The Mechanism Of Wnt10b In Lung Epithelial Cells’ Epithelial-Mesenchymal Transition Induced By Paraquat

ObjectiveTo investigate the role and mechanism of Wntlob in regulating epithelial-mesenchymal transition of paraquat-induced pulmonary fibrosis.MethodsThe rats were treated with paraquat by intraperntoneal injecting,to construct pulmonary fibrosis model.The occurrence of epithelial-mesenchymal transition(EMT)and activation of wnt/α-catenin signaling pathway was detected.In vitro,16HBE cells were treated with paraquat(PQ,50 μmol/ml)or Wnt10b recombinated protein(100 ng/ml)for 72 h,and the protein and gene expression levels of epithelial markers E-cadherin,fibroblast markers including Vimentin and alpha SMA,and Wnt10b were detected,respectively.Furthermore,16HBE was transfected with Wnt10b-siRNA by using lentivirus vector,then treated with paraqua and cultured for 72 hs.The expression of EMT relatived factors and key proteins of wnt/β-catenin pathway was estimated.Results1.In vivo,Paraquat induced epithelial-mesenchymal transition in rats and activated the wnt/β-catenin signaling pathway.HE staining and Masson’s staining showed alveolar structure significantly damaged,pulmonary interstitial thickened,in which collagen deposition apparently increased in lung tissue of paraquat group.Behinds,compared with the control group,the expression of E-cadherin significantly decreased both in protein and mRNA level,whereas the expression of Vimentin and alpha SMA significantly increased,verifing the occurrence of epithelial-mesenchymal transition,and further pulmonary fibrosis.At the same time,qRT-PCR results showed that compared with the control group,the expression levels of β-catenin and MMP-2 in PQ group were significantly increased,and the mRNA levels of Cyclin D1,Axin2 and MMP-7,the proteins on the downstream of Wnt signaling pathway,were significantly increased,verifying activation of wnt/β-catenin signaling pathway.2.In vitro,paraquat induced epithelial-mesenchymal transition in human lung epithelial cells(16HBE),and the expression of Wnt10b was significantly increased.Compared with the control group,the protein expression and mRNA levels of E-cadherin were significantly decreased after paraquat treatment,while the protein expression and mRNA levels of Vimentin and alpha SMA was elevated,indicating that paraquat can induce epithelial-mesenchymal transition in HBE cells.In addition,the results of Western Blot and qRT-PCR showed that the expression of Wnt10b in the PQ group was significantly higher than that in the control group,the difference was statistically significant.3.Wnt10b can activate the wnt/β-catenin signaling pathway to induce epithelial-mesenchymal transition in lung epithelial cells;after targeted down-regulation of Wntlob,the activation of wnt/β-catenin signaling pathway induced by PQ is inhibited,while paraquat-induced epithelial-mesenchymal transition was reversed,indicating that Wnt10b may participate in the regulation of paraquat-induced pulmonary fibrosis by activating the wnt/p-catenin signaling pathway.Wnt10b may be a potential target for drug treatment of paraquat poisoning.Conclusion1.Intraperitoneal injection of paraquat can induce epithelial-mesenchymal transition in rats,and the activation of wnt/β-catenin signaling pathway is found.2.Paraquat can induce epithelial-mesenchymal transition in lung epithelial cells,and trigger the wnt/β-catenin signaling pathway.3.Wntlob can induce epithelial-mesenchymal transition and trigger wnt/β-catenin signaling pathway in HBE.Knocking down Wnilob,the process of epithelial-mesenchymal transition induced by paraquat can be reversed,as well as wnt/β-catenin signaling pathway was inhibited,indicating that Wntlob is involved in the development and progression of paraquat-induced pulmonary fibrosis,which may be regulated by the wnt/p-catenin signaling pathway and may be a therapeutic target for paraquat poisoning.