藁本内酯抗药物诱导的斑马鱼血栓的作用研究

The Antithrombotic Effect of Ligustilide on Chemical-Induced Zebrafish Thrombosis Model

目的旨在采用苯肼(phenyl hydrazine,PH)和盐酸肾上腺素(adrenaline hydrochloride,AH)诱导稳定的斑马鱼血栓模型,并研究藁本内酯(ligustilide,LIG)的抗血栓作用。方法采用正交实验设计方法优化PH及AH诱导斑马鱼血栓模型建立的条件,包括诱导剂浓度、诱导时间、染色时间、鱼龄4个方面;将斑马鱼随机分为空白对照组、诱导剂组(PH/AH处理)、阳性对照组(PH/AH+阿司匹林处理)和给药组(PH/AH+20/40/60μmol/L LIG)进行给药处理。对于实验结果的判断,主要通过在进行给药处理后,用邻联茴香胺染色,并用Image-Pro Plus 6.0计算尾部染色面积的方法,定量分析血栓形成情况。结果苯肼的最优诱导条件为浓度0.75μmol/L,诱导时间24 h,染色时间35 min,诱导时鱼龄2 dpf(days post-fertilization);盐酸肾上腺素的最优诱导条件为浓度15μmol/L,诱导时间16 h,染色时间30 min,诱导时鱼龄4 dpf。LIG对斑马鱼血栓形成有一定的抑制作用,最佳浓度在20~40μmol/L范围时,可以从尾静脉处观察、检测到血栓生成显著减少。结论LIG在斑马鱼体内具有一定的抗血栓作用,最佳浓度为20~40μmol/L。

Objective This study aimed to induce a stable zebrafish thrombosis model with Phenyl Hydrazine(PH)and Adrenaline Hydrochloride(AH),and to study the antithrombotic effects of Ligustilide(LIG)with this model.Methods Parameters of PH/AH induced zebrafish thrombosis model including PH/AH concentration,treatment duration,staining time,and days post-fertilization(dpf)at the time of induction were optimized by orthogonal experimental design.The zebrafish were randomly divided into different treatment groups:blank control group,PH/AH treatment group(PH/AH treatment),positive control group(PH/AH+aspirin treatment),and LIG treatment groups(PH/AH+20/40/60μmol/L LIG treatment).The thrombosis was evaluated by calculating the total area of the red spots of the zebrafish tail with Image-Pro Plus 6.0 after o-Dianisidine Dihydrochloride staining,and the result was achieved by comparing the data in each group using statistical analysis.Results The optimized parameters were as follows:for PH induced zebrafish thrombosis model,the PH concentration was 0.75μmol/L;the treatment duration was 24 h;the staining time was 35 min;and the dpf was 2 at the time of induction.For AH induced zebrafish thrombosis model,the AH concentration was 15μmol/L;the treatment duration was 16 h;the staining time was 30 min;and the dpf was 4 at the time of induction.LIG exhibited a certain degree of inhibitory effect on zebrafish thrombosis at the concentration of 20~40μmol/L,the decrease of thrombosis could be significantly observed and determined from the tail vein.Conclusion LIG has a certain antithrombotic effect in zebrafish at the concentration of 20~40μmol/L.