| ObjectWe previously found that the intervention of epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)could significantly inhibit bleomycin-induced pulmonary fibrosis in mice,down-regulate the activity of EGFR/Akt signaling pathway,and make the expression of α-smooth muscle actin(α-SMA)which is the interstitial characteristic protein of epithelial-mesenchymal transition(EMT)decreased,with increased activation of Forkhead box protein o3a(Foxo3a),in the meantime.It suggests that Foxo3 a has protective effect on pulmonary fibrosis in mice,but the related downstream signaling pathways and specific mechanisms are still indistinct.In this study,bleomycin(BLM)solution(3mg/kg)was injected into trachea of C57BL/6 mice to establish model of pulmonary fibrosis,and the representative drug of EGFR-TKI,Gefitinib,was given to intervene in order to explore the changes of downstream signaling pathways,clarifying the possible mechanism of Foxo3 a in EMT in pulmonary fibrosis.Thus,we hope to find the key target of pulmonary fibrosis and provide novel ideas for the treatment of it.Methods1.The Mechanism of EGFR/Foxo3a/Snail1 Pathway in Bleomycin-induced Pulmonary Fibrosis in MiceThirty C57BL/6 mice aged 6 weeks in half genders were randomly divided into these three groups: a control group,a BLM group and a gefitinib-intervened group.After anaesthetized successfully,the mice in each group were fixed on the operation table.Then cervical pedicle skin flaps were incising longitudenally to expose trachea.The mice in the BLM group and gefitinib-intervened group were injected with 100μl bleomycin solution at a dose of 3 mg/kg via trachea.The mice in the control group were injected with equal amounts of saline via trachea.After that,the mice were rotated upright immediately to make the liquid evenly distributed in their lungs,meanwhile,mice in each group were consecutively given intragastric administration for 14 days.The mice in the gefitinib-intervened group were injected with bleomycin at a dose of 3 mg/kg via trachea and then gastrically perfused with gefitinib solution(20mg/kg gefitinib dissolved in 100 μl 5%glucose solution).The mice in the control group and BLM group were gastrically perfused with 100 μl 5% glucose solution.14 days after the treatment,all the mice were sacrificed and anatomized for collecting lung tissues.The lung of mice in each group was immersed in 4% polyformaldehyde solution.48 hours after fixation,lung tissues were removed for dehydration,paraffin embedding and sectioning,preparing for Hematoxylin-eosin(HE)staining and Masson’s trichrome staining.The method of Mikawa was used to estimate inflammatory injury score of lungs,and the method of Ashcroft was for evaluating score of fibrosis degree.The m RNA level of α-SMA,E-Cadherin,Foxo3 a,Forkhead box protein m1(Foxm1)and Snail1 were determined by RT-PCR,in the meantime,the expression of α-SMA,E-Cadherin,Foxo3 a,Foxm1,Snail1 protein as well as phosphorylation ratio of Foxo3 a in lung tissues were detected by Western Blot.2.Statistical methodsAll the experimental dada was statistically analyzed with SPSS20.0.When data accorded with test of normality,it could be showed as mean±standard deviation.All of the data was deal with test of normality and homogeneity test of variance.When samples accorded with test of normality and homogeneity test of variance,analysis of variance(ANOVA)and Bonferroni test were applicable to multiple comparisons between groups.The difference was statistically significant when P was less than 0.05.While samples conformed to test of normality but did not meet homogeneity test of variance ANOVA,Kruskal-Wallis H test of non-parametric test was used for comparisons among multiple groups.Dunn’s multiple comparative test was used comparisons between two groups.When P was less than adjusted significant level,the difference was statistically significant.Results1.The Mechanism of EGFR/Foxo3a/Snail1 Pathway in Bleomycin-induced Pulmonary Fibrosis in Mice1.1 HE staining and scores of inflammation injury degree of lung tissues in miceThe scores of inflammation injury degree of lung tissues in mice showed significant difference(F=90.087,P=0.000).In the control group,the alveolar structure of lung tissues of mice was complete and clear,with thin alveolar walls,meanwhile,inflammatory cells were seen between interstitial tissues.In the BLM group,the lung tissues of mice showed destruction of the alveolar structure,with thick alveolar walls,in the meantime,more inflammatory cells were found in alveoli,and many fibroblasts also appeared between interstitial tissues.The pathological damage degree of lung tissues in the gefitinib-intervened group was between the control group and the BLM group.Refer to Mikawa’s method,inflammation injury degree of lung tissues was estimated.The pathological scores of lung tissues of mice in each group were1.80±0.83,8.60±0.89 and 2.60±0.89 in proper order.The pathological score of lung tissues in the BLM group was higher than that in the control group(P<0.01).The pathological score of lung tissues in the gefitinib-intervened group was lower than that in the BLM group(P<0.01),but when compared with the control group,it went up(P=0.522).1.2 Masson’s trichrome staining and fibrosis scores of lung tissues in miceFibrosis scores of lung tissues in mice showed significant difference(F=38.111,P=0.000).In the control group,the alveolar structure of lung tissues of mice was clear,and no evident inflammatory cells and collagen deposition were observed.In the BLM group,the lung tissues of mice demonstrated disordered structure,with a number of inflammatory cells as well as massive collagen deposition.In the gefitinib-intervened group,although alveolar walls of mice slightly thickened with a small quantity of inflammatory cells,the alveolar structure of lung tissues kept complete basically with a small amount of blue collagen deposition.According to the method of Ashcroft,fibrosis scores of three group were evaluated.Fibrosis scores of lung tissues of mice in three group were successively 1.40±0.55,5.60±1.14 and 2.80±0.45.The fibrosis score of lung tissues in the BLM group was higher than that in the control group(P<0.01).The fibrosis score of lung tissues in the gefitinib-intervened group was lower than that in the BLM group(P<0.01),but higher than that in the control group(P<0.05).1.3 α-SMA m RNA level of lung tissues in miceα-SMA m RNA level of lung tissues in mice showed significant difference(F=15.690,P=0.001).The α-SMA m RNA level of lung tissues in the BLM group was higher than that in the control group(1.72±0.44,0.80±0.12,P<0.01).Theα-SMA m RNA level of lung tissues in the gefitinib-intervened group was decreased when compared to that in the BLM group(0.80±0.10,1.72±0.44,P<0.01),and while compared with the control group,it was also lower(0.80±0.10±,0.80±0.12,P=1.000).1.4 E-Cadherin m RNA level of lung tissues in miceE-Cadherin m RNA level of lung tissues in mice showed significant difference(F=16.665,P=0.001).The E-Cadherin m RNA level of lung tissues in the BLM group was lower than that in the control group(0.13±0.02,0.31±0.08,P<0.01),while the E-Cadherin m RNA level of lung tissues in the gefitinib-intervened group was increased compared with that in the BLM group(0.33±0.05,0.13±0.02,P<0.01).The E-Cadherin m RNA level of lung tissues in the gefitinib-intervened group was also higher than that in the control group(0.33±0.05,0.31±0.08,P=1.00).1.5 Foxo3 a m RNA level of lung tissues in miceFoxo3a m RNA level of lung tissues in mice showed no significant difference(F=0.575,P=0.582).The Foxo3 a m RNA level of lung tissues in the BLM group was lower than that in the control group(0.60±0.32,0.74±0.39,P=1.000).The Foxo3 a m RNA level of lung tissues in the gefitinib-intervened group was rising when compared to that in the BLM group(0.97±0.69,0.60±0.32,P=0.950).The Foxo3 a m RNA level of lung tissues in the gefitinib-intervened group was also higher than that in the control group(0.97±0.69,0.74±0.39,P=1.000).1.6 Foxm1 m RNA level of lung tissues in miceFoxm1 m RNA level of lung tissues in mice showed no significant difference(F=0.648,P=0.546).The Foxm1 m RNA level of lung tissues in the BLM group was higher than that in the control group((0.22±0.16,0.13±0.09,P=1.000).The Foxm1 m RNA level of lung tissues in the gefitinib-intervened group was ascending when compared to that in the BLM group(0.26±0.24,0.22±0.16,P=1.000),in the meantime,the Foxm1 m RNA level of lung tissues in the gefitinib-intervened group was higher than that in the control group as well(0.26±0.24,0.13±0.09,P=0.887).1.7 Snail1 m RNA level of lung tissues in miceSnail1 m RNA level of lung tissues in mice showed significant difference(F=33.487,P=0.000).The Snail1 m RNA level of lung tissues in the BLM group was higher than that in the control group(3.22±0.52,1.35±0.24,P<0.01).The Snail1 m RNA level of lung tissues in the gefitinib-intervened group was decreased when compared to that in the BLM group(1.15±0.38,3.22±0.52,P<0.01).The Snail1 m RNA level of lung tissues in the gefitinib-intervened group was also lower than that in the control group(1.15±0.38,1.35±0.24,P=1.000).1.8 α-SMA protein expression of lung tissues in miceα-SMA protein expression of lung tissues in mice showed significant difference(F=19.882,P=0.000).The α-SMA protein expression of lung tissues in the BLM group was higher than that in the control group(2.09±0.30,0.86±0.06,P<0.01).The α-SMA protein expression of lung tissues in the gefitinib-intervened group was reduced when compared to that in the BLM group(1.34±0.37,2.09±0.30,P<0.01),meanwhile,the α-SMA protein expression of lung tissues in the gefitinib-intervened group was higher than that in the control group(1.34±0.37,0.86±0.06,P=0.112).1.9 E-Cadherin protein expression of lung tissues in miceE-Cadherin protein expression of lung tissues in mice showed significant difference(F=8.937,P=0.007).The E-Cadherin protein expression of lung tissues in the BLM group was lower than that in the control group(0.56±0.13,1.09±0.14,P<0.01),while the E-Cadherin protein expression of lung tissues in the gefitinib-intervened group was increased compared with that in the BLM group(0.98±0.26,0.56±0.13,P<0.05).However,the E-Cadherin protein expression of lung tissues in the gefitinib-intervened group was lower than that in the control group(0.98±0.26,1.09±0.14,P=1.000).1.10 Foxo3 a protein expression of lung tissues in miceFoxo3a protein expression of lung tissues in mice showed no significant difference(F=1.130,P=0.365).The Foxo3 a protein expression of lung tissues in the BLM group was lower than that in the control group(0.62±0.18,0.78±0.17,P=0.759).The Foxo3 a protein expression of lung tissues in the gefitinib-intervened group went up when compared to that in the BLM group(0.80±0.23,0.62±0.18,P=0.612).The Foxo3 a protein expression of lung tissues in the gefitinib-intervened group was also higher than that in the control group(0.80±0.23,0.78±0.17,P=1.000).1.11 Phosphorylation ratio of Foxo3 a of lung tissues in micePhosphorylation ratio of Foxo3 a of lung tissues in mice showed significant difference(F=12.490,P=0.003).Phosphorylation ratio of Foxo3 a of lung tissues in the BLM group was higher than that in the control group(0.14±0.04,0.05±0.01,P<0.01).Phosphorylation ratio of Foxo3 a of lung tissues in the gefitinib-intervened group was lessened when compared to that in the BLM group(0.06±0.03,0.14±0.04,P<0.05),while phosphorylation ratio of Foxo3 a of lung tissues in the gefitinib-intervened group was higher than that in the control group(0.06±0.03,0.05±0.01,P=1.000).1.12 Foxm1 protein expression of lung tissues in miceFoxm1 protein expression of lung tissues in mice showed no significant difference(F=0.065,P=0.938).The Foxm1 protein expression of lung tissues in the BLM group was higher than that in the control group(1.45±0.51,1.35±0.53,P=1.000).The Foxm1 protein expression of lung tissues in the gefitinib-intervened group was ascendant when compared to that in the BLM group(1.46±0.41,1.45±0.51,P=1.000),in addition,the Foxm1 protein expression of lung tissues in the gefitinib-intervened group was also higher than that in the control group(1.46±0.41,1.35±0.53,P=1.000).1.13 Snail1 protein expression of lung tissues in miceSnail1 protein expression of lung tissues in mice showed significant difference(H=7.539,P=0.023).The Snail1 protein expression of lung tissues in the BLM group was higher than that in the control group(0.60±0.09,0.23±0.11,P<0.05).The Snail1 protein expression of lung tissues in the gefitinib-intervened group was depressed when compared with that in the BLM group(0.27±0.22,0.60±0.09,P<0.05),while the Snail1 protein expression of lung tissues in the gefitinib-intervened group was higher than that in the control group(0.27±0.22,0.23±0.11,P=0.695).ConclusionIncreased activity of Foxo3 a can down-regulate Snail1,which decreases the expression of α-SMA and increases the expression of E-Cadherin,thereby attenuating epithelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis in mice. |
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