Establishment Of A Lipid-lowering Drug Screening Platform In Zebrafish By ApoEb Knockout

BackgroundThe incidence of cardiovascular diseases in China remains high,and diseases such as hyperlipidemia,atherosclerosis,coronary heart disease and other diseases are still major threats to the health of Chinese residents.The occurrence and development of many cardiovascular diseases are based on disorders of lipid metabolism.Most studies on diseases related to these disorders use classic Apo E and LDLR knockout mice as disease models.However,the mouse model has natural shortcomings.On the one hand,when the animals are sacrificed to perform the development of the disease,the potential impact of a series of stress reactions that may exist during the sacrifice on the experimental results cannot be ruled out.On the other hand,after gene knockout and high fat diet,it is impossible to observe the behavior patterns of cells closely related to the occurrence and development of atherosclerosis in vivo after eating.In addition,the construction of mouse models is time-consuming and laborious,which is not conducive to large-scale application.Due to the advantages of transparent whole body,large breeding volume and low cost,zebrafish larvae have gradually become a new model animal for cardiovascular disease research in recent years.In this study,the Apo Eb knockout zebrafish lipid metabolism disorder model was established to provide more options for cardiovascular disease research and lipid-lowering drug research.Materials and methods1.Construction of Apo Eb knockout zebrafish lineFirstly,the expression pattern of Apo Eb gene in the early development of zebrafish larvae was detected by real-time quantitative PCR and in situ hybridization.Then,g RNAs were designed according to the reported Apo Eb gene sequence.g RNAs and Cas9 m RNA were synthesized in vitro,and microinjected into the 1-cell zebrafish fertilized eggs,and the Apo Eb gene was knocked out by the CRISPR/Cas9 system.Then,genotyping and real-time quantitative PCR detection of m RNA were used to determine the wiped out of Apo Eb expression.After mutant screening and hybridization,we finally obtained a stable genetic Apo Eb knockout zebrafish line.In addition,the early vascular development of knockout larvae was observed in vivo to rule out the potential interference ofdevelopmental defects on subsequent experimental results.2.Construction of a lipid-lowering drug screening platformRefer to how the Apo E knockout lipid metabolism disorder mice model was constructed and the methods previously reported,Apo Eb knockout zebrafish larvae were incubated with 0.1% egg yolk suspension as high-fat diet for 2 days,and then Oil Red O staining was used to determine intravascular lipid deposition.Then,1.5-month-old adult fish were fed with high-fat diet bought from manufacturer for 12 weeks,and the plasma lipid level was measured at 18-week-old.At the same time,the high-fat diet larvae were collected and homogenized.The lipid level in the homogenate was then determined.Next,in the established lipid metabolism disorder model,the lipid-lowering effect and suitable concentration of simvastatin on zebrafish larvae were also observed by Oil Red O staining and tissue homogenate lipid detection.3.The preventive effect of Xuezhikang on atherosclerosisIn the Tg(flk1:m Cherry;lyz:e GFP)transgenic fish line and the wild type fish line,the lipid-lowering drug screening platform was constructed according to the aforementioned method.When exploring the mechanism of Xuezhikang's lipid-lowering and anti-atherosclerosis effect,simvastatin was used as a positive control drug to reduce the lipid deposition in vessels.The intracellular lipid accumulation was observed by Oil Red O staining,and the behavioral patterns of intravascular neutrophils under the same conditions were observed by fluorescence microscopy,and the relationship between Xuezhikang's effect on neutrophils and lipid accumulation was explored.Results1.Construction of Apo Eb knockout zebrafish lineA zebrafish line with a large deletion of Apo Eb gene(?542 bp)was constructed.The results of real-time quantitative PCR showed that there was no more Apo Eb m RNA expression.After genotyping and hybridization,it was confirmed that the knockout of Apo Eb could be stably inherited,and obvious abnormality of the early vascular development was not seen in the mutant.2.Construction of a lipid-lowering drug screening platformApo Eb knockout larvae were given 0.1% egg yolk suspension for 2 days(egg yolk powder contains 20% crude fat and 4% cholesterol),and high-fat dry feed(contains 24%crude fat and 5% cholesterol)was given to adult fish for 12 weeks.Through these ways,stable models of lipid metabolism disorder could be successfully constructed.Tissue homogenate and plasma lipid detection showed the levels of total cholesterol,triglyceride and low-density lipoprotein cholesterol were elevated respectively.Simvastatin treatment revealed that larvae had a better survival rate with a significant lipid-lowering effect at a simvastatin concentration of 0.4 ?g/m L,which could be used as a positive control in subsequent studies.3.The preventive effect of Xuezhikang on atherosclerosisIntravascular lipid accumulation was significantly reduced after Xuezhikang treatment,and the recruitment of neutrophils in the blood vessels was significantly inhibited,suggesting that Xuezhikang may has a similar mechanism to statins,that prevents atherosclerosis through promoting lipid metabolism and inhibiting the recruitment of neutrophils.ConclusionsApo Eb knockout combined with high-fat diet can construct a model of lipid metabolism disorder in zebrafish,which can be used for large-scale lipid-lowering drug screening and pharmacology and toxicology research.Xuezhikang has a preventive effect on atherosclerosis by promoting lipid metabolism and inhibiting the recruitment of neutrophils in blood vessels.