| Objectives:To investigate the effect of FTY-720 on bleomycin-induced pulmonary fibrosis in mice and its mechanism of action.Methods:Sixty healthy male Balb/c mice of specific pathogen-free grade were randomly divided into control group(control group),bleomycin group(BLM group),saline+FTY-720 control group(NS+FTY-720 group),and bleomycin+FTY-720 treatment group(BLM+FTY-720),with 5 mice in each group,and three batches were housed in parallel,and the mice were sacrificed on days 7,14,and 28 of treatment.HE and Masson staining were used to observe the changes of inflammation and fibrosis in lung tissues of mice,respectively;and the inflammation score,fibrosis score and lung index were calculated;the white blood cells in BALF were stained and counted by Swiss-Apodemsa staining solution;the total protein content in BALF supernatant was determined by BCA method;the expression levels of inflammatory factors IL-I p,IL-6 and TNF-α in BALF supernatant were determined by ELISA;the positive expression levels of COL1A1 in lung tissues were detected by immunohistochemistry;and the expression levels of COL1A1,MMP9,TGF-β1,p-TA K1 and p-P38 MAPK in the whole lung homogenates of mice in each group were detected by Western blot.Results:1.Pathological changes of lung tissue:After HE staining,the structure of lung tissue in the control group was basically normal;compared with the control group,the structure of lung tissue in the BLM group was disorganized and inflammatory cells were infiltrated on the 7th day,the fibrous exudation of lung tissue was observed on the 14th day,while the inflammatory cell infiltration was improved,the alveolar septum of lung tissue was significantly thickened on the 28th day,and a large number of fibrous tissue was formed in the alveolar interstitium;after FTY-720 treatment,the above changes were relieved;Masson staining showed that BLM had a large number of blue collagen fibers and pink cellulose exudation from the 14th day;after FTY-720 treatment,the blue fibrous tissue was reduced.2.Alveolar inflammation and pulmonary fibrosis score:compared with the control group,the alveolar inflammation score in the lung tissue of mice on the 7th day was the highest(p<0.01),and the alveolar inflammation induced by FTY-720 injection was significantly reduced(p<0.05);The degree of pulmonary fibrosis was significantly reduced(p<0.01).3.Lung index:The lung index of BLM group was significantly higher than that of control group(p<0.05),while the lung index of mice decreased significantly after FTY-720 treatment(p<0.05).4.Protein content and white blood cell count in BALF:The protein content in BALF of BLM group mice was significantly higher than that of control group(p<0.01),and FTY-720 significantly decreased the protein content in BALF of BLM-induced mice(p<0.01);the white blood cell count of BLM model group mice reached the peak value on the 7th day(p<0.01),and the white blood cell count of model group gradually decreased on the following 14 and 28 days,while the white blood cell count was significantly decreased after FTY-720 administration(p<0.01).5.Determination of IL-1 β,IL-6 and TNF-α:Compared with the control group,the cytokines IL-1β,IL-6 and TNF-α in BALF of mice in the model group were significantly increased at 7 days,14 days and 28 days(p<0.01),and FTY-720 decreased the contents of IL-1β,IL-6 and TNF-α(p<0.01).6.Expression of COL1A1:Immunohistochemical results showed that COL1A1 expression was observed in the lung tissues of mice in the model group at 7 days,14 days,and 28 days,and the positive area of COL1A1 expression was significantly reduced after treatment with FTY-720.7.TGF-β1 expression levels:TGF-β1 expression was significantly increased in the model group at 7 days,14 days,and 28 days compared with the control group(p<0.01),and FTY-720 could reduce TGF-β1 protein expression levels(p<0.01).8.Expression of COL1A1,MMP9:The expression of COL1A1 and MMP9 in the lung tissue of mice in the model group was significantly increased compared with the control group at 7 days,14 days,and 28 days,and FTY-720 decreased COL1A1 and MMP9 protein expression levels.9.The expression of p-TAK1 and p-P38 MAPK:The expression of p-TAK1 and p-P38 MAPK in the model at 7 days,14 days and 28 days was significantly increased compared with the control group,and FTY-720 decreased the expression levels of β-TAK1 and p-P38 MAPK protein.Conclusions:1.FTY-720 has a protective and antifibrotic effect on bleomycin-induced pulmonary fibrosis in mice;2.FTY-720 inhibits the development of pulmonary fibrosis by inhibiting the TGF-β1/TAK1/P38 MAPK signaling pathway. |
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