The Roles Of Rho/Rock Signaling Pathway In Silica-induced Epithelial-mesenchymal Transition In Human Bronchial Epithelial Cells

Objective: To investigate whether Silica dust could induce epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBE) in vitro and the role of Rho/ROCK signaling pathway in this process.Methods: (1) The morphological changes of HBE induced by Silica dust were observed by microscope. (2) HBE were treated by Silica dust with various concentrations (0, 50, 100, 200, 300μg/ml). After 72 hours, Immunocytochemistry and Western Blot were used to detect the protein expression of E-cadherin (E-cad), Vimentin,α-smooth muscle actin (α-SMA). (3) The changes of migration ability of HBE induced by Silica dust were investigated by wound -healing assay in vitro. (4) HBE were exposed to 200μg/ml Silica dust at different time points (0, 1, 2, 6, 12, 24h), and we performed GST pull-down assay to evaluate Rho activity. (5) HBE were pretreated by Y-27632 with different concentrations (10, 20, 30μM), furthermore, Immunocytochemistry and Western Blot were used to detect the protein expression of E-cad, Vimentin,α-SMA induced by Silica dust. Meanwhile, wound-healing assay was used to detect the changes of migration ability in vitro.Reslut: (1) Incubation with Silica dust, HBE transited to myofibrob last-like cells morphologically. (2) Immunocytochemistry demonstrated that Silica dust(200μg/ml) exposure leaded to a dcreased E-cad and an increased Vimentin andα-SMA expression. (3) Under the treatment of Silica dust, the expression of E-cad protein decreased in a concentration -dependent manner(100μg/ml,200μg/ml,300μg/ml P<0.05). And the expression of Vimentin protein increased in a concentration-dependent manner(200μg/ml,300μg/ml P<0.05), it reached peak at 300μg/ml, (7.30±3.02) times of the control group. Similarly, the expression ofα-SMA protein were increased by Silica dust(100μg/ml, 200μg/ml, 300μg/ml, P<0.05), peaking at 200μg/ml, (5.09±1.98) times of the control group. Then the expression ofα-SMA protein decreased slightly at 300μg/ml, significantly higher than those in the control group (P<0.05). (4) Wound-healing assay In vitro showed that the areas of wound-healing of the test groups were larger than those in control group at 6,12 and 24h respectively(P<0.05). (5) Silica dust treatment could induce a robust increase in the activity of Rho,which was (3.09±0.30) times of the control group at 2h(P<0.05). And at 6h Rho activity increased to maximum, (7.36±1.08) times of that in the control group(P<0.05).(6) In prevention experiment, immunocytochemistry showed that Y-27632(20μM) upregulated E-cad and downregulated Vimentin andα-SMA expression. (7) Western Blot showed that under the prevention of Y-27632 (20, 30μM), the expression of E-cad increased and it were (1.98±0.38) times and (2.17±0.51) times of those in the SiO₂(200μg/ml) group(P<0.05). However, the expression of Vimentin andα-SMA were inhibited by Y-27632 (20,30μM).The inhibition ratio were (64.32%,69.45% and 70.30 %, 78.37%) (P<0.05). (8)Wound-healing assay in vitro showed that the areas of wound-healing were prevented by Y-27632 (20,30μM) (P<0.05).Conclusion: (1) Silica dust could induce epithelial-mesenchymal transition of HBE in vitro.(2) Rho/ROCK signaling pathway in HBE could be activated by Silica dust.(3) Rho/ROCK signaling pathway is involved in the regulation of EMT in HBE induced by Silica dust.(4) Y-27632 prevented Silica dust induced EMT in HBE.