STAT3-RORγt-IL-17/IL-23 Signaling Pathway Mediates Asthma Aggravating In Rats Exposed To Traffic-related PM_2.5and Its Different Components

Objective:To observe the effects of traffic-related PM_2.5 on the methylation of gene promoters in asthmatic rats,and then influence the regulation of Th17 cells and their cytokines in asthmatic aggravation.To provide a new basis for aggravating the onset of asthma.Methods:PM_2.5 sampling was carried out along the traffic road in Yingze District of Taiyuan City from November to December 2017,and the organic and water-soluble components were extracted from the collected PM_2.5,and then configured to the corresponding concentration of the venom.Ninety SD rats were randomly divided into normal saline group（group A）,asthma group（group B）,PM_2.5 whole-particle 3 mg/kg·bw group（group C）,ovalbumin（OVA）+low water-soluble components 1.8 mg/kg·bw group（group D）,OVA+high water-soluble components 7.2 mg/kg·bw group（group E）,OVA+DMSO solvent control group（group F）,OVA+low organic components 0.6mg/kg·bw group（G group）,OVA+high organic components 2.4 mg/kg·bw group（group H）,5-Aza+OVA+high organic components 2.4 mg/kg·bw group（group I）,10rats in each group.OVA and aluminum hydroxide suspensions were intraperitoneally injected to induce sensitization,and OVA inhalation challenge was used to establish a rat asthmatic model.The water-soluble and organic components of the extracted particles were used to infuse tracheal instillation in rats exposed to the poisoned group.The sensitization,challenge,and exposure of the normal saline control group were all replaced with physiological saline,and the DMSO control group was instilled with DMSO（<0.1%）as a solvent control group.In group I,5-Aza was intraperitoneally injected 15 minutes before each exposure.The number of leukocytes in alveolar lavage fluid（BALF）was counted by a cell counter.Changes in body weight and organ coefficient were analyzed.Pathological changes in rat lungs were observed by HE staining.Pyrosquensing detection of methylation of CPG islands were used in the promoter region of STAT3 and RORγt genes in lung tissue.The relative expression levels of STAT3,P-STAT3 and RORγt in lung tissue were detected by Western Blot.The relative expression levels of STAT3,P-STAT3 and RORγt in lung tissue were detected by immunohistochemistry（IHC）.The percentage of peripheral blood Th17cells in CD4⁺T lymphocytes was detected by Flow cytometry.The levels of IL-23 and IL-17 in BALF were detected by enzyme-linked immunosorbent assay（ELISA）.Results:1.The total number of cells and the percentage of eosinophils（Eos）in the alveolar lavage fluid（BALF）of the asthmatic group were higher in C,E,and H groups than in A,B and their low-dose groups,while the PM_2.5 water-soluble components mainly caused the increase of the percentage of neutrophils in BALF（P<0.05）,but there was no significant difference in the organic components exposure group（P>0.05）;the organic components exposure group caused the basophilicity in BALF.The percentage of granulocytes increased（P<0.05）,but there was no significant difference between the water-soluble components（P>0.05）,both components caused a decrease in the percentage of macrophages in BALF（P<0.05）.2.The mean value of STAT3 gene methylation in lung tissue of rats in group B,C,D,E and H was lower than that in group A.The mean value of STAT3 gene methylation in lung tissue of group I was higher than that in group H（P<0.05）.The methylation of RORγt gene in rat lung tissue showed that the degree of methylation of pos.1-9 in CPG islands decreased with the increase of PM_2.5 and its components,and the methylation rate of pos.1-7,9 in H group was significantly lower than that in the C group（P<0.05）.3.The expression of STAT3 in each asthmatic model group was higher than that in group A.The expression of STAT3 in group E,H and I was higher than that in group B and C（P<0.05）.The expression of P-STAT3 protein in group C,E,H and I was significantly higher than that in group A and B,and there was a dose-effect relationship in each group（P<0.05）.The expression of RORγt protein in group C,E and H was significantly higher in group A and group B.The expression of RORγt protein in group H was significantly higher than that in group F,G and I（P<0.05）.The results of immunohistochemistry were consistent with those of Western Blot.P-STAT3 protein was mainly expressed in the nucleus of pulmonary bronchial epithelial cells,while RORγt was expressed in cytoplasm.4.The ratio of Th17 cells in CD4⁺T cells in E and H groups were higher than that in group A,B,D and F groups（P<0.05）.The Th17 cells of group I was lower than that of H group（P<0.05）.5.The expression of IL-17 in BALF of group C,E and H were higher than that of group A.The expression of IL-17 in group E and H were higher than that in group D and F,G and I（P<0.05）.The expression of IL-23 in BALF of group C,E and H were higher than that of group A,and the expression of IL-23 in group C was higher than that of group B（P<0.05）.6.The methylation rate of STAT3/P-STAT3 gene CPG islands at the pos.1 and pos.6 in H and I groups were negatively correlated with STAT3/P-STAT3 protein expression,and the lung tissue RORγt gene CPG island pos.3-5,pos.7,pos.10 were negatively correlated with the expression of RORγt protein（P<0.05）.In the other groups,the methylation rate of STAT3 gene CPG islands in the lung of rats were negatively correlated with STAT3 protein expression.P-STAT3 gene CPG islands were methylated at pos.1,pos.3,pos.6.There was also a negative correlation between protein expression and gene methylation.Besides,the pos.1-12 of CPG island of RORγt gene in lung tissue were negatively correlated with the expression of RORγt protein（P<0.05）.Conclusion:PM_2.5 particles and its water-soluble and organic components can activate the STAT3-RORγt-IL-17/IL-23 signaling pathway to aggravate asthma attacks.STAT3-RORγt regulates its protein expression through gene methylation,and it can promote the differentiation of Th17 cells and release IL-17 and IL-23,thereby aggravating asthma attacks.