斑马鱼血栓模型的建立与冠心宁片的干预作用

Establishment of a zebrafish model of thrombosis and the intervention effect of Guanxinning tablet

目的采用3种诱导剂分别建立斑马鱼血栓模型,观察冠心宁片的抗血栓作用。方法分别采用1.5μmol/L苯肼、80μmol/L花生四烯酸和5 mg/L普纳替尼建立活体斑马鱼血栓模型,并用冠心宁片、氯吡格雷或阿司匹林等药物干预,观察尾静脉血栓形成情况,并用邻联茴香胺染色法对心脏红细胞染色强度进行定量分析,计算预防血栓药效。结果苯肼、花生四烯酸和普纳替尼诱导后斑马鱼尾静脉血栓明显增加和心脏红细胞染色强度显著降低(P<0.001),同时冠心宁片均能显著对抗这三种模型的血栓形成,其心脏红细胞染色强度和预防血栓药效均明显增加(P<0.001),具有一定的剂量效应关系,IC50分别为44.32、138.5和459.5 mg/L。结论苯肼、花生四烯酸和普纳替尼均能建立不同形成机理的斑马鱼血栓模型,而冠心宁片具有明显的抗血栓作用,且其抗血栓机制存在多靶点效应。

Objective To establish a zebrafish model of thrombosis induced by three kinds of inducers and observe the anti-thrombotic effect of a Chinese traditional medicine,Guanxinning tablet（ GXN）. Methods The zebrafish models of thrombosis was induced by using 1. 5 μmol / L phenyl hydrazine,80 μmol / L arachidonic acid and 5 mg / L ponatinib,respectively,and were treated with various concentration of GXN,clopidogrel or asprin. The thrombus in the tail vein was observed under microscope,Erythrocytes in the zebrafish heart were stained with o-dianisidine and the erythrocyte staining intensity was assessed with a NIS-Elements DTM image analyzer,and the anti-thrombolic effect of GXN was calculated.Results Venous thrombus was significantly increased and the staining intensities of erythrocytes in the heart were significantly decreased after induction by phenyl hydrazine,arachidonic acid or Ponatinib（ P 〈 0. 001）,respectively. At the same time,GXN showed an incresing anti-thrombolic effect in the zebrafish models（ P 〈 0. 001） in a dose-effect manner,with a IC50 of GXN of 44. 32 mg / L,138. 5 mg / L and 459. 5 mg / L,respectively. Conclusions The zebrafish models of thrombosis are successfully established by phenyl hydrazine,arachidonic acid or Ponatinib,respectively,by different formation mechanisms. GXN has been shown to have an anti-thrombosis effect,probably,by multiple target effects.