| Objective:In recent years,the exposure of some public health events has made the harm of environmental pollutants to human health a growing concern.Phthalate esters(PAEs)are one of the most widely used plasticizers at present,while dibutyl phthalate(DBP),as a classical diester compound in the PAEs family,is widely exposed to the environment with the progress of human life and production.And the exposure routes are diversified.Some studies have shown that DBP can cause toxicity to human digestive system,endocrine system,nervous system,reproductive system,urinary system and other systems,and ultimately induce the occurrence of related diseases.Moreover,DBP can cross the placental barrier and cause fetal dysplasia through maternal exposure.Based on the national conditions of rapid industrialization in China and the extensive exposure of DBP and the diverse characteristics of damage to organisms,people are increasingly concerned about the potential hazards of DBP to human body.Although DBP has been proved to cause multi-system damage to the human body,at present,the toxicity of DBP to blood vessels still remains in the data tracking of epidemiological investigations and simple in vitro experiments,and the number is extremely scarce.There is no relevant in vivo experimental study on the harm of DBP to blood vessels and the possibility of its mechanism.Therefore,we take zebrafish as the model animal to simulate the process of fetal development after DBP exposure.To observe the changes in the vascular development stage of DBP,and preliminarily clarify its mechanism of action,so as to provide experimental basis for revealing the vascular system toxicity of DBP.Methods:1.Zebrafish of vascular green transgenic fish TG(FLK1: GFP)and wild-type(WT)AB were fed to adulthood and their embryos were obtained;2.The zebras with wild-type(WT)AB were exposed to fish DBP environment,and the mortality of zebrafish at different concentrations was obtained and the death curve was obtained,and the appropriate low,medium and high concentration groups were set according to the death curve;3.Observe the changes of DBP on the general physiological indexes of zebrafish development after exposure of wild AB zebrafish according to concentration gradient;4.Use the vascular green transgenic fish TG(FLK1: GFP)for concentration gradient exposure,observe its influence on vascular development and count the changes of related indexes;5.The alkaline phosphatase staining method was used to verify whether the vascular development changes of wild-type AB zebrafish were consistent with those of vascular transgenic fish;6.According to the time window of vascular development,different time nodes were selected for DBP exposure,and the sensitive period of zebrafish to DBP was observed and the changes of relevant indicators were counted;7.Zebrafish is selectively evacuated from the exposed environment for a period of time after DBP exposure,and whether vascular development is saved through the acquired compensation mechanism of the body is observed and statistically analyzed;8.using qRT-PCR technology to detect the expression of vascular development related genes;9.Detection of oxidative stress expression in zebrafish by using DCFH-DA fluorescent probe and enzyme activity kit;10.Targeted attempt rescue experiment with small molecular reagents;11.Statistical methods: All data were analyzed by GraphPad Prism8.0 software,and each parallel data was expressed as the average standard deviation of at least three independent experiments.Two-way ANOVA and T-test were used to compare the differences between the two groups to evaluate the statistical differences between the control group and the treatment group.P<0.05 represented by * is statistically significant,P<0.01 represented by * * is extremely significant,P<0.001 represented by * * is supersignificant,and the error bar represents ± SE.Results:1.DBP exposure has developmental toxicity on zebrafishAfter DBP exposure to zebrafish,compared with the control group,heart rate of zebrafish decreased,body length became shorter,and egg yolk absorption area decreased,which indicated that the development of zebrafish after DBP exposure was affected.2.DBP exposure has adverse effects on the intestinal vasculature of zebrafishAfter the zebrafish was exposed according to the concentration gradient,the use of vascular transgenic fish could significantly observe the intestinal vascular development errors of zebrafish,which were manifested as increased sprouting of abnormal blood vessels,less area of blood vessels under the intestine,and abnormal morphology of blood vessels under the intestine.The results of alkaline phosphatase staining with wild-type zebrafish were consistent with those of the transgenic fish,and two-way validation showed that DBP could produce adverse effects on the blood vessels under the intestine of zebrafish.3.Effects of 3.DBP exposure on the sub-intestinal blood vessels of zebra fish run through the whole development time windowThe exposure was conducted in different stages and at different times according to the development time window of blood vessels under zebra fish intestine.The results showed that compared with the control group,DBP could induce developmental errors of blood vessels under zebra fish intestine in early,middle and late stages of the development time window.Proving that zebrafish blood vessels are highly sensitive to the DBP holder.At the same time,the phenotype caused by early exposure and late exposure was relatively alleviated compared with that caused by concentration comparison,proving that the adverse effects of DBP on blood vessels may be caused by affecting the development of blood vessels and it has played a role in the early stage.4.The vascular toxicity of 4.DBP exposure to zebrafish cannot be saved through acquired compensationTo determine whether the vascular developmental toxicity of DBP exposure in zebrafish could be reversed by the acquired compensation mechanism of the body,a drug withdrawal experiment was conducted.After drug withdrawal,it was observed that the DBP-induced incorrect pattern of vascular development in the gut of zebra fish could not be reversed through the body’s compensation.5.DBP changes the expression of vascular development related genesAfter DBP exposure,there were changes in gene expression related to vascular development when compared with controls.Specific manifestations include:increased expression of VEGF pathway-related genes,increased expression of HIF-αpathway-related genes,decreased expression of BMP pathway-related genes,and decreased expression of NOTCH pathway-related genes.6.DBP can increase the level of oxidative stress in zebrafishAfter detection with fluorescent probes and related enzyme activity kits,it was observed that compared with the control group,DBP exposure caused a significant increase in oxidative stress level,as well as a significant decrease in the activities of catalase and superoxide dismutase in zebrafish.7.Astaxanthin and ZM-306416 can salvage DBP-induced zebrafish vascular toxicityA rescue experiment of zebrafish after DBP exposure was conducted using ZM-306416 an inhibitor of the antioxidants astaxanthin and VEGF.The results showed that the antioxidants astaxanthin and ZM-306416 could,to a certain extent,save the outcome of sub-intestinal vascular development in zebra fish.Conclusion:1.DBP exposure can induce ontogeny disorder of zebrafish embryos2.2.DBP has vascular development toxicity,and can cause vascular development abnormalities under the intestine of zebra fish.3.DBP may exert vascular development toxicity by enhancing the oxidative stress level in zebrafish,inducing the activation of HIF-α pathway and interfering with the correct expression of downstream vascular development-related pathways. |
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