The Mechanism Of JAK2/STAT3/RORγt Signaling Pathway And Its Gene Methylation In Inducing Jurkat T Cells To Release Inflammatory Cytokines From Traffic-Related PM_2.5 And Its Different Components

Objective:The purpose of this study was to investigate the role of JAK2/STAT3/RORγt signaling pathwaysin the regulation of traffic-related fine particulate matter(PM_2.5)and its different components on releasing inflammatory factors from Jurkat T cells,and the regulatory role of promoter region methylation of STAT3 and RORγt genes in JAK2/STAT3/RORγt signaling,so as to provide a basis for further exploring the immune damage of PM_2.5.Methods:1.Regulation of JAK2/STAT3/RORγt signaling in the induction of inflammatory cytokines by PM_2.5and its different components in Jurkat T cells.（1）PM_2.5was collected at traffic intersections in Taiyuan,from which soluble and organic components were extracted,and then configured with the required concentration of poison.Jurkat T cells were set with PBS control group（PBS）,PM_2.5low dose group(LPM_2.5),PM_2.5high dose group(HPM_2.5),the PM_2.5water-soluble components of low-dose group（WSL）,the PM_2.5water-soluble components of high-dose group（WSH）,DMSO solvent control group,the PM_2.5organic-soluble components of low-dose（OEL）,the PM_2.5organic-soluble components of high-dose（OEH）.All the groups were applied to the Jurkat T cells for 48 hours,then detect the protein expression of JAK2,p-JAK2, STAT3,p-STAT3,and RORγt by Western blot and the gene levels of JAK2,STAT3,and RORγt by RT-PCR.Measure the content of IL-17 and IL-23 in cell supernatant by enzyme-linked immunosorbent assay（ELISA）.（2）Inhibition of JAK2 expression by AG490,the Jurkat T cells were set to PBS control group（PBS）,tyrosine phosphorylation inhibitor group（AG490）,PM_2.5all particles group(PM_2.5),PM_2.5+AG490 group(PM_2.5+AG490),PM_2.5water-soluble components group（WSE）,WSE+AG490 group（WSE+AG 490）,DMSO solvent group（DMSO）,DMSO+AG490 group（DMSO+AG 490）,PM_2.5organic components group（OE）,OEH+AG490 group（OE+AG 490）,a total of 10 groups were included.The Jurkat T cells were infected for 48 h,and the protein expression levels of JAK2,p-JAK2,STAT3,p-STAT3,and RORγt were measured by Western blot,the gene expression levels of JAK2,STAT3,and RORγt measured by RT-PCR.The content of IL-17 and IL-23 in the cell supernatant were measured by ELISA.（3）Small interfering RNA（si-STAT3）was transfected into Jurkat T cells,set as:negative control NC group（si-NC）,transfection group（si-STAT3）,PM_2.5negative control group(PM_2.5+si-NC),PM_2.5transfection group(PM_2.5+si-STAT3),PM_2.5water-soluble component negative control group（WSE+si-NC）,PM_2.5water-soluble component transfection group（WSE+si-STAT3）,organic solvent control group（DMSO+si-NC）,organic solvent transfection control group（DMSO+si-STAT3）,PM_2.5organic component negative control group（OE+si-NC）,PM_2.5organic component transfection group（OE+si-STAT3）,a total of 10 groups.After 48 h acted on Jurkat T cells,the gene expression levels of STAT3 and RORγt genes were measured by RT-PCR,the protein expression levels of STAT3,p-STAT3,and RORγt were measured by Western blot,and the content of IL-17 and IL-23 in cell supernatant was measured by ELISA.2.The affect of methylation of STAT3/RORγt gene in the induction of inflammatory cytokines by traffic-related PM_2.5and its different components.Set up ten groups:PBS control（PBS）,methylation inhibitor 5-Aza（5-Aza）,PM_2.5whole particle(PM_2.5),PM_2.5methylation inhibition(PM_2.5+5-Aza),PM_2.5water soluble components（WSE）,PM_2.5water soluble component methylation inhibition（WSE+5-Aza）,DMSO solvent（DMSO）,DMSO methylation inhibition（DMSO+5-Aza）,PM_2.5organic components（OE）,OE methylation inhibitor（OE+5-Aza）.The expression level of methylation of STAT3 and RORγt in Jurkat T cells was detected by pyrosequencing,measured the protein levels of STAT3,p-STAT3,RORγt in Jurkat T cells by Western blot and the gene expression of STAT3 and RORγt measured by RT-PCR.Analyzed the relationship between the expression levels of STAT3 and RORγt methylation and gene expression,then measured the content of IL-17 and IL-23 in cell supernatants by ELISA.Results:1.Regulation of JAK2/STAT3/RORγt signaling in the induction of inflammatory cytokines by PM_2.5and its different components in Jurkat T cells.（1）With the increase of the toxic concentration of PM_2.5,PM_2.5water-soluble components and PM_2.5organic components,the Jurkat T cell activity gradually decreased.After 48 h,JAK2,STAT3,RORγt genes in LPM_2.5group were higher than PBS control group;JAK2 and STAT3 genes in WSH group were higher than PBS control group,and JAK2,STAT3,and RORγt in OEL and OEH group were higher than in DMSO control group.The expression of p-JAK2 increased in HPM_2.5,WSL and WSH,while STAT3increased in LPM_2.5,WSL and WSH,and RORγt increased significantly in PM_2.5and WSE.The IL-23 release was increased in the WSH group,and the IL-23 release was increased in the OEH group（P<0.05）.（2）After addition of JAK2 inhibitor AG490,JAK2,STAT3 and RORγt gene and protein expression in the PM_2.5+AG490 group were decreased compared with the PM_2.5group,the STAT3 and RORγt genes and the protein level of JAK2 and STAT3 in WSH group were decreased with the WSH+AG490 group,and JAK2 and STAT3 protein expression in OEH+AG490 group lower than OE group.In addition,the expression of cytokines IL-17 and IL-23 in WSH+AG 490 group were reduced,which reversed the trend of increased expression after infection.（3）After 48 h infection with PM_2.5and its related components,STAT3,RORγt gene and protein expression in PM_2.5+si-STAT3,WSE+si-STAT3,and OE+si-STAT3 were decreased compared with the respective infected groups.The expression of IL-17 in PM_2.5+si-STAT3 group,the IL-23 in WSE+si-STAT3 group,and IL-17 and IL-23 in OE+si-STAT3 were decreased compared with the respective infected groups alone.2.The affect of methylation of STAT3/RORγt gene in the induction of inflammatory cytokines by traffic-related PM_2.5and its different components.（1）The methylation levels of STAT3 and RORγt promoter regions in PM_2.5,WSE and OE groups were decreased compared with the control group,while the protein and gene expression of STAT3 and RORγt were higher than those of the control group.Methylation of the STAT3,RORγt promoter regions was inversely correlated with related protein expression,with the addition of the methylation inhibitor 5-Aza reducing protein gene expression and decreased IL-17 and IL-23 expression in the cell supernatant.（2）Methylation levels at Pos.1 of STAT3 and Pos.6 were positively correlated with STAT3 gene expression（rs:0.422,0.391,P<0.05）and Pos.2 and Pos.3（rs=-0.400,-0.0.05）;Cp G island levels of Pos.3 and Pos.5 with RORγt gene expression（rs=-0.354,-0.404,respectively,P<0.05）.Conclusion:Traffic-related PM_2.5and its different components can cause Jurkat T cell damage,and regulate the secretion of pro-inflammatory factors IL-17 and IL-23 by JAK2/STAT3/RORγt signaling pathway,and reduce the methylation level of STAT3promoter region in Jurkat T cells.It provides a scientific basis for the immune damage caused by PM_2.5and its different components.