Study On Differential Effects Of Ozone And Fine Particulate Matter Exposure On Methylation Modification Of RNA M~6A

Recently,China has made great achievements in controlling air pollution,but the problem of air pollution still cannot be ignored.The synergistic prevention and control of ozone（O₃）and fine particulate matter(PM_2.5)have become the key to air pollution control.O₃ and PM_2.5 are harmful to the cardiovascular system as they enter the body through the respiratory system.They weaken the defense function of the respiratory tract and cardiovascular system,then result in impaired lung function and an increased risk of cardiovascular diseases,including bronchial asthma,chronic obstructive pulmonary disease,emphysema,ischemic heart disease,stroke,thrombosis,and etc.Epidemiological studies have shown that both lead to approximately the same cardiopulmonary health outcomes.However,O₃ and PM_2.5 have different physicochemical properties,as PM_2.5 is a solid-liquid mixture with a small particle size,while O₃ is a strongly oxidizing gaseous pollutant.Therefore,the biological processes leading to diseases and the exposure-response relationships of key biological indicators should be different between the two pollutants.As an important bridge between environmental factors and genomic information,epigenetic modification has become a key method to study the regulatory mechanisms of adverse health outcomes caused by air pollutants.In eukaryotic cells,N⁶-methyladenosine（m⁶A）is the most abundant and commonly occurring post-transcriptional modification mode.This modification mode affects several types of RNA,including m RNA,r RNA,t RNA and micro RNA.The regulation of RNA m⁶A methylation modification is dynamic and reversible,and it is regulated by various enzymes.It plays a crucial role in regulating downstream molecular events and biological functions such as RNA splicing,transport,stability,and translation at the post-transcriptional level.Recently,numerous studies have demonstrated that exposure to PM_2.5 may can lead to the onset or exacerbation of lung diseases by modifying RNA m⁶A.Our research team has discovered that exposure to PM_2.5and O₃ may also be related to RNA m⁶A modification.To gain a deeper understanding of the regulatory mechanism,this paper continued to explore the impact of PM_2.5 and O₃ exposure on RNA m⁶A methylation and their differences based on previous studies,providing important clues for exploring the biological mechanism of air pollutants affecting human health.Human type II lung epithelial cell A549 and SD rat were selected as the study object,and different O₃ concentrations and time were set for exposure.Trypan blue staining,HE staining,q PCR,ELISA methods were used to preliminarily explore the effects of O₃ exposure on RNA m⁶A methylation and its potential regulatory pathway;explore the differences between PM_2.5 and O₃ on the methylation of RNA m⁶A.The main results are as follows:（1）Cell level.O₃ exposure resulted in decreased cell survival,significantly increased RNA m⁶A levels,significantly increased RNA methyltransferase METTL3,METTL14 and WTAP expression levels,significantly decreased RNA demethylase FTO and ALKBH5 at gene expression levels,and no significant changes in protein levels.TGF-βand Smad2 expression levels were significantly increased,while Smad3expression did not change significantly.（2）Animal level.O₃exposure caused significant lesions in the lung,the l,significantly increased RNA m⁶A levels,significantly increased the expression level of RNA methyltransferase METTL3,METTL14 and WTAP,decreased the expression level of RNA demethylase FTO,ALKBH5,and not changed TGF-βand downstream key factors Smad2 and Smad3 expression levels.（3）Combined with the previous results of the PM_2.5 study,we found that at the cell level,both O₃ and PM_2.5 resulted in a significant increase in RNA m⁶A levels,RNA methyltransferase and TGF-β.The difference is that Smad2 expression levels were significantly increased after O₃ exposure,and Smad3 expression levels were significantly increased after PM_2.5 exposure;at the animal level,both O₃ and PM_2.5exposure resulted in a significant increase in RNA m⁶A levels.However,RNA demethylase FTO and ALKBH5 gene expressions were significantly down-regulated after O₃ exposure,and RNA methyltransferases METTL3,METTL14,WTAP and key factors in the TGF-β/Smad signaling pathway were significantly up-regulated after PM_2.5 exposure.In conclusion,O₃ exposure mainly affects RNA m⁶A modification by changing the expression level of RNA demethylase,while PM_2.5 affects RNA m⁶A by changing the expression level of methyltransferase;PM_2.5 changes the expression of TGF-βto regulate the interaction between downstream factors Smad2/3 and RNA methyltransferase complex,and then regulate RNA m⁶A methylation modification,while the effect of O₃ on TGF-βsignaling pathway is not robust and needs to be further explored.