The Role Of TSLP And Its Regulation In Airway Inflammation Of TDI-induced Asthma

?Objective? 1.The method of triggering the tracheal instillation was used to improve the model of TDI asthma mouse model,and the establishment of the model was evaluated.2.To observe the expression of ROR-?t,Foxp3,IL-17 A and TSLP m RNA and the methylation of promoter region in TDI asthma,and to explore the role of TSLP and Th17/Treg cell balance in the pathogenesis of TDI.3.To observe the therapeutic effect of TSLP neutralizing antibody intervention on TDI asthma and the interaction between TSLP and Th17/Treg cell balance,and to explore the pathogenesis of TDI asthma.?Methods? 1.Establishment of animal model BALB/c mice aged 8-10 weeks of 20 with SPF were selected and randomly divided into TDI group(TDI group)and solvent control group(AOO group)for 1 week.TDI mice were incubated with 20 ?L of 0.3% TDI sensitized(solvent: acetone: olive oil = 2: 3)at day 1 and day 1,and 20 ?L of 0.01% TDI was stimulated by tracheal instillation(solvent Acetone: olive oil = 1: 4).AOO group of mice,according to the above method to give the same amount of solvent.Two groups of mice were housed under the same feeding conditions and observed the general growth status of mice,measuring body weight per day.Mice were sacrificed 24 hours after the end of TDI challenge.Six mice in each group were tested for the total number of cells and cell count in bronchoalveolar lavage fluid(BALF)and the inflammatory cells of BALF were incubated with enzyme-linked immunosorbent assay(IL-4)and IFN-? levels were measured by ELISA.The hematoxylin-eosin(HE)staining was observed after partial perfusion of 4 mice.Pathological changes in lung tissue.2.Changes of TSLP expression and Th17/Treg cell balance in TDI asthma and its associated gene promoter region methylation change pattern BALB/c mice aged 8-10 weeks of 32 with SPF were selected and adapted to the environment for 1 week and then randomly divided into TDI and AOO groups.TDI of asthma mouse model was established as described above.Mice were sacrificed 24 hours after the end of TDI challenge.Six mice in each group were tested for the total number of cells and cell counts in BALF.Four groups of paraformaldehyde were perfused and fixed in part.Lung tissue was observed by HE staining.And lung histopathological changes.The expression of ROR-?t,IL-17 A,Foxp3 and TSLP m RNA and gene promoter in lung tissue of TDI group and AOO group were detected by real-time quantitative reverse transcription RT-PCR and Mass Array flight mass spectrometry,respectively Region methylation.3.The protective effect of TSLP neutralizing antibody on inflammatoryof TDI response BALB/c mice of 48 with SPF grade were randomly divided into TDI group,AOO group and TSLP neutralizing antibody intervention group(TSLP group),TDI group mice(solvent was acetone: olive oil = 2: 3),and 20 ?L of 0.01% TDI was stimulated by tracheal instillation(solvent was acetone: olives oil = 1: 4).The TSLP neutralizing group was given intraperitoneal injection of TSLP neutralizing antibody(15 mg/kg)and AOO mice 30 min before challenge,and the same amount of solvent was administered according to the above method.Three groups of mice were housed under the same feeding conditions and observed the general growth status of mice.The mice were sacrificed 24 hours after the end of TDI challenge.The serum levels of Ig E were detected in 6 mice in each group.The changes of IL-4,IFN-? and IL-13 were observed in lung homogenate The histopathological changes of trachea and lung tissues in mice were observed by HE staining in 4 mice,and the lung tissue of 6 mice in each group was treated with Western blot to expression the TSLP protein,other with real-time quantitative reverse transcription RT-PCR the m RNA levels of ROR-?t,Foxp3 and IL-17 A were detected in the lung tissue.?Results? 1.Establishment of animal model(1)After TDI sensitization challenge,10 mice in TDI group showed obvious wheezing,shortness of breath,nod breathing,arch,forelimb and other asthma symptoms,AOO group mice no obvious symptoms.Compared with the AOO group,there was no significant difference between the TDI group and the body weight before and after the experiment(P> 0.05).(2)The number,classification and smear of inflammatory cells in BALF of two groups of mice: Compared with AOO group,the total number of white blood cells,neutrophils, lymphocytes,mononuclear cells,eosinophils The percentage of neutrophils and the percentage of eosinophils were significantly increased,the percentage of lymphocytes and the percentage of mononuclear cells decreased,the difference was statistically significant(P< 0.05).Compared with AOO group,the proportion of macrophages and lymphocytes in BALF smear of TDI group increased by 4.6 times and 4 times,respectively.(3)The levels of IL-4 and IFN-? in BALF of two groups were significantly higher than those in group AOO(P<0.01).(4)Pathological changes of lungs in two groups of mice: AOO group of lung tissue bronchial cavity no exudate,the wall and the surrounding tissue without inflammatory cell infiltration,alveolar septum no thickening,no inflammatory cell infiltration,alveolar There was no exudate in the cavity of the TDI group,and the bronchial wall was damaged in the lung tissue of the TDI group.There was a lot of inflammatory cell infiltrat ion in the bronchial lumen and alveolar cavity.2.Changes of TSLP expression and Th17/Treg cell balance in TDI asthma and its associated gene promoter region methylation change pattern(1)After TDI sensitization challenge,16 mice in TDI group showed obvious wheezing,shortness of breath,nod breathing,bow,forelimb and other asthma symptoms,AOO group mice no obvious symptoms.(2)The number of inflammatory cells in BALF of two groups was compared with that of AOO group.The percentage of white blood cells,the percentage of neutrophils and the percentage of eosinophils in TDI group were increased,the percentage of lymphocytes The percentage of mononuclear cells decreased,the difference was statistically significant(P <0.01).(3)The histopathological changes of trachea and lung in the two groups were: the tracheal lumen of AOO group was intact,no exudate,no inflammatory cell infiltration,no degeneration and necrosis of mucosal epithelial cells;Narrow,thickening of the wall,smooth muscle hyperplasia,a large number of inflammatory cell infiltration,inflammatory cells were agglomerated,mucosal epithelial cell necrosis;AOO group lung tissue alveolar septum no thickening,no inflammatory cell infiltration,alveolar cavity No exudate.There was no exudate in the bronchial cavity of the lung,and no inflammatory cell infiltration was found in the wall and the surrounding tissue.The alveolar stroma of TDI group showed hyperemia and edema.Pulmonary bronchial lumen and alveolar cavity there are a lot of inflammatory cell infiltration,mainly in eosinophils,neutrophils infiltration mainly.(4)The m RNA expression levels of ROR-?t,IL-17 A,Foxp3 and TSLP m RNA in the lung tissue of the two groups were compared with those of the AOO group,and the expression of Foxp3 m RNA in the lung tissue of TDI group decreased.ROR-?t,IL-17 A and TSLP m RNA level increased,the difference was statistically significant(P<0.01).(5)Comparison of methylation levels of ROR-?t,IL-17 A,Foxp3 and TSLP gene promoter groups in lung tissue of two groups: Compared with AOO group,the degree of methylation of Cp G sites 3,4,5,6,8,11 and 12 in the promoter region of ROR-?t gene decreased in lung tissue of TDI group(P<0.05),and the degree of methylation of Cp G sites 6 and 7 in the promoter region of IL-17 A gene decreased(P<0.01),the degree of methylation of Cp G sites 1 and 10 in the promoter region of Foxp3 gene increased(P<0.01),the difference was statistically significant.But the Cp G of TSLP gene promoter region in TDI group was not significantly changed.3.The protective effect of TSLP neutralizing antibody on inflammatoryof TDI response(1)After TDI sensitization challenge,16 mice in TDI group showed obvious wheezing,shortness of breath,shortness of breath,bow,forelimb and other asthmatic symptoms.TSLP neutral group had lighter asthma symptoms than TDI group.AOO group mice had no obvious symptoms.(2)The levels of Ig E in serum,IFN-?,IL-4 and IL-13 in lung tissue homogenate of three groups were different.Compared with AOO group,there was no significant difference in IFN-? and IL-4 between lung tissue homogenate(P>0.05),but the levels of Ig E in serum and IL-13 in lung tissue in TSLPgroup were increased(Z=-2.324,-2.324)(P<0.05).The levels of Ig E in serum,IFN-?,IL-4 and IL-13 in lung tissue in TDI group were significantly higher than those in control group(Z=-2.882,-2.882,-2.882,-2.882)(P<0.05).Compared with TSLP group,the levels of Ig E in serum,IFN-?,IL-4 and IL-13 in lung homogenate of TDI group were increased(Z=-2.324,-2.324,-2.324,-2.324)there was significant difference(P<0.05).(3)Three groups of mice trachea,lung pathological examination results: AOO group of mice tracheal lumen integrity,no exudate,no inflammatory cell infiltration,mucosal epithelial cells without degeneration and necrosis;TDI group tracheal lumen changes Narrow,thickening of the wall,smooth muscle hyperplasia,a large number of inflammatory cell infiltration,inflammatory cells were agglomerated,mucosal epithelial cell necrosis;relative to the TDI group,TSLP group of mice can significantly improve the tracheal infiltration of inflammation,Smooth muscle hyperplasia;AOO group of lung tissue alveolar septum no thickening,no inflammatory cell infiltration,alveolar cavity no exudate.There was no exudate in the bronchial cavity of the lung,and no inflammatory cell infiltration was found in the wall and the surrounding tissue.The alveolar stroma of TDI group showed hyperemia and edema.Pulmonary bronchial lumen and alveolar cavity there are a lot of inflammatory cell infiltration,mainly in eosinophils,neutrophils infiltration mainly.TSLP neutralization group compared with the TDI group,alveolar stroma congestion and edema,pulmonary bronchial lumen and alveolar cavity inflammatory cells within the degree of infiltration.(4)Western blot was used to detect the expression of TSLP in the lungs of three groups.Compared with the AOO group,the relative expression of TSLP level in TDI group was significantly higher than that in the control group,and the level of TSLP was significantly decreased after the neutralization antibody intervention.(P<0.05).(5)The expression of ROR-?t,IL-17 A and Fox P3 m RNA in the lung tissue of the three groups were different.Compared with the AOO group,there was no significant difference in the expression of Fox P3 m RNA in lung tissue(P>0.05).The expression of ROR-?t,IL-17 A m RNA in lung tissue was significantly higher in TSLP group(Z=-2.324,-2.324)(P<0.05)and TDIgroup(Z=-2.882,-2.882)(P<0.01).Compared with TSLP group,there was no significant difference in the expression of Fox P3 m RNA in lung tissue(P>0.05),but the expression of ROR-?t,IL-17 A m RNA was significantly higher in TDIgroup(Z=-2.324,-2.324)(P<0.05).?Conclusions? 1.TDI asthmatic mouse model was improved by the method of tracheal instillation,which ensured the stability and reproducibility of TDI asthma model.2.The imbalance of TSLP and Th17/Treg cells involved in the pathogenesis of TDI asthma.The methylation abnormalities of ROR-?t,IL-17 A and Foxp3 gene promoter regions may be involved in the regulation of Th17/Treg cell imbalance.3.After the intervention of TSLP neutralizing antibody,the symptoms of asthma in mice were relieved obviously,the expression of IL-17 A and ROR-?t m RNA in the intervention group was significantly lower than that in the TDI group,which indicated that TSLP could be used as a target for TDI asthma treatment,And this therapeutic effect may play a role by inhibiting the expression of IL-17.