Study On The Mechanism Of IL-9 Promoting EMT In PM_2.5-Induced Pulmonary Fibrosis By Activating STAT3

Objective:To explore the role of IL-9-p-STAT3-Snail/TWIST1 signal pathway in promoting EMT in PM_2.5-induced pulmonary fibrosis,and reveal the role of PM_2.5in pulmonary fibrosis and some molecular mechanisms.Methods:The study is divided into two parts.Part I:Thirty-two male SD rats were randomly divided into 4 groups（n=8 per group）,and fed in cages.Nonexposed intratracheal instillation was performed by using 0.9%normal saline（1 m L/kg.bw）or low,medium and high（1 mg/kg.bw,5 mg/kg.bw,25mg/kg.bw）doses of PM_2.5once a week for 12 weeks.The pathological changes of lung tissues were observed 24 hours after the last exposure.The level of IL-9in serum and BALF was detected by ELISA.Western blot method was used to detect the protein expression levels of IL-9,STAT3,p-STAT3,Snail,TWIST1,E-Cadherin,Vimentin,COL I andα-SMA.PartⅡ:（1）The relative survival rate of BEAS-2B cells was detected by CCK-8 reagent after 12,24 and 48 h of exposure to different concentrations of PM_2.5suspension;（2）After treating BEAS-2B cells with 0,50,100,150 and 200μg/m L PM_2.5suspension for 24 h,western blot was used to detect the protein expression levels of IL-9,STAT3,p-STAT3,Snail,TWIST1,E-Cadherin,Vimentin,COL I andα-SMA.（3）Use STAT3 inhibitor Stattic and 100μg/m L PM_2.5suspension co-treating BEAS-2B cells for 24 h,western blot was used to determine the expression levels of those proteins.（4）Using IL-9 neutralizing antibody and 100μg/m L PM_2.5suspension co-treating BEAS-2B cells for 24h,western blot was used to determine the expression levels of those proteins.Results:Part I:（1）Compared with the control group,low,medium and high doses of PM_2.5caused different degrees of inflammatory cell infiltration,widening and breaking of lung septum,alveolar fusion and alveolar structure destruction.Different degrees of collagen fiber deposition were also observed in the PM_2.5exposed groups.（2）The levels of cytokine IL-9 in serum and BALF of rats exposed to different doses of PM_2.5were higher than those of the control group in a dose-dependent manner.The results of immunohistochemistry showed that the expression of IL-9 and IL-9R in the lung tissues of rats exposed to PM_2.5was higher than that in the control group.（3）The protein expression levels of IL-9,p-STAT3,Snail,TWIST1,Vimentin,COL I andα-SMA in PM_2.5exposed groups were significantly higher than those of the control group,and the protein expression levels of E-Cadherin were significantly lower than the control group.PartⅡ:（1）The prolongation of PM_2.5exposure time and the increase of exposure dose can lead to the decline of the relative survival rate of BEAS-2B cells.（2）With the increase of PM_2.5concentration,the protein expression levels of IL-9,p-STAT3,Snail,TWIST1,Vimentin,COL I andα-SMA in cells were significantly increased compared with the control group,and the expression of E-Cadherin significantly decreased.（3）In the cells co-treated with Stattic and PM_2.5,the expression levels of those proteins were significantly decreased,and the expression of E-Cadherin protein were increased.（4）In the cells co-treated with IL-9neutralizing antibody and PM_2.5,the protein expression levels of p-STAT3,Snail,TWIST1,Vimentin,COL I andα-SMA decreased and the expression of E-Cadherin increased.Conclusion:（1）Chronic PM_2.5exposure can lead to pulmonary fibrosis in rats.（2）PM_2.5can promote the expression of IL-9,lead to the phosphorylation of STAT3,upregulate Snail and TWIST1,induce EMT in lung epithelial cells,and mediate the occurrence and development of pulmonary fibrosis.