Inhibition Of EGFR-TGFβ Attenuating Bleomycin-induced Pulmonary Fibrosis In Mice

BackgroundInterstitial lung diseases (ILD) refer to a collection of disorders characterized by varying degrees of inflammation and fibrosis in the lung interstitium. The cause of ILD is sometimes known (e.g., environmental factors, drugs, collagen vascular disease, and sarcoidosis). When the cause is unknown, it is labeled as idiopathic and further classified into the currently recognized single disease entities. Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause, occurring primarily in older adults, and limited to the lungs. Research on pathogenesis has been conducted mostly in the context of the most frequent idiopathic ILD, idiopathic pulmonary fibrosis. There is still lack of specifically effective treatments to prevent or reverse IPF.In the currently accepted paradigm, a major factor in IPF pathogenesis is thought to be recurrent alveolar epithelial cell (AEC) injury. Initial injury leads to aberrant activation of AECs, creating a profibrotic environment with accumulation of collagen-producing fibroblasts and myofibroblasts. To date,(myo) fibroblasts have been suggested to accumulate via three mechanisms:proliferation and differentiation of resident lung fibroblasts; epithelial-to-mesenchymal transition (EMT), in which AECs undergo transition to (myo) fibroblasts; and transition of bone marrow-derived fibrocytes or circulating progenitors to fibroblasts. The significance and relative contribution of each pathway to fibrosis have not been definitively established, hampering development of novel therapies. EMT converts epithelial cells into migratory and/or invasive mesenchymal cells, and is well established during development and carcinogenesis, with its role in fibrosis being more controversial. The associations between EMT and epithelial cell repairment have been found in both IPF patients and mice with bleomycin-induced lung fibrosis. The mechanism has not yet fully understood, but the recent study confirmed some related signal pathways (e.g., EGFR/Akt, TGF-β/Smad Nox/ROS signaling pathways).Epidermal growth factor receptor (EGFR) exists on the cell surface and is activated by binding of its specific ligands, including epidermal growth factor(EGF) and transforming growth factor α(TGF-α)(a full list of the ligands able to activate EGFR). EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity. These downstream signaling proteins initiate several signal transduction cascades, principally the MAPK, Akt and JNK pathways, leading to DNA synthesis and cell migration, adhesion, and proliferation. EGFR and its ligands were increased in the lung, after the exposure to inflammation and hyperoxia. TGF-α content also significantly increased in bronchial alveolar lavage fluid of patients with IPF. A number of experimental studies support a role for EGFR participation in EMT processes. EGFR signal not only promotes the epithelial cell differentiation, but also prolongs the lifetime of mesenchymal cells, eventually aggravatting the degree of fibrosis. EGFR tyrosine kinase inhibitors (EGFR-TKI) inhibit EGFR tyrosine kinase activity to disrupt EGFR signaling function. It was demonstrated that EGFR-TKI prevented and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury. It was showed that induction of EMT may contribute to the decreased efficacy of therapy in primary and acquired resistance to gefitinib. Thus, EGF and its receptor play a key role in the occurrence and development of interstitial lung disease. It is also an important factor for EMT.TGF-β is a cytokin that controls proliferation, cellular differentiation, and other functions in most cells. Proapoptotic effects of TGF-β1have been described as occurring via smad-dependent mechanisms. TGF-β1has been implicated as a key mediator of EMT in fibrotic diseases. It was demonstrated that EGF inhibited apoptosis and facilitated TGF-β1induction of EMT by increasing proliferation and accentuating E-cadherin loss. Another study of squamous cancer cells has found that although the TGF-β1is not EGFR’s ligand, but it indirectly activated EGFR and activated the downstream and collagen synthesis related genes, to promote the synthesis and secretion of collagen and fibroblasts.We previously found that gefitinib inhibited bleomycin-induced lung fibrosis and significantly decreased the scores of lung inflammation and fibrosis. Phosphorylated EGFR and Akt were decreased; meanwhile TGF-β1expression and the level of α-SMA were also decreased. In the process of pulmonary fibrosis, is there an interaction between EGFR and TGF-β? How do they control each other?To identify the regulating mechanism of EGFR and TGF-β on lung fibrosis EMT, we built the mouse model with bleomycin-induced lung fibrosis and detected the epithelial and mesenchymal indicators. By using EGFR-TKI and TGF-β receptor I inhibitor (TGF-β RI inhibitor), we explored the inhibition of EGFR/Akt pathway and TGF-β/Smad pathway to clarify the mechanism of EGFR-TGFβ attenuating bleomycin-induced pulmonary fibrosis in mice. We focused on the ongoing controversy of how EMT contributed to lung fibrosis and inhibition of EMT would be benefit for the therapy.Method1Gefitinib blocks bleomycin-induced pulmonary fibrosisThirty KM female mice were randomLy divided into three groups:the control group (received normal saline intratracheally), the bleomycin group (received bleomycin intratracheally,3mg/kg), and the bleomycin plus gefitinib group (received bleomycin intratracheally and gefitinib orally,20mg/kg). All the mice were sacrificed14days after the treatments. Pulmonary histological changes were evaluated by Hematoxylin-eosin stain and Masson’s trichrome stain. a-SMA、 E-cadherin、Fibronectin in lung tissue were determined by Western blotting.2EGFR inhibition attenuates TGF-β and Smad2/3ExpressionThirty KM female mice were randomLy divided into three groups:the control group (received normal saline intratracheally), the bleomycin group (received bleomycin intratracheally,3mg/kg), and the bleomycin plus gefitinib group (received bleomycin intratracheally and gefitinib orally,20mg/kg). All the mice were sacrificed14days after the treatments. Serum TGF-β1was evaluated by ELISA. Phosphorylation of EGFR, Erk, Smad2/3in lung tissue were determined by Western blotting.3TGF-β RI inhibitor significantly attenuates EGFR ExpressionThirty KM female mice were randomLy divided into three groups:the control group (received normal saline intratracheally), the bleomycin group (received bleomycin intratracheally,3mg/kg), and the bleomycin plus TGF-β RI inhibitor group (received bleomycin intratracheally and TGF-β RI inhibitor intraperitoneally,10mg/kg). All the mice were sacrificed14days after the treatments. Pulmonary histological changes were evaluated by Hematoxylin-eosin stain and Masson’s trichrome stain. Serum TGF-β1was evaluated by ELISA. Phosphorylation of Smad2/3、c-Src、EGFR in lung tissue were determined by Western blotting.4Statistical AnalysesData are presented as mean±S for at least three separate experiments. ANOVA and LSD tests were used for multiple group comparisons. P<0.05compared with controls was considered statistically significant.Results1Gefitinib blocks bleomycin-induced pulmonary fibrosis EMT1.1Pathological scores of lung tissue demonstrated significant difference (F=38.750, P<0.001). The control group had clear alveolar structure, thin alveolar walls and less inflammatory cells between interstitial tissues. While the bleomycin group had destructive alveolar structure, thick alveolar walls, more inflammatory cells between interstitial tissues. Pathological damage of the bleomycin plus gefitinib group was less than the bleomycin group. The pathological score of the bleomycin group was significantly higher than the control group (n=5, P<0.001). After gefitinib treatment, pathological score was significantly decreased (n=5, P<0.01).1.2Fibrosis scores of lung tissue demonstrated significant difference (F=31.971, P<0.001). The control group had complete alveolar structure, barely any inflammatory cells and collagen deposition. While the bleomycin group had destructive alveolar structure, lots of inflammatory cells and collagen deposition. Pathological damage of the bleomycin plus gefitinib group was less than the bleomycin group. The fibrosis score of the bleomycin group was significantly higher than the control group (n=5, P<0.001). After gefitinib treatment, fibrosis score was significantly decreased (n=5, P<0.01). 1.3EGFR protein phosphorylation level of lung tissue demonstrated significant difference (F=23.607, P<0.001). EGFR protein phosphorylation level of the bleomycin group was significantly increased (n=4, P<0.001), while it was significantly reduced in the bleomycin plus gefitinib group (n=4, P=0.001).1.4a-SMA protein content of lung tissue demonstrated significant difference (F=7.581, P<0.05). a-SMA of the bleomycin group was significantly increased (n=4, P<0.01), while it was significantly reduced in the bleomycin plus gefitinib group (n=4, P<0.05).1.5E-Cadherin protein content of lung tissue demonstrated significant difference (F=13.561, P<0.01). E-Cadherin of the bleomycin group was significantly reduced (n=4, P=0.001), while it was significantly increased in the bleomycin plus gefitinib group (n=4, P=0.01).1.6Fibronectin protein content of lung tissue demonstrated significant difference (F=31.726, P<0.001). Fibronectin of the bleomycin group was significantly increased (n=4, P<0.001), while it was significantly reduced in the bleomycin plus gefitinib group (m=4, P<0.001).2EGFR inhibition attenuates TGF-β and Smad2/3Expression2.1EGFR protein phosphorylation level of lung tissue demonstrated significant difference (F=23.607, P<0.001). EGFR protein phosphorylation level of the bleomycin group was significantly increased (n=4, P<0.001), while it was significantly reduced in the bleomycin plus gefitinib group (n=4, P=0.001).2.2The serum concentration of TGF-β1demonstrated significant difference (F=14.391, P=0.001). TGF-β1of the bleomycin group was significantly increased (n=5, P<0.001), while it was significantly reduced in the bleomycin plus gefitinib group (n=5, P<0.01). 2.3Erk protein phosphorylation level of lung tissue was statistically significant (F=12.78, P<0.01). Erk protein phosphorylation level of the bleomycin group was significantly increased (n=4, P<0.001), while it was significantly reduced in the bleomycin plus gefitinib group (n=4, P<0.05).2.4Smad2/3protein phosphorylation level of lung tissue demonstrated significant difference (F=17.7, P=0.001). Smad2/3protein phosphorylation level of the bleomycin group was significantly increased (n=4, P<0.001), while it was significantly reduced in the bleomycin plus gefitinib group (n=4, P<0.05).3TGF-P RI inhibitor significantly attenuates EGFR Expression3.1Pathological scores of lung tissue demonstrated significant difference (F=80.987, P<0.001). The control group had clear alveolar structure, thin alveolar walls and less inflammatory cells between interstitial tissues. While the bleomycin group had destructive alveolar structure, thick alveolar walls, more inflammatory cells between interstitial tissue. Pathological damage of the bleomycin plus Ly364947group was a little more than the bleomycin group. The pathological score of the bleomycin group was significantly higher than the control group (n=4, P<0.001). After Ly364947treatment, pathological score was also increased (n=4, P>0.05).3.2Fibrosis scores of lung tissue demonstrated significant difference(F=39.328, P<0.001). The control group had complete alveolar structure, barely any inflammatory cells and collagen deposition. While the bleomycin group had destructive alveolar structure, lots of inflammatory cells and collagen deposition. Pathological damage of the bleomycin plus Ly364947group was a little more than the bleomycin group. The fibrosis score of the bleomycin group was significantly higher than the control group (n=4,.P<0.001). After Ly364947treatment, fibrosis score was also increased (n=4, P>0.05). 3.3The serum concentration of TGF-β1of lung tissue demonstrated significant difference (F=5.446, P<0.05). TGF-β1of the bleomycin group was significantly increased (n=4, P<0.001), while this phenomenon was not found in the bleomycin plus Ly364947group (n=4, P>0.05).3.4Smad2/3protein phosphorylation levels of lung tissue demonstrated significant difference (F=15.351, P=0.001). Smad2/3protein phosphorylation levels of the bleomycin group was significantly increased (n=4, P<0.001), while it was significantly reduced in the bleomycin plus Ly group (n=4, P<0.01).3.5c-Src protein phosphorylation level of lung tissue demonstrated significant difference (F=25.950, P<0.001). c-Src protein phosphorylation level of the bleomycin group was significantly increased (n=4, P<0.001), while it was significantly reduced in the bleomycin plus Ly364947group (n=4, P<0.001).3.6EGFR protein phosphorylation level of lung tissue demonstrated significant difference (F=26.559, P<0.001). EGFR protein phosphorylation level of the bleomycin group was significantly increased (n=4, P<0.001), while it was significantly reduced in the bleomycin plus Ly364947group (n=4, P<0.001).Conclusion1EGFR inhibition blocks suppresses bleomycin-induced pulmonary fibrosis EMT.2EGFR inhibition attenuates TGF-β/Smad2/3signal pathway activation and downregulates the epithelial-mesenchymal transition.3TGF-P RI inhibitor (Ly364947) significantly attenuates Smad2/3Expression and inhibits EGFR Expression. Inhibition of EGFR-TGFβ attenuates bleomycin-induced pulmonary fibrosis in mice.