The Neurodevelopmental Toxicity Effect And Molecular Mechanism In Response To Ultrafine Particulate Matter Exposure

In recent years,the health problems caused by air pollution have been the focus of worldwide attention and scientific research.Among these,ultrafine particles(PM_0.1,aerodynamic diameter≤100 nm)may be the main contributor to health effects.Existing studies suggest an association between PM_0.1 exposure and neurological diseases.However,the route of ingestion of particulate matter and whether it has neurodevelopmental toxic effects are unclear.Mouse embryonic stem cells（mESCs）are developmentally totipotent and can be induced to differentiate into neurons.They are used to study the early embryonic developmental toxicity and functional toxicity of various environmental pollutants.Therefore,this topic aims to elucidate the uptake behavior of ultrafine particles and their effects on neuronal development and related molecular mechanisms using embryonic stem cells.1.Given the complexity of the atmospheric environment and its pollutants,in this part of the study,quantum dots（QDs）are selected as model ultrafine particles to investigate their uptake and efflux behavior in mESCs.First,we characterized the morphology,zeta potential,hydrated particle size,spectral characteristics and stability of QDs by transmission electron microscopy（TEM）,dynamic light scattering,fluorescence spectrophotometer and inductively coupled plasma mass spectrometry（ICP-MS）.It was found that QDs were spherical particles with a particle size of about 10 nm,which were negatively charged in water and culture medium.The optimal emission peak wavelength of QDs was 646 nm,and it was not easy to release metal ions in aqueous solution;the sublethal concentration of QDs was screened by cell viability.On this basis,the uptake and efflux of QDs in mESCs were investigated using the fluorescence signals of QDs,flow cytometry and fluorescence imaging.The results showed that the uptake of QDs by mESCs was time-and dose-dependent.At the same time,QDs were excreted by the cells and some of the QDs taken up by the cells were present in the cells.2.Brain development is a complex and precisely regulated process,and the directed induction of mESCs neural differentiation provides a strong basis for studying of neural development.Therefore,this part of the study aims to establish a mESCs monolayer neural differentiation model to investigate the potential neurodevelopmental toxicity of PM_0.1.We first characterized the morphology,zeta potential and hydration particle size of PM_0.1.It was found that PM_0.1 was an irregular aggregate,its hydration particle size was larger than the TEM particle size,and it was negatively charged in water and culture medium.Based on sublethal exposure,it was found that PM_0.1 exposure induced oxidative stress and affected the self-renewal ability of mESCs.After mESCs were directed to induce neural differentiation,the effects of PM_0.1 on neuronal morphology,pluripotency,key factors of neurogenesis,intracellular Ca²⁺level and cell cycle were investigated by light microscopy,RT-q PCR and Fluo-3 AM fluorescent probe.The results showed that PM_0.1 exposure could affect the number of neurofilaments,change the morphology of neurons,induce the expression of neurogenesis related factors Map2,Dcx,Neurod1,Pax6,Snai2,Msx2 to decrease and the expression of pluripotent factor Sox2 to increase,thus inhibiting the differentiation of mESCs.At the same time,PM_0.1 exposure increased the intracellular calcium levels,inhibited cell proliferation,and blocked the G1 and S phases of cells.3.Previous research has shown that RNA methylation plays an important regulatory role in neurogenesis and neurodevelopment.In this part of the study,we investigated the molecular mechanism of PM_0.1-mediated neural differentiation abnormalities using methylated RNA immunoprecipitation sequencing（MeRIP-seq）.First,the m RNA expression of N⁶-methyladenine（m⁶A）modified methyltransferase,demethylase and recognition protein was detected by RT-q PCR after PM_0.1 exposure.It was found that PM_0.1induced an increase in the expression of methyltransferase（Mettl3,Mettl14,Wtap）,a decrease in the expression of demethyltransferase（Fto）,and an abnormal expression of recognition protein（Ythdc1,Ythdc2,Ythdf2,Hnrnpa2b1,Eif3a）.Functional enrichment analysis（GO analysis）revealed that m RNAs with differential m⁶A methylation were mainly enriched in synaptic organization,axonogenesis,dendritite development,regulation of neurogenesis,forebrain development and other neurodevelopmental processes.Further analysis of MeRIP-seq and RNA-seq showed that the change of 92 m RNA expression in PM_0.1 treatment group might be related to m⁶A modification.Through the GO enrichment analysis of these differential genes using Clue GO,it was found that the cluster with the most obvious change was the brain development process.After verification,it was found that after PM_0.1 exposure,the expression of Sall1,Pou4f1,Bcl11b,Zic1,Hoxa2,Nr2e1 might be reduced by m⁶A modification,thus affecting the neural development process.In conclusion,this study investigated the uptake and efflux behavior of ultrafine particles in mESCs,and further analyzed the potential neurodevelopmental toxicity mechanism of PM_0.1 by inducing neural differentiation model in vitro.The experimental data provided a theoretical basis for the health risk assessment of PM_0.1.