清热活血方对斑马鱼胚胎血管新生的影响及安全性  

Study on Effects of Qingre Huoxue Recipe (清热活血方) on Angiogenesis in Zebrafish Embryos and Its Safety

作　　者：王海霞 智恺 马志强 武子英 刘立玲 燕美彤 果彤 曹炜 WANG Haixia;ZHI Kai;MA Zhiqiang;WU Ziying;LIU Liling;YAN Meitong;GUO Tong;CAOWei(Guang'anmen Hospital,China Academy of Chinese Medical Sciences,Beijing,100053;China Academy of Chinese Medical Sciences;Beijing University of Chinese Medicine)

机构地区：[1]中国中医科学院广安门医院 [2]北京中医药大学

出　　处：《中医杂志》2020年第1期74-78,共5页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81373592)

摘　　要：目的观察清热活血方对斑马鱼胚胎血管新生的抑制作用并探讨其安全性。方法选取受精后2天的斑马鱼胚胎120个,置于12孔板中,每孔10个。分为空白组、阳性药组和清热活血方50μg/ml、100μg/ml、150μg/ml、200μg/ml组,阳性药组给予浓度为50μg/ml瓦他拉尼(PTK787)3ml,清热活血方各浓度组给予相应浓度清热活血方3ml,各组连续给药72h,每24h更换暴露液,在体式显微镜下观察统计斑马鱼胚胎体节间血管指数及镜下荧光点数。通过预实验确定清热活血方最大非致死浓度(MNLD)为802μg/ml和最小全致死浓度(MTLD)为2560μg/ml,在此区间设定8个药物浓度,建立致死曲线,计算半数致死浓度(LD50)。选用1/10 MNLD(80μg/ml)、1/3 MNLD(267μg/ml)、MNLD 3个浓度作为毒性指标观察的给药浓度,给予药物暴露液3ml,连续给药72h,显微镜下计数斑马鱼胚胎心率,并记录各浓度清热活血方对各个脏器系统及其他表型指标产生的影响,包括斑马鱼胚胎卵黄、肠腔、肠褶皱、尾鳍等。结果与空白组比较,阳性药组及清热活血方100μg/ml、150μg/ml、200μg/ml组斑马鱼胚胎体节间血管指数及镜下荧光点数均降低(P<0.01)。清热活血方100μg/ml组、150μg/ml组、200μg/ml组之间比较,随着中药浓度的增加斑马鱼胚胎体节间血管指数显著降低(P<0.05),而各组斑马鱼胚胎镜下荧光点数仅有减少趋势而差异无统计学意义(P>0.05)。根据清热活血方致死曲线可得LD50为1200μg/ml,安全指数为5.1。与空白浓度比较,清热活血方267μg/ml、802μg/ml浓度干预时斑马鱼胚胎心率显著增快(P<0.01),80μg/ml浓度对斑马鱼胚胎心率影响不明显(P>0.05),267μg/ml浓度与802μg/ml浓度对斑马鱼胚胎心率增快的程度差异无统计学意义(P>0.05)。当浓度高于267μg/ml时,清热活血方对斑马鱼胚胎的发育表现出实质性的毒性作用,如肠褶皱消失、肠变性等。结论浓度为100μg/ml的清热活血方对�Objective To observe the inhibitory effect of Qingre Huoxue Recipe(清热活血方)on angiogenesis of zebrafish embryos and to explore its safety.Methods A total of 120 zebrafish embryos2 days after fertilization were selected and placed in 12-well plates with 10 in each well.They were divided into a blank group,a positive drug group and a Qingre Huoxue Recipe 50μg/ml,100μg/ml,150μg/ml,200μg/ml group.The positive drug group was given Vatarani(PTK787)3 ml at concentration of 50μg/ml.Each concentration group of Qingre Huoxue Recipe was given 3 ml of the corresponding drug.All groups were continuously administered for 72 h,and the exposure solution was changed every 24 h.The zebrafish embryonic intersegmental vascular index and the number of fluorescence points under the microscope were observed and counted under a stereomicroscope.The maximum non-lethal concentration(MNLD)of Qingre Huoxue Recipe was determined to be 802μg/ml and the minimum total lethal concentration(MTLD)was 2560μg/ml through pre-experiments,and 8 drug concentrations were set in this interval to establish a lethal curve and calculate the half-lethal concentration(LD50).Three concentrations of 1/10 MNLD(80μg/ml),1/3 MNLD(267μg/ml),and MNLD were selected as the drug concentration for observation of toxicity indicators.Drug exposure solution 3 ml was administered for 72 h.The embryo heart rate of zebrafish was counted under microscope,and the effects of Qingre Huoxue Recipe on various organ systems and other phenotypic indicators were recorded,including zebrafish embryo yolk,intestinal cavity,intestinal folds,and tail fins.Results Compared with the blank group,the intersegmental vascular index and microscopic fluorescence points of the zebrafish embryos in the positive drug group and Qingre Huoxue Recipe 100μg/ml,150μg/ml,and 200μg/ml groups were decreased(P<0.01).Comparing among Qingre Huoxue Recipe 100μg/ml,150μg/ml,and 200μg/ml groups,the intersegmental vascular index of the zebrafish embryonic body decreased significantly with the

关 键 词：类风湿关节炎 清热活血方 血管新生 胚胎毒性 斑马鱼 

分 类 号：R285.5[医药卫生—中药学]