The Roles Of MAPK Signaling Pathway In Silica-induced Epithelial-mesenchymal Transition In Human Bronchial Epithelial Cells

Objective:To explore whether SiO2 could induce epithelial-mesenchymal transition (EMT) in human bronchial epithelial (HBE) cells in vitro and the role of MAPK signaling pathway in this process.Methods:(1)The morphological changes of HBE induced by SiO2 were observed by microscope. (2)HBE cells were treated by SiO2 with various concentrations (0,50,100,200,300μg/ml) after 72 hours, Western blotting were used for detecting E-cadherin (E-cad), Vimentin and smooth muscle actin (α-SMA) expression. (3)HBE cells were exposed to 200μg/ml Silica dust at different time periods (0,0.5,1,2,4, 8h), and assessed the activation status of ERK1/2 and p38 MAPK by immunoblotting. (4) HBE cells were pretreated by U0126 and Sb203580 with different concentrations(10,20,30μM) after 1 hour, and assessed alterations in the protein expression of E-cadherin,α-SMA and vimentin. (5) HBE cells were pretreated by ERK inhibitor U0126 together with the p38 MAPK inhibitor SB203580, Western Blot were evaluated for E-cadherin, vimentin andα-SMA expression.Reslut:(1)After incubation of HBE cells with 200μg/ml SiO2, cells were lose the paving stone changes, and acquired a spindle-shaped. (2)HBE cells were treated by SiO2 with different concentrations. Using western blot,we observed that the expression of E-cadherin decreased after 72h, while the expression of vimentin andα-SMA increased in HBE cells after continuous exposure to SiO2. When the concentration of SiO2 were 200μg/ml, E-cadherin expression levels(1.42±0.10) significantly lower than that control (3.74±0.95) (P<0.05), and the lowest at 300μg/ml(100,200,300μg/ml P<0.05); And then the expression ofα-SMA were increased by the stimulation of SiO2, (5.09±1.98) times of the control (P<0.05), andα-SMA expression decreased slightly at 200～300μg/ml, but higer than those of the control; Similarly, the expression of vimentin were increased by the stimulation of SiO2(200,300μg/ml P<0.05), (7.30±3.02) times compared with the control, with peak at 300μg/ml. (3) Western Blot show that the activity of ERK and p38 at different time points, SiO2 at 200μg/ml significantly increased phosphorylated ERK1/2 and p38 MAPK form 30min,and then decreased at 60min and 30min respectively. (4) Western Blot showed that SiO2 (200μg/ml) induced downregulation of E-cadherin was effectively prevented by pretreatment with (20,30μM)U0126, and the E-cadherin expression were (2.52±0.87) times and (3.17±0.88)times of those SiO2 (200μg/ml) groups (P<0.05); Pretreatment with 30μM U0126, effectively inhibited SiO2 induced upregulation of a-SMA and vimentin expression. The inhibition ratio were 40.43% and 60.33%(P<0.05). Pretreatment with 30μM SB203580, an p38 MAPK inhibitor effectively inhibited SiO2 (200μg/ml) induced downregulation of E-cadherin expression, and the E-cadherin expression was (1.77±0.26) times compared with the SiO2 (200μg/ml) group (P<0.05); However 30μM SB203580 prevented SiO2 induced changes in a-SMA and vimentin expression, and the inhibition ratio were 34.67% and 55.44%(P<0.05). (5) HBE cells were pretreated with the MEK inhibitor U0126(30μM), combined with the p38 MAPK inhibitor SB203580(30μM), while immunoblotting show that the protein expression of E-cadherin, a-SMA and vimentin were non-significant differences compared with those of the individual kinase inhibitors (P>0.05).Conclusion:(1) SiO2 could induce epithelial-mesenchymal transition in HBE cells. (2) During SiO2-induced EMT in HBE cells, enhanced activation of ERK1/2 and p38 MAPK. (3) SiO2-induced EMT in HBE cells, is modulated by ERK1/2 and p38 MAPK signaling pahways.