted from 2 dpf to 9 dpf and LC50 was calculated. Components of EE and WD ware analyzed by HPLC method. RESULTS Both EE（0.1 mg crude drug·L-1） and WD（1.0 mg crude drug·L-1） groups could increase the staining area and optical density values of zebrafish skeleton compared with prednisolone group（P〈0.01）, indicating the increase in bone mineralization; the OPG m RNA expression in both EE and WD（1.0 mg crude drug·L-1） groups increased, while the RANKL m RNA expression decreased（P〈0.01） and the ratio of OPG/RANKL improved obviously（P〈0.01）.Embryos exposed to EE, WD, psoralen and bakuchiol showed swelling of the heart and yalk sac, and decrease in GOT. The LC50 of WD and psoralen was 58 and 521 times that EE and bakuchiol,respectivel

关 键 词：斑马鱼 补骨脂 骨骼发育 

分 类 号：R285.1[医药卫生—中药学]