The Effect Of Wnt3a In Paraquat-Induced Epithelial-Mesenchymal Transformation Of Alveolar Epithelial Cell

ObjectiveTo investigate the role of Wnt3a in paraquat-induced epithelial-mesenchymal transformation of alveolar epithelial cell and its molecular mechanism.MethodsUsing paraquat(50μmol/L)to induce human alveolar epithelial cells(HBE),Western Blot and q-PCR were used to detect the expression of epithelial cell markers E-cadherin,Occludin and fibroblast markers a-SMA,Vimentin,to verify the establishment of human alveolar EMT model induced by paraquat.Stimulating HBE with Wnt3a human recombinant cytokine(100μg/ml),Western Blot and q-PCR techniques were used to detect the expression levels of epithelial markers E-cadherin,Occludin and fibroblast markers α-SMA,Vimentin and Wnt/β-catenin pathway key protein,verifying activation of Wnt/β-catenin signaling pathway.Design and construct siRNA fragments and the negative control for Wnt3a,transfect HBE cells,down-regulate Wnt3a protein expression level,at the same time,use paraquat to induce HBE,detect the expression of E-cadherin,Occludin,α-SMA and Vimentin.Observe the effect of inhibition of Wnt3a protein expression on EMT of pulmonary alveolar epithelial cells in paraquat-induced pulmonary fibrosis.Results1.Paraquat can promote the EMT process and activate Wnt/β-catenin signaling pathway in human alveolar epithelial cells.Compared with the control group,E-cadherin and Occludin expression were decreased in PQ group,α-SMA and Vimentin expression were significantly increased in PQ group,suggesting that PQ can induce epithelial-mesenchymal transformation in human lung epithelial cells,at the same time,the expression level of β-catenin increased,the difference was statistically significant,which indicates the activation of Wnt/β-catenin signal pathway.Western Blot and q-PCR results showed that Wnt3a expression in paraquat group was significantly higher than that in control group.2.Wnt3a is involved in the regulation of paraquat-induced epithelial-mesenchymal transition and the activation of Wnt/β-catenin signaling pathway.The expression of E-cadherin and Occludin in HBE cells treated with Wnt3a recombinant protein was significantly lower than that in control group,while the expression of a-SMA and Vimentin in the fibroblast was significantly increased,the expression of β-catenin,the Wnt/β-catenin pathway key protein,increased significantly,which suggested that Wnt3a could induce EMT process of HBE and activate Wnt/β-catenin signal pathway3.Wnt3a siRNA inhibits the EMT process induced by PQ and inhibits the Wnt/β-catenin activated by PQ after down-regulating Wnt3a expression.Transfection of HBE with Wnt3a-siRNA to interfere with the target gene,after treated with paraquat 50μmol/L for 72 hours,the expression of β-catenin in Wnt3a-siRNA group was significantly lower than that of the PQ group,indicating that the Wnt/β-catenin signaling pathway activated by PQ could be inhibited by Wnt3a siRNA.The expression of E-cadherin and Occludin in epithelial cells was increased,while that of SMA and Vimentin in fibroblasts was decreased,which indicated that the expression of Wnt3a was down-regulated,and the paraquat induced EMT process of HBE was blocked.Conclusion1.Paraquat can promote the epithelial-mesenchymal transformation of human alveolar epithelial cells and activate Wnt/β-catenin signaling pathway,inducing Wnt3a expression significantly.2.Wnt3a can activate Wnt/β-catenin signal pathway and induce epithelial-mesenchymal transition in human alveolar epithelial cells.3.Down-regulating the expression of Wnt3a by siRNA,the epithelial-mesenchymal transformation of HBE induced by Paraquat was blocked,and the activated Wnt/β-catenin pathway was inhibited,which suggested Wnt/β-catenin signal pathway might be activated by Wnt3a,Wnt3a may be a potential molecular target for clinical treatment of pulmonary fibrosis induced by paraquat poisoning,which may be involved in the regulation of the development of paraquat induced pulmonary fibrosis.