Effect Of PM2.5 On Respiratory Microecology Of Rats And Induction Of Cell Cycle Arrest

ObjectiveIn recent years,along with rapid industrial development,the problem of air pollution has become more serious,and smog weather has frequently occurred.The concentration of air fine particles is closely related to the occurrence of haze weather.Therefore,research on fine particles such as PM2.5(Particulate Matter 2.5)in the air has become a hot spot.An this study,we collected fine particles with PM2.5 diameter in the atmosphere,prepared PM2.5 aerosolized Wistar rat model and PM2.5 suspension to treat human lung epithelial cells H1299,H292 cell model,and studied PM2.5 pairs of rat upper respiratory flora composition changes and human lung epithelial cells due to PM2.5 caused by cycle arrest.High-throughput sequencing was used to detect the difference of upper respiratory tract flora in Wistar rats before and after dusting by Western blotting,and cyclinDl,TS and mTOR in lung tissue,H1299 and H292 cells were detected by high-throughput sequencing,P70S6K1 expression level.The corresponding indicators were used to investigate the effects of PM2.5 on the composition of upper respiratory flora and the induction of cell cycle arrest in human lung epithelial cells.Methods1.Collect the fiber filter paper of PM2.5 and prepare a concentration(750 ?g/m3)of PM2.5 suspension to expose the Wistar rat model according to the national environmental quality secondary standard 10 times.2.16S rDNA was used to detect the changes of oropharyngeal flora in rats at different time points(2W,4W)before and after dusting.3.Cell cycle changes of two passage cell lines(human lung epithelial H292 and H1299 cells)before and after PM2.5 treatment were detected by flow cytometry.4.Western blots were used to detect the expression of TS,cyclinDl,mTOR and P70S6K1 proteins in the lung tissues of the two cell lines and after 2 and 4 weeks of exposure.5.Transient transfection:Transfection of H292 cells with TS siRNA inhibitor to inhibit the expression of TS molecules6.Statistical analysis:The first part of the 16S rDNA high-throughput sequencing results,using R Studio software to complete the statistical analysis of the results,the heterogeneity of respiratory flora using cluster analysis;P value calculation based on Wilcoxon rank sum test,and FDR correction(P<0.05),the difference was statistically significant.The second part of the results were analyzed by SPSS 22.0 software,and the statistical significance was determined by analysis of variance(P<0.05),which was considered statistically significant.Results1.The high-throughput sequencing of 16S rDNA showed that the abundance and diversity of respiratory bacteria groups changed with the increase of dust time in rats,which reduced the dominant bacteria in the respiratory flora and the abundance of conditional pathogens,increase.2.After treatment with H2.5 and H1299 cells for 48 h,the cell cycle arrest of H292 and H1299 cells was in S phase,and the expression of cyclinD1 was down-regulated(P<0.05).When the concentration of PM2.5 was 200 ?g/mL,the percentage of cells in S phase reached 50.35%and 56.20%,respectively.To analyze PM2.5-induced lung injury,exposure to Wistar rat experiments was performed.The results showed that consistent with the cell exposure experiment,the expression of cyclinDl protein was down-regulated in the PM2.5 exposed group(P<0.05).3.TS protein expression was down-regulated with PM2.5 treatment of H292 and H1299 cells(P<0.05).After exposure to Wistar rats in vivo,the expression of TS protein in the exposed tissues was also down-regulated(P<0.05).4.Inhibition of TS molecule expression in H292 cells resulted in more pronounced cell cycle arrest and downregulation of cyclin D1 protein expression after PM2.5 exposure(P<0.05).5.The expression of p-mTOR and p-P70S6K1 was down-regulated in H1299 and H292 cells with increasing PM2.5 concentration(P<0.05).Wistar rats were exposed in vivo to prolong the exposure time(2,4 weeks),and the expression of p-mTOR and p-P70S6K1 was significantly down-regulated(P<0.05).Conclusion1.Exposure to PM2.5 changed the abundance and diversity of the flora in the respiratory tract,breaking the balance of respiratory flora.2.After PM2.5 treatment,the lung tissue biosynthesis of rats was impaired,and the cell cycle arrest of human H292 and H1299 was induced.The mTOR/P70S6K1 signaling pathway plays a key role in its mediated biological effects,and it is speculated that TS is Targets that regulate the cell cycle process after PM2.5 exposure.