基于斑马鱼的香青兰总黄酮整体发育急性毒性评价研究

Study on overall developmental acute toxicity evaluation of total flavonoids of Dracocephalum moldavica L.based on zebrafish

目的利用模式生物斑马鱼研究香青兰总黄酮(TFDM)整体发育急性毒性。方法采用发育至48 h的斑马鱼暴露于5、10、20、40、42、44、46、48、50、100μg·mL^(−1)的TFDM,分别于暴露后24、48 h(24、48 hpe),计算死亡率、半数死亡浓度(LC_(50))值;测量每组斑马鱼幼鱼的体长,进行形态学观察并评分;显微镜下观察斑马鱼心脏形态并拍照,记录心率,使用Image-Pro Plus 5.1测量斑马鱼静脉窦-动脉球(SV-BA)距离;显微镜下观察各组斑马鱼是否有体侧水肿来判断TFDM对肾脏的影响并拍照;应用肝脏标记绿色荧光的转基因斑马鱼Tg(L-FABP∶EGFP),通过检测肝脏荧光强度和面积,观察TFDM对肝脏毒性的影响。结果TFDM的24 hpe LC_(50)为50μg·mL^(−1),48 hpe LC_(50)为48μg·mL^(−1),100μg·mL^(−1)TFDM组斑马鱼幼鱼全部死亡。与空白对照组比较,20μg·mL^(−1)以下的TFDM对斑马鱼的形态和心、肝、肾各脏器无影响;20μg·mL^(−1)浓度的TFDM处理斑马鱼48 h导致个别斑马鱼鱼鳔体积减小或缺失,对其他脏器无显著影响;40μg·mL^(−1)的TFDM导致斑马鱼出现轻微的心包水肿,处理48 h以后斑马鱼体长显著减小(P<0.01),形态评分显著下降(P<0.01),斑马鱼的肝脏形态出现轻微变化,但肝脏荧光强度和肝脏荧光面积无显著性变化,对肾脏无影响;暴露在50μg·mL^(−1)的TFDM中24 h,斑马鱼出现心包水肿,心率显著下降(P<0.05),肝脏荧光强度和面积显著减小(P<0.05),肾脏无明显变化。结论TFDM对斑马鱼的毒性较小,低浓度(≤10μg·mL^(−1))的TFDM对斑马鱼无毒性;中浓度(20μg·mL^(−1))下TFDM对斑马鱼的毒性微弱,仅导致部分斑马鱼鱼鳔体积减小或缺失,对其他各脏器无毒性;高浓度(≥40μg·mL^(−1)及以上)下有轻微的心脏毒性和肝脏毒性,在临床应用中有必要合理控制用量。

Objective To study the effects of total flavonoids of Dracocephalum moldavica L.(TFDM)on overall developmental acute toxicity of zebrafish.Methods Zebrafish embryos at 48 hpf(hours post fertilization)were exposed to different concentrations(5,10,20,40,42,44,46,48,50,100μg·mL^(−1))of TFDM.The mortality rate and half death concentration(LC_(50))were recorded at 24 and 48 hpe(hours post exposure).The body length of each group of juvenile zebrafish was measured,morphological observation and scoring were carried out.The heart morphology of zebrafish was observed and photographed under a microscope,and the heart rate was recorded.Image-pro Plus 5.1 was used to measure the distance between zebrafish venous sinus and arterial sphere(SV-BA).The influence of TFDM on kidney was determined by observing lateral edema of zebrafish in each group under the microscope and taking pictures.Transgenic zebrafish Tg(L-FABP∶EGFP)labeled with green fluorescence in liver was used to observe the effect of TFDM on liver toxicity by detecting the intensity and area of liver fluorescence.Results The 24 hpe L_(C50) of TFDM was 50μg·mL^(−1) and the 48 hpe LC_(50) was 48μg·mL^(−1) and all the juvenile zebrafish in TFDM 100μg·mL^(−1) group died.Compared with blank control group,TFDM under 20μg·mL^(−1) had no effect on morphology and organs of heart,liver and kidney of zebrafish.TFDM treatment with 20μg·mL^(−1) concentration for 48 h resulted in decreased or lost swim bladder volume of individual zebrafish,but had no significant effect on other organs.After treatment for 48 h,the body length and morphology score of zebrafish were significantly decreased(P<0.01),and the liver morphology of zebrafish was slightly changed,but the liver fluorescence intensity and liver fluorescence area had no significant change,and had no effect on kidney.After exposure to 50μg·mL^(−1) TFDM for 24 h,pericardium edema was observed,heart rate decreased significantly(P<0.05),liver fluorescence intensity and area decreased significantly(P<0.05),and kidney did not change significantly.Conclusion In this study,TFDM was found to be less toxic to zebrafish.At low concentration(≤10μg·mL^(−1)),TFDM had no toxicity to zebrafish.TFDM showed weak toxicity to zebrafish at medium concentration(20μg·mL^(−1)),which only reduced or lost the swim bladder of some zebrafish,and had no toxicity to other organs.Slight cardiotoxicity and hepatotoxicity were observed at high concentrations(≥40μg·mL^(−1)).Therefore,it is necessary to strengthen the management and control the dosage in clinical application.