冰片对斑马鱼胚胎发育的安全性评价

Safety Evaluation of Borneol in the Development of Zebrafish Embryos

目的:研究冰片对斑马鱼胚胎发育的影响。方法:以斑马鱼胚胎为模型,二甲基亚砜为助溶剂,制备冰片溶液(33.6、67.1、134.3、268.5、537.0、1 074.0μmol/L,即冰片①、②、③、④、⑤、⑥组),并且设空白对照组,以受精后小时(hpf)为时间段,对3hpf胚胎进行暴露染毒处理,每24 h更换1次冰片溶液,分别在8、24、48、72、96 hpf时间点以显微镜观察班马鱼胚胎发育形态,测定24 hpf斑马鱼胚胎自主抽动次数、48 hpf心率、孵化率、畸形率和死亡率。实时荧光定量聚合酶链反应法检测斑马鱼胚胎Sepn1基因表达的情况。结果:与空白对照组比较,冰片④、⑤、⑥组斑马鱼胚胎48 hpf出现脊柱弯曲、心囊水肿、卵黄囊水肿,游泳异常的毒性症状;冰片②、③、④、⑤、⑥组斑马鱼胚胎24 hpf自动抽动次数减少;冰片④、⑤、⑥组斑马鱼胚胎24 hpf心率减少;在48 hpf时,冰片①、②、③、④、⑤、⑥组斑马鱼胚胎孵化率降低;在72 hpf时,冰片②、③、④、⑤、⑥组斑马鱼胚胎孵化率降低;在96 hpf时,冰片⑤、⑥组斑马鱼胚胎孵化率降低;冰片④、⑤、⑥组斑马鱼胚胎96 hpf畸形率增加;24 hpf后,冰片④、⑤、⑥组斑马鱼胚胎死亡率降低;冰片④组Sepn1 mRNA表达增强,差异有统计学意义(P<0.01或P<0.05)。结论:高剂量冰片对斑马鱼胚胎发育有明显影响,且呈现一定的时间依赖性。建议婴幼儿、孕妇和哺乳期的妇女长时间或大剂量使用含冰片的药物均应谨慎。

OBJECTIVE： To study the effects of bomeol on the development of zebrafish embryos. METHODS： Dimethyl sulf- oxide （DMSO） as solubilizing agent was used to prepare borneol solution （33.6,67.1,134.3,268.5,537.0, 1 074.0 pmol/L, boneol ①、②、③、④、⑤、⑥group）. Control group was established. With zebrafish embryos as a model, embryos were exposed to borneol solutions 3 hours post fertilization （hpf） ; the drug solution was replaced every 24 h. The morphology of embryos was observed un- der microscope at 8, 24, 48, 72 and 96 hpf. 24 hpf spontaneous movements, 48hpf heart rate, hatching rate, embryo deformity rate and mortality rate were detected. The expression of Sepnl gene was detected with real-time quantitative PCR technique. RE- SULTS ： The times of 24 hpf spontaneous movements was 0. Spinal curvature, perlcardial edema, yolk sac edema, abnormal swim- ming were observed at 48hpf; there was statsitical signifcance between 2 groups（P〈O.O1）. 48hpf heart beats of zebrafish was de- creased; there was statistical significance between 2 groups（P〈0.05）. When the concentration of borneol was 1 074 μmol/L, hatch- ing rate of zebrafish decreased at 96 hpf; there was statistical significance （P〈0.01）. When the concentration of borneol was〉 268.5 μmol/L, deformity rate of zebrafish increased at 96 hpf; there was statistical significance（P〈0.01 ）. The mortality showed time-and concentration-dependent relationship. The expression of Sepnl gene in borneol groups increased after expose to bomeol; there was statistical significance btween 2 groups（P〈0.05）. CONCLUSIONS： High-dose borneol has marked effect on the develop- ment of zebrafish embryos in a time-and concentration-dependent manner. It is forbidden for infants, pregnant and lactation women using bomeol or the preparation of borneol for long periods or high-dose. Compared with normal control group, Spinal curvature, pericardial edema, yolk sac edema, abnormal swimming at 48 hpf were observed in borneol④、⑤、⑥group, heart beats of zebrafish was decreased at 24 hpf in borneol ④、⑤、⑥ group;hatching rate of zebrafish decreased at 96 hpf in borneol⑤、⑥group;deformity rate of zebrafish increased at 96 hpf in borneol ④、⑤、⑥group; after 24 hpf, rate of embryonic death decreased in borneol④、⑤、⑥group,the expression of Sepnl mRNA increased in bomeol④group,here was statistical significance btween 2 groups（P〈0.01,P〈 0.05）. CONCLUSIONS： Bomeol had marked effect on the development of zebrafish embryos and present a time-and concentra- tion-dependent relationship. It is forbidden for infants, pregnant and lactation women using borneol or the preparation of borneol for long periods of time or high dose.