The Role Of The Icotinib In The Treatment Of Pulmonary Fibrosis In Silicosis

Objective To investigate the effect of epidermal growth factor receptor tyrosine kinase inhibitor, Icotinib, on the expression of E-cadherin, α-SMA and EGFR in A549cell with epithelial-mesenchymal transition (EMT) induced by silica. To study the roles of EGFR signaling pathway in silica-induced EMT in Human A549cells in vitro.Methods By making the cell model method of silicosis in vitro, With50ug/ml SiO2stimulate AM, The supernatants were collected after3,6,12,18,24,36h and then to detect the TGF-β1protein expression by ELISA, respectively. With the expression of TGF-β1peak time points (Tm) of AM culture supernatant as a condition of supernatant. A549cells were cultured by AM supernatant and stimulated with indicated doses of silica (0,50,100,200ug/ml SiO2) for48h. The dust lung epithelial cells were intervene by Icotinib. The cells were devided into three groups:control (N, SiO2Oug/ml), silica (F, SiO2200ug/ml), and silica plus Icotinib (A1, SiO2200ug/ml+low dose20umol/L; A2, SiO2200ug/ml+middle dose40umol/L; A3, SiO2200ug/ml+high dose80umol/L). After48h, the cells morphology changes were observed under phase-constrast microscope. RT-PCR and immunnoflurescence were used to detect the expression level of mRNA and protein of E-cadherin, α-SMA, and EGFR.Result After stimulated by SiO2, the TGF-β1protein expression at each time point was significantly higher in AM supernatants than the control group. With the extension of action time was reduced after the first rise trend, and the highest level was in18h, the difference was statistically significant (P<0.05). After exposure to silica, A549cells induced mesenchymal characterized by cells morphological changes, such as displayed a spindle-shape, fibroblast-like morphology under phase-constrast microscope, especially in200ug/ml group. After the intervention of Icotinib, All cells has stopped to change to the mesenchymal phenotype, as evidenced by the shorter of pseudopodia significantly and a generally round shape. Compared with the control, the E-cadherin mRNA and protein expression in silica-induced was significantly down-regulated, especially in200ug/ml group (P<0.05); The α-SMA, EGFR mRNA and protein expression in silica-induced was significantly up-regulated, especially in200ug/ml group (P<0.05). The difference between the experimental group (50,100,200ug/ml SiO2) and the control group was statistically significant (P<0.05). After intervened by Icotinib for48h, E-cadherin mRNA and protein expression was no significant change. The difference between F group and intervention group (A1, A2, A3) was no statistically significant (P>0.05), The difference between N group and intervention group (A1, A2, A3) was statistically significant (P<0.05); α-SMA mRNA and protein expression was significantly reduced; the signal transduction protein EGFR mRNA and protein expression gradually lowered its expression. Both the expression α-SMA and EGFR were associated with the concentration of Icotinib. Each group was statistically significant (P<0.05).Conclusions After stimulated to AM by SiO2in vitro, the expression of TGF-β1protein levels were significantly increased with in a time-effect relationship. The AM supernatant together with SiO2can induce lung epithelial cell to mesenchymal transition, which may be related to EGFR signaling pathway. Icotinib can inhibit lung epithelial cell to mesenchymal transition after silica stimulated, which may be associated to reduce the α-SMA expression and inhibition of EGFR activity. Thus, inhibition of EGFR activation may provide new ideas for the clinical treatment of pulmonary fibrosis.