Regulation Of JAK2/STAT5/Foxp3 Signaling Pathway On Secretion Of Inflammatory Factors In Jurkat T Cells Induced By Traffic-related PM_2.5 And Different Components

Objective:To study the effects of traffic-related Fine particulate matter(PM_2.5)and its different component extracts on the secretion of inflammatory mediators in Jurkat T cells,and the roles of STAT5 and Foxp3 gene methylation in regulating JAK2/STAT5/Foxp3 signaling pathway in cell inflammation,so as to provide scientific reference for enriching the immunotoxicity caused by PM_2.5and its different components.Methods:Using JAK2 inhibitors（AG490）to intervene in Jurkat T cells,and randomly divided the cells into normal saline group（NS）,PM_2.5whole particle group(PM_2.5),PM_2.5water-soluble group（WSI）,normal saline+AG490 group（NS+AG490）,PM_2.5whole particle+AG490 group(PM_2.5+AG490),PM_2.5water-soluble component+AG490 group（WSI+AG490）,DMSO group（DMSO）,PM_2.5organic component group（OE）,DMSO+AG490 group（DMSO+AG490）,PM_2.5organic component+AG490 group（OE+AG490）.After PM_2.5,its water-soluble components and organic components were exposed for 24h,RNA was extracted,and the gene transcription of JAK2,STAT5 and Foxp3 in cells was determined by q RT-PCR method.The cell protein was extracted,and the protein of JAK2,p-JAK2,Foxp3,STAT5,p-STAT5 was determined by Western blot（WB）method.In addition,the protein of p-JAK2,p-STAT5 and Foxp3 was determined by immunofluorescence method.Determination of TGF-β、IL-10 content in Cell Culture Supernatant by ELISA Kit.The percentage of Treg cells was detected by flow cytometry.Plasmid STAT5 was transfected into Jurkat T cells,and Jurkat T cells were randomly divided into empty plasmid control group,plasmid control group,PM_2.5and its different components+empty plasmid exposure group,and PM_2.5and its different components+plasmid exposure group.After 24 hours of exposure,relevant experimental indicators in Jurkat T cells were detected using q RT-PCR and WB methods.Methylation inhibitor（5-Aza）intervened Jurkat T cells and randomly divided the cells into normal saline group（NS）,PM_2.5whole particle group(PM_2.5),PM_2.5water-soluble group（WSI）,normal saline+5-Aza group（NS+5-Aza）,PM_2.5whole particle+5-Aza group(PM_2.5+5-Aza),PM_2.5water-soluble component+5-Aza group（WSI+5-Aza）,DMSO group（DMSO）,PM_2.5organic component group（OE）,DMSO+5-Aza group（DMSO+5-Aza）,PM_2.5organic component+5-Aza group（OE+5-Aza）.After PM_2.5,its water-soluble components and organic components were treated for 24h,the relevant experimental indicators in Jurkat T cells were detected by q RT-PCR and WB.Pyrophosphate sequencing was used to detect the methylation level of CPG island in STAT5 and Foxp3 gene promoter regions.Determination of TGF-β、IL-10 content in Cell Culture Supernatant by ELISA Kit.Results:1.（1）Compared with the control group,the expression levels of JAK2 and p-JAK2 protein in cells exposed to PM_2.5,its water-soluble components and organic components were significantly higher（P<0.05）,and the expression levels of STAT5,p-STAT5 and Foxp3 protein were significantly lower（P<0.05）;After the intervention of AG490,the expression levels of JAK2 and p-JAK2 protein in cells decreased significantly（P<0.05）,and the expression levels of STAT5,p-STAT5 and Foxp3 protein increased significantly（P<0.05）.（2）Compared with the control group,the expression of JAK2 gene in cells exposed to PM_2.5,its water-soluble components and organic components increased significantly（P<0.05）,and the expression of STAT5 and Foxp3 genes decreased significantly（P<0.05）;After AG490 intervention,JAK2 gene expression level was significantly reduced（P<0.05）,STAT5,Foxp3 gene expression level was significantly increased（P<0.05）.（3）Compared with the control group,the contents of TGF-βand IL-10 in the supernatant of cell culture decreased after exposured to PM_2.5,its water-soluble components and organic components（P<0.05）;After the intervention of AG490,the contents of TGF-βand IL-10 increased（P<0.05）.（4）After treatment with PM_2.5and its water-soluble components,the percentage of Treg cells decreased significantly compared with the control group（P<0.05）;AG490 significantly increased the percentage of Treg cells after intervention.After STAT5 plasmid transfection,the expression levels of STAT5,Foxp3 gene and protein were significantly higher than those of the empty group（P<0.05）.2.Compared with NS or DMSO control group,PM_2.5and its water-soluble and organic components can cause a decrease in the expression of STAT5 and Foxp3 genes and proteins in cells（P<0.05）.After 5-Aza treatment,the expression of STAT5 and Foxp3 genes and proteins is significantly increased（P<0.05）.Both PM_2.5and its water-soluble and organic components can cause an increase in the methylation level of the Foxp3 gene site in cells（P<0.05）.After 5-Aza treatment,the methylation level of the STAT5 and Foxp3 gene sites significantly decreased（P<0.05）.PM_2.5and its water-soluble and organic components in cell culture supernatant TGF-β、IL-10 level decreased（P<0.05）,after 5-Aza intervention,TGF-βThe content increased（P<0.05）.Conclusions:1.Traffic-related PM_2.5and different components aggravate inflammatory response in Jurkat T cells;2.JAK2/STAT5/Foxp3 signaling pathway promotes the secretion of inflammatory cytokines in Jurkat T cells induced by traffic-related PM_2.5and different components;3.Traffic-related PM_2.5and different components could reduce the proportion of Treg cells and the contents of TGF-βand IL-10 in Jurkat T cells;4.Methylation of STAT5 and Foxp3 genes is involved in the regulation of STAT5-Foxp3gene and protein expression in Jurkat T cells caused by traffic-related PM_2.5and different components.