基于斑马鱼模型评价注射用益气复脉（冻干）的心血管和肝脏安全性研究

目的 应用斑马鱼模型评价注射用益气复脉（冻干）（YQFM）的心血管毒性和肝毒性。方法 受精后48 h的野生型AB系斑马鱼随机分为空白对照组（未注射组）、溶剂对照组（注射0.9%氯化钠注射液10 nL）和YQFM 10、20、100、200、400、600、800和1 000 ng组，iv给药4 h后统计各剂量组斑马鱼死亡数量，计算最大非致死剂量（MNLD）和10%致死剂量（LD₁₀）值。心血管毒性和肝毒性评价剂量设置为MNLD/10（20 ng）、MNLD/3（60 ng）、MNLD（200 ng）和LD₁₀（400 ng）；在显微镜下观察YQFM对斑马鱼心率的影响，观察斑马鱼有无心包水肿、血流速度变化、血液流失、心包瘀（出）血、血栓等，计算各表型发生率；在显微镜下对肝脏进行观察，对典型表型进行拍照，利用分析软件NIS-ElementsD 3.10分析肝脏面积、肝脏不透光度和卵黄囊面积。结果 YQFM MNLD和LD₁₀分别为198.06 ng（约为200 ng）和397.12 ng（为400 ng）；与溶剂对照组比较，YQFM 20、60 ng组斑马鱼未见异常，没有观察到心血管毒性，未见心包水肿，未见心率减慢，未见心律不齐；YQFM 200 ng组斑马鱼出现发生率较低的心血管毒性，表现为心包水肿（发生率仅为3.3%），未见心率减慢，未见心律不齐；400 ng组斑马鱼出现较轻微的心血管毒性，表现为心包水肿（发生率为13.3%）、血流变慢（发生率为6.7%）、血流缺失（发生率3.3%），未见心率减慢，未见心律不齐。与溶剂对照组比较，YQFM各剂量（20、60、200、400 ng）组斑马鱼相对肝脏大小、肝脏不透光度平均值和卵黄囊延迟吸收倍数均无统计学差异。结论 YQFM在MNLD剂量下，不诱发斑马鱼心血管毒性和肝脏毒性。

Evaluation of cardiovascular and hepatic safety of Yiqi Fumai Lyophilized Injection in zebrafish model in vivo

Objective The cardiotoxicity and hepatotoxicity of Yiqi Fumai Lyophilized Injection (YQFM) were evaluated by zebrafish model in vivo experiment. Methods At 48 h after fertilization, wild type AB zebrafish were randomly divided into blank control group (no injection group), solvent control group (injection of 0.9% sodium chloride injection 10 nL) and YQFM 10, 20, 100, 200, 400, 600, 800 and 1 000 ng groups. 4 h after iv administration, the death number of zebrafish in each dose group was counted, and the maximum non-lethal dose (MNLD) and 10% lethal dose (LD₁₀) values were calculated. The evaluation doses of cardiovascular toxicity and hepatotoxicity were set as MNLD/10 (20 ng), MNLD/3 (60 ng), MNLD (200 ng) and LD₁₀ (400 ng). The effects of YQFM on the heart rate of zebrafish were observed under a microscope, and the incidence of each phenotype was calculated. The liver was observed under a microscope and the typical phenotypes were photographed. The liver area, liver impermeability and yolk sac area were analyzed using nIS-Elements D 3.10 software. Results The MNLD and LD₁₀ of YQFM were 198.06 ng (about 200 ng) and 397.12 ng (400 ng), respectively. Compared with solvent control group, zebrafish in YQFM 20 and 60 ng groups showed no abnormalities, no cardiovascular toxicity, no pericardium edema, no slow heart rate, and no arrhythmia. 200 ng zebrafish showed low incidence of cardiovascular toxicity, such as pericardium edema (3.3%), no slow heart rate, no arrhythmia. In the 400 ng group, zebrafish showed mild cardiovascular toxicity, including pericardium edema (13.3%), hemorheology slowness (6.7%), loss of blood flow (3.3%), and no slow heart rate or arrhythmia. Compared with solvent control group, there were no significant differences in relative liver size, mean value of liver impermeability and delayed absorption ratio of yolk capsule of zebrafish in YQFM (20, 60, 200 and 400 ng) groups. Conclusion YQFM did not induce cardiotoxicity and hepatotoxicity in zebrafish at the MNLD dose.