Establishment Of Zebrafish Thrombosis Model And Its Application In Screening Of Traditional Chinese Medicine Activit

The formation of thrombus is the main reason for cardiovascular diseases,and the disease has become one of the worldwide highest lethality diseases.At the same time,the number of patients is gradually increased.Activating Blood Circulation Herbs have significant antithrombotic activity,and have been widely applied and verified in clinical practice.Screening of chemical composition with significant antithrombotic activity from traditional Chinese medicine is has become the main way to carry out new drug research and development,which has attracted widespread attention in the world.However,the characteristics of traditional Chinese medicine(such as complex of multi-chemical composition,multiple targets,etc.)and the disadvantages of traditional anti-thrombotic drug screening models[1-2]lead to the problem that the majority of traditional Chinese medicine antithrombotic material base is difficult to explain.Naturally,its mechanism of activity can’t be interpreted in modern scientific language,which makes it difficult to obtain international recognition.Therefore,the development of new thrombosis model[3]and rapid,accurate analysis method of thrombosis has great significance for anti-thrombotic activity screening and mechanism research of drugs,especially Activating Blood Circulation Herbs.The unique advantage of zeprafish makes it a new animal model.The study shows[4]that 26206 protein-coding genes have been detected in zebrafish.And the zebrafish genome has a higher specific gene compared to other mammals.Zebrafish embryo or juvenile fish is so small that can realize high-throughput screening in the porous plate.And transparent early juvenile makes it possible to intuitively observe pharmacological response of body organs under the microscope.Naturally,the experimental operation is simple.Three day-post-fertilization Zebrafish heart,liver,kidney and other major organs have fully developed,and each organ development process is clear.Therefore,the ending of experiment is clear,and experiment period is short.In cardiovascular research,the dominance of zebrafish is more pronounced.For example,the clotting factor involved in human thrombosis is almost always found in the genome sequence of zebrafish,and relative to the mammals,zebrafish model is more suitable for qualitative and quantitative analysis.Epinephrine hydrochloride is one of the commonly exploited inducer of rat and mouse thrombosis models,and the mechanism of thrombosis caused by it is clear.Based on the above reasons,this experiment is to utilize the adrenaline hydrochloride as an inducer to establish the zebrafish thrombosis model,and try to exploit this model to screen and research antithrombotic activity of Activating Blood Circulation Herbs.The main contents of the experiment include three parts.Firstly,the study trys to set up zebrafish thrombosis model induced by epinephrine hydrochloride.Then,we utilize this model to screen and research antithrombotic activity of Activating Blood Circulation Herbs.Finally,the zebrafish thrombosis model was used to evaluate the antithrombotic activity of Salvia miltiorrhiza in different habitats.The following content will separately discuss the three sections.The first part:set up zebrafish thrombosis model induced by adrenaline hydrochlorideFirst,study on the condition of zebrafish thrombosis model induced by epinephrine hydrochloride.The influences of the concentration of epinephrine hydrochloride and the time of induction on the zebrafish were investigated by single factor analysis.The concentration of the inducer and the time of induction were decided according to the result.Based on the development of zebrafish,the age of the zebrafish was selected.Orthogonal experiments were carried out to screen the optimal conditions of zebrafish thrombosis model.Degree of these models was quantitatively evaluated based on the dyeing strength of zebrafish tail vein red blood cells stained with O-dianisidine staining.This part tried to establish a stable and reliable zebrafish thrombosis model.Second,verify the zebrafish thrombosis model.We utilized antithrombotic drugs aspirin,warfarin as positive control drugs to verify this model.The platelet fluorescence transgenic zebrafish was exploited to track the aggregation of platelet fluorescence after the intervention of epinephrine hydrochloride.Lastly,the pathologic examination was utilized to further determine whether the zebrafish thrombosis model was successful.The second part:Application of zebrafish thrombosis model in screening antithrombotic activity of traditional Chinese medicineFirst,Adaptability of zebrafish thrombosis model in antithrombotic activity screening of traditional Chinese medicine The study used the zebrafish thrombosis model induced by adrenaline hydrochloride as the living screening model,and intervene the zebrafish with different levels of traditional Chinese medicine(Chinese medicine monomer composition,single herb,traditional Chinese medicine compound).Dyeing intensity of zebrafish heart red blood cells stained with O-dianisidine staining was considered as a quantitative index for evaluating antithrombotic efficacy.And we qualitatively analyzed the antithrombotic activity by observing the platelet aggregation of transgenic zebrafish with fluorescent markers to study the adaptability of zebrafish thrombosis model in antithrombotic activity screening of traditional Chinese medicine.Second,screen the antithrombotic activity of traditional Chinese medicine by zebrafish thrombosis model.The experiment selected 9 species of Activating Blood Circulation Herbs which have been studied with antithrombotic activity,and 2 kinds of Chinese medicine which have not yet been found antithrombotic effect to intervene zebrafish thrombosis model with the purpose of screening antithrombotic activity of traditional Chinese medicine.The third part:Evaluate the antithrombotic activity of Salvia miltiorrhiza in different habitats by the zebrafish thrombosis modelSelect 10 origins of salvia miltiorrhiza and prepare 75%methanol extract.The zebrafish thrombosis model was intervened with different concentrations of extract to draw amount-effect curve and select the appropriate concentration.Compare antithrombotic activity of Salvia miltiorrhiza in different producing area,and inspect the method.Results:First,Through the extreme difference analysis and variance analysis of orthogonal test,it is concluded that the optimum conditions for the zebrafish thrombosis model induced by adrenaline hydrochloride is that zebrafish at 3 dpf treated with adrenaline hydrochloride at a concentration of 15 μmol·L-1 for a time period of 16 h.Second,Compared with model,heart red blood cells of aspirin and warfarin group zebrafish was obviously enhanced.That is,the two antithrombotic agents showed significant antithrombotic activity.We found that zebrafish blood flow slowed down and platelet aggregation was significantly increased after intervened with adrenaline hydrochloride under microscope,which is consistent with the literature[5-6],At the same time,pathologic diagnosis showed that model group zebrafish tail vein erythrocyte aggregation was obviously increased,which further verify that adrenaline hydrochloride can induce the tail vein thrombosis.From the above,it can be speculated that zebrafish thrombosis model induced by adrenaline hydrochloride can better simulate the human thrombosis in the state of disease,and can be used in the research of thrombosis in vivo.Third,compared with model group,Salvianolic acid B at 100 μg·mL-1 can significantly inhibit zebrafish thrombus(P<0.01).When the water extracts of Salvia miltiorrhiza at 300μg·mL-1,95%ethanol extracts at 45 μg·mL-1,75%ethanol extracts at 40 μg·mL-1 were administrated to zebrafish embryo,there were significant differences compared with control group(P<0.01).Xiaoshuan Tongluo Tablets showed higher inhibitory activity,when the concentration was 1200 μg·mL-1.From the above results we can see that the zebrafish thrombosis model has good applicability in antithrombotic activity screening of traditional Chinese medicine.Four,in experiment of screening prevention and treatment of thrombosis of traditional Chinese medicine,9 kinds of Activating Blood Circulation Herbs showed varying degrees preventive and therapeutic effects on this zebrafish thrombosis model.However,under two experimental conditions,Glycyrrhiza uralensis Fisch and Codonopsis pilosula(Franch.)Nannf did not display the antithrombotic activity on the zebrafish thrombosis model.Five,the effects of Salvia miltiorrhiza in different habitats on the zebrafish thrombosis were different,and the inhibitory effect of Salvia miltiorrhiza from Shandong,Anhui,Henan and Sichuan on the zebrafish thrombosis was stronger,and the antithrombotic activity of Salvia miltiorrhiza in Yunnan was the least.Then,we investigated the reaction system.The results showed that the zebrafish thrombosis model induced by adrenaline hydrochloride could be used to evaluate the antithrombotic activity of Salvia miltiorrhiza in different habitats,and it was of good reliability.Conclusion:In the study,we successfully set up zebrafish thrombosis model induced by adrenaline hydrochloride.This model has good reproducibility and applicability in the screening of antithrombotic activity of traditional Chinese medicine.Therefore,this experiment provides a simple,high-throughput,and accurate thrombosis model for antithrombotic activity screening,which is helpful for the screening and research of antithrombotic activity of traditional Chinese medicine and provides a feasible way to establish the quality evaluation system of Activating Blood Circulation Herbs based on biological activity.