The Effects Of Montelukast On The Expression Of β-catenin And TGF-β1 In Experimental Rat Pulmonary Fibrosis

objective:To observe the expression changes of β-catenin and TGF-β1 in bleomycin-caused pulmonary fibrosis of rat lung tissues,as well as the impact of montelukast on their expressions.Thereby,pathogenesis of pulmonary fibrosis can be further understood,and the treatment of montelukast in pulmonary fibrosis can be further investigated to provide a theoretical basis for clinical applications of montelukast.Methods:Seventy five healthy and clean male SD rats were divided into five groups randomly,including normal saline group(NS group),bleomycin group(BLM group),and low doses of montelukast group(A1 group 5mg/kg),moderate doses of montelukast group(A2 group 10mg/kg)and high doses of montelukast group(A2 group 20mg/kg).Each group contained 15 rats.Apart from NS group,a one-time injection of NS-diluted bleomycin 0.2ml(5mg/kg)through rat tracheal to develop pulmonary fibrosis model;NS group was given the injection of NS with the same amount.After 2 days of the model development,daily gavage intervention was given to every group at same time,wherein A1,A2 and A3 group were given with montelukast of 5mg/kg,10mg/kg and 20mg/kg respectively and BLM group were given with equal amount of NS.5 rats from each group were sacrificed at 7th,14 th,and 28 th days after gavage intervention.During the experiment,behavioral performances of rats were observed;gross morphology of lung tissue was observed,and liver tissues were stored for Hematoxylin–eosin(HE)staining and Masson staining to observe alveolitis and pulmonary fibrosis changes,then severity scores were rated;bronchoalveolar lavage was performed and Bronchial Alveolar Lavage Fluid(BALF)was collected,then Enzyme-linked immunosorbent assay(ELISA)was used to detect the TGF-β1 content in Lavage fluid in rats;immunohistochemistry(IH)was conducted to detect the β-catenin expression in lung tissues in rats.Results:(1)animal general condition observation: rats in NS group were lively,their fur was bright,feeding was normal,weight was gaining,and breathing was smooth.Rats in BLM group were less lively,they were depressed and sluggish,fur was messy and dull,feeding was reduced,weight was losing,and breathing was rapid.Rats in A1 group were similar to BLM group.With the extension of the treatment time,rats in A2 and A3 group recovered their vitality,fur was messy and bright,feeding was increased,mental conditions were improved,and breathing became stable.(2)rats in NS group had normal lung tissue structure,alveoli form was complete,and no thickening of the alveolar septa,no inflammatory cell infiltration,and no deposition of collagen fibers lung parenchyma was found.At 7th day,alveolar space could see great number of inflammatory cells infiltration,alveolar septal thickening,intralesional alveolar collapse and inconspicuous pulmonary fibrosis were observed in rats alveolar cavity in BLM group;at 14 th day,alveolitis was still observable with decreased number,alveolar septal thickening and fibroblast proliferation,Fibrotic area was observable;the inflammatory cells of alveolar cavity were obviously lower at 28 th day,alveolar structure was severely damaged,large numbers of fibroblasts were appeared in interstitial lung,large numbers of collagen deposition and severe fibrosis were observed.Severity of alveolitis and pulmonary fibrosis in A1 group was similar to BLM group(p>0.05);in A2 and A3 group,Lung tissue and alveolar structure were integral,early inflammatory infiltration,advanced fibrosis area and collagen deposition level were significantly improved when compared with BLM and A1 group(p<0.05).(3)the TGF-β1 protein concentration in BALF: ELISA results: TGF-β1 concentration was the lowest in NS group,and its concentration in each group at every time point were higher obviously than NS group(P<0.05);the concentration of TGF-β1 in BALF had no significant difference between A1 group and BLM group(P>0.05).A2 and A3 groups had lesser TGF-β1 concentration at every time point than BLM group and A1 group(P<0.05);(4)β-catenin expression in lung tissue: immunohistochemistry result: β-catenin in rat lung tissue of NS group had low expression,and no significant change was observed at different time points;compared to NS group,β-catenin expressions in lung tissue from every other group were obviously increased(P<0.05);the expression of β-catenin of lung tissue had no significant difference between A1 group and BLM group(P>0.05);A2 and A3 groups had lesser β-catenin expression at different time points than BLM group and A1 group(P<0.05).(5)By the single factor linear correlation analysis,there was a positive correlation between the the degree score of Alveolar inflammation and the expression of β-catenin,at 7th,14 th,and 28 th days,r value was 0.77,0.81,0.64 respectively(p<0.05);and also there was a positive correlation relationship between the β-catenin expression and the degree score of pulmonary fibrosis,at 7th,14 th,and 28 th days,r value was 0.68,0.74,0.70 respectively(p<0.05).Conclusion:1.The success of the experiment through endotracheal perfusion BLM induced pulmonary fibrosis in rats model.2.The expression of TGF-β1 and β-catenin was high in pulmonary fibrosis formation process.3.Montelukast gavage could alleviate the alveolitis and pulmonary fibrosis in pulmonary fibrosis rats,Its mechanisms may be related to the Wnt/β-catenin signaling pathway and TGF-β1 related signaling pathway.