Developmental Toxicity Evaluation Of Atmospheric Particulates And Prevent Mist Haze Active Ingredients Screening Study

Objective: 1.To investigate the general developmental toxicity of atmospheric particulates to zebrafish embryos/larvae,and to compare the general toxicity of different particle sizes of atmospheric particulates(TSP,PM₁₀,PM_2.5).2.The developmental toxicity of atmospheric particulates on the cardiovascular system of zebrafish embryos/larvae was studied by using zebrafish as a model organism,and the mechanism of its toxicity was discussed.3.In this paper,the neurodevelopmental toxicity of atmospheric particulates to zebrafish embryos/larvae was studied by using zebrafish as a model organism,and the mechanism of its toxicity was discussed.4.The medicine and food homologous traditional Chinese medicine with the function of preventing haze damage was screened.Thus,the functional food with the function of preventing haze damage in the later stage was developed.Methods: 1.Study on the general developmental toxicity of zebrafish embryos/larvae caused by atmospheric particulates(TSP,PM₁₀,PM_2.5): Normally developed 4 hpf fertilized eggs?wild-type AB zebrafish?were selected under a stereo microscope.Fresh embryo culture water was used as a blank control group,and the concentrations of TSP,PM₁₀ and PM_2.5 were 25,50,100,200,and 400 ?g/m L.Each group was treated for 5 days.At 24,48,72,96,and 120 hpf,the effects of atmospheric particles on the mortality,hatching rate,deformity rate,body length and morphological changes of zebrafish embryos/larvae were observed.2.In this study,the wild-type AB strain and the Tg?cmcl2:EGFP?,Tg?flk1:EGFP?and Tg?CD41:EGFP?transgenic strains were used to study the effects of atmospheric particulates(TSP,PM₁₀,PM_2.5)on heart morphology,sinus venosus and bulbus arteriosus?SV-BA?distance,heart rate,vascular subintestinalis,blood flow,platelet fluidity and reactive oxygen species?ROS?level in zebrafish.Furthermore,expression changes in genes involved in endoplasmic reticulum stress?ERS?,Nrf2 and Wnt pathway-related genes were detected via Reverse transcription Polymerase Chain Reaction?RT-PCR?.3.This paper studied the effects of atmospheric particulates(TSP,PM₁₀,PM_2.5)on the behavior,brain waves,dopamine ganglia,neurovascular development,and cerebral hemorrhaging of zebrafish using wild-type AB zebrafish and Tg?vmat2:EGFP?and Tg?flk1:EGFP?transgenic zebrafish.Additionally,the expression of CNS-related genes was detected by reverse transcription-polymerase chain reaction?RT-PCR?.4.In this paper,18 kinds of traditional Chinese medicine with the same source of medicine and food?mulberry leaf,fruit of seabuckthorn,chrysanthemum flower,bitter apricot seed,eucommia bark,honeysuckle flower,white hyacinth bean,fruit of chinese wolfberry,root of coastal glehnia,common yam rhizome,trichosanthes kirilowii maxim,fresh ginger,bulb of greenish lily,platycodon root,seed of jobstears,indian bread,jujube chinese date,rice bean?were analyzed by network pharmacology.The target genes of each traditional Chinese medicine are predicted,and the biological processes and pathways corresponding to all predicted target genes are enriched.Then,this mechanisms that have been studied in this paper are compared and analyzed to find out the biological processes and pathways of intersection.Thus,screening out the medicine and food homologous traditional Chinese medicine that can prevent and alleviate the toxicity of haze.Results: 1.Atmospheric particulates(TSP,PM₁₀,PM_2.5)significantly increased the mortality and deformity rate of zebrafish,significantly decreased the hatching rate,and significantly shortened the body length of zebrafish.Atmospheric particulates caused embryonic developmental malformations,mainly manifested as diminished or missing swim bladder,yolk sac absorption delay,pericardial sac edema,and embryonic developmental delays.In addition,the general developmental toxicity of atmospheric particles with different particle sizes is also very different.The comparison of general developmental toxicity of atmospheric particles in this study: PM₁₀ > PM_2.5 > TSP.2.Atmospheric particulates(TSP,PM₁₀,PM_2.5)caused a significant increase in pericardial sac area and SV-BA distance,a decrease in heart rate,inhibition of vascular subintestinalis growth,blood flow obstruction,atmospheric particulates,and other cardiovascular toxicities.Atmospheric particulates induced an increase in the ROS level,and atmospheric particulates(PM₁₀)induced significant increases in the expression levels of ERS signalling pathway factors and Nrf2 signalling pathway factors.The expression levels of the Wnt pathway-related genes also showed significant changes.Furthermore,ROS inhibitor NAC could ameliorate the cardiovascular toxicity of atmospheric particulates(PM₁₀)in zebrafish larvae.3.The results showed that in zebrafish larvae,atmospheric particulates(TSP,PM₁₀,PM_2.5)caused neurotoxicity,such as reduced behavioral performance,abnormal brain waves,shortened dopamine ganglia,inhibition of neurovascular development,and cerebral hemorrhaging.And atmospheric particulates(PM₁₀)resulted in a significant decrease in the expression levels of CNS-related genes?such as th1,dat,drd1,drd2 a,drd3,drd4 b,mbp,syn2 a,?1-tubulin,and elavl3?.4.The corresponding biological processes and pathways of these 18 kinds of traditional Chinese medicine?mulberry leaf,fruit of seabuckthorn,chrysanthemum flower,bitter apricot seed,eucommia bark,honeysuckle flower,white hyacinth bean,fruit of chinese wolfberry,root of coastal glehnia,common yam rhizome,trichosanthes kirilowii maxim,fresh ginger,bulb of greenish lily,platycodon root,seed of jobstears,indian bread,jujube chinese date,rice bean?have different degrees of intersection with the toxic mechanism of atmospheric particles studied in this paper.Among them,The number of intersections of mulberry leaf,fruit of seabuckthorn,chrysanthemum flower,bitter apricot seed,eucommia bark and honeysuckle flower more than 20.The number of intersections of white hyacinth bean,fruit of chinese wolfberry,root of coastal glehnia,common yam rhizome,trichosanthes kirilowii maxim,fresh ginger,bulb of greenish lily and platycodon root are 10-20.Conclusion: 1.Atmospheric particulates(TSP,PM₁₀,PM_2.5)can cause general developmental toxicity of zebrafish embryos/larvae,and PM₁₀ is the most toxic,followed by PM_2.5,while TSP is relatively weak.These studies provide a basis for further study of the impact of atmospheric particles on environmental toxicity.2.Atmospheric particles(PM₁₀)may result in cardiovascular toxicity by inducing higher ROS levels in the body,which could then induce ERS and lead to defects in the expression of genes related to the Wnt signalling pathway.The Nrf2 signalling pathway was activated as a stress compensatory mechanism during the early stage of atmospheric particles(PM₁₀)-induced cardiovascular injury.However,it was insufficient to counteract the atmospheric particles(PM₁₀)-induced cardiovascular toxicity.Therefore,the disorder of gene expression related to ERS,Nrf2 and Wnt signaling pathway may be an important cause of cardiovascular development toxicity of zebrafish induced by atmospheric particulates.3.Atmospheric particulates(PM₁₀)may affect the development and function of the dopaminergic neurons in the CNS,inhibit synapse formation,disrupt cytoskeleton formation,and inhibit neuron differentiation in zebrafish larvae by causing disorders in the expression of CNS-related genes,thereby causing neurodevelopmental toxicity.4.The biological processes/pathways of 18 kinds of traditional Chinese medicine with the same source of medicine and food intersect with the toxic mechanism of atmospheric particulate matter studied in this paper.Through the screening of drugs and food homologous traditional Chinese medicine with anti haze functional activity,we can provide a reasonable basis for the follow-up targeted research and development of functional food with anti haze damage.