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The Effects and Mechanism of Wnt/β-catenin Signaling Induces Lung Fibrosis

Author: SongPing
Tutor: ZhengJinXu
School: Jiangsu University
Course: Clinical Laboratory Science
Keywords: lung fibroblast wnt1 β-catenin proliferation epithelial-mesenchymaltransition
CLC: R563.9
Type: PhD thesis
Year: 2014
Downloads: 14
Quote: 0
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Abstract


Objective:Idiopathic pulmonary fibrosis(IPF) is a lethal lung disease of unknown etiology. It is characterized by alterations of the alveolar epithelium, the expansion of the fibroblast/myofibroblast population and extracellular matrix (ECM)deposition, which results in an irreversible distortion of the lung architecture. Recent studies showed that abnormal activation of the Wnt/β-catenin signaling pathway occurs in lung tissue of patients with IPF. In the present study, we explored the role of wntl in human embryo pulmonary fibroblast (HEPF) proliferation, activation and myofibroblast differentiation of fibroblasts. A549cells were treated by Wntl and transfected by β-catenin plasmid cells to investigated the possible role of the Wnt signaling pathway in inducing epithelial-mesenchymal transitions (EMT) in lung epithelial cells. Pulmonary fibrosis was induced in mice by intraltracheal instillation of bleomycin,to explore the relationship with β-catenin. HEPF and A549cells were cultured by bronchoalveolar fluid (BALF) which conducted from bleomycin-induced murine model of pulmonary fibrosis, to investigate the effects on the cell proliferation and phenotype transformation, which were correlated with Wnt/β-catenin signaling pathway;The recombinant lentiviruses which express siRNA targeting β-catenin gene were transfected into HEPF and A549cells, and then cultured cells by BALF,to explore the effects on the cell phenotype transformation.Methods:HEPF cells were stimulated with various concentrations of Wntl, the proliferation of HEPF cells observed by MTT method.Western blot analysis and qRT-PCR revealed that alpha-smooth muscle actin (a-SMA), vimentin and collagen I mRNA and protein expression. A549cells were stimulated with various concentrations of Wntl, western blot testing and qRT-PCR revealed that E-cadherinc (E-cad), vimentin, a-SMA, collagen I mRNA and protein expression. We used β-catenin plasmid-transfected A549cells,the cellular morphologic changes were observed by light microscope.Western blot analysis and qRT-PCR revealed that the expression of epithelial phenotypic marker E-cad and the mesenchymal marker a-SMA, vimentin and collagen I. Furthermore, we used intratracheal instillation of bleomycin as a model of pulmonary fibrosis,on the3rd,7th,14th and28th day after treatment with bleomycin, lung were dissected out, separated from other tissues. Hematoxylin and Eosin (H&E), Masson’s Trichrome staining were used to detect the degrees of acute inflammation and fibrosis;Hydroxyproline (HYP) was tested by lung tissue alkaline hydrolysis. Western blot and immunohistochemical method was adopted to detect the protein of β-catenin. HEPF and A549cells cultured by BALF which was conducted on days7after bleomycin administration. qRT-PCR and westen blot were used to determine the mRNA and protein expression of E-cad, a-SMA, vimentin, collagen I and β-catenin.To clarify further the role of β-catenin in fibrosis, we constructed recombinant lentiviral vector expressing siRNA targeting β-catenin gene. The most effective sequence of siRNA was screened by efficiency of β-catenin gene knock-down in transfected HEPF cells. The recombinant lentiviruses were transfected into HEPF and A549cells, and then cultured cells by BALF coming from Mouse models. qRT-PCR and westen blot were used to determine the mRNA and protein expression of E-cad, a-SMA, vimentin, collagen I.Results:The present study revealed that cell proliferation improved following stimulation using different concentrations of Wntl in a concentration-dependent manner. When the concentration exceeded20μg/l, cell proliferation was significant (P<0.05) and the cell expression of a-SMA, vimentin and collagen I mRNA, as well as protein expression, significantly increased (P<0.05); A549cells were treated by Wntl, the results showed that the mRNA and protein expression of vimentin, a-SMA and collagen I gradully increased, meanwhile those of E-cad gradully decreased in a concentration dependent manner. A549were transfected by β-catenin plasmid cells, showing a changes their phenotype from pebble-shaped to fusiform. The mRNA and protein expression of vimentin, a-SMA and collagen I increased significantly, whereas those of E-cad decreased significantly. We used intratracheal instillation of bleomycin as a model of acute lung injury and fibrosis, the expressions of β-catenin had the consistent increasing trends, increaseing on day7and peaking around day14. To explored that lung fibrosis induced by abnormal wound healing in response to alveolar epithelial cell injury were relationship with the Wnt/β-catenin signaling pathway. BALF was obtained from bleomycin-induced models of pulmonary fibrosis. HEPF cells were cultured with Dulbecco’s modified Eagle’s medium plus BALF. The mRNA and protein expression of a-SMA, vimentin and collagen I significantly increased and these increases were associated with β-catenin. Furthermore, following being infected with the lentivirus expressing β-catenin shRNA, HEPF cells were cultured with BALF. However, the mRNA and protein expression of a-SMA, vimentin and collagen I did not increase significantly. We also incubated A549cells by BALF from the intratracheal bleomycin mice model. We observed an increased expression of mesenchymal markers (a-SMA, vimentin and collagen I), a concomitant decreased expression of epithelial markers (E-cad), corresponding to an increased expression of P-catenin. When A549cells were infected with a lentivirus expressing P-catenin shRNA, the expression of mesenchymal cell markers did not increase significantly and E-cad expression did not decrease.Conclusion:These findings show that the activation of the Wnt/β-catenin signaling pathway can increases the number of myofibroblasts and promotes fibroblasts to change into myofibroblasts,which leading to excessive ECM deposition. It also suggests that the activation of the Wnt signaling pathway can increase the number of myofibroblasts in pulmonary fibrosis through epithelial-mesenchymal transition. The Wnt/β-catenin signaling pathway is involved in the occurrence and development of pulmonary fibrosis,and the inhibition of Wnt/β-catenin signaling pathway suppresses the cell phenotype changed and pulmonary fibrosis.Furthermore, these results also demonstrated that the activation of a biological repair response and persistence at the injury site is a key factor in the formation of pulmonary fibrosis. The Wntl/β-catenin signalling pathway is important in the formation of fibrotic disease in lung injury and may provide opportunities for treatment and intervention in IPF.

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CLC: > Medicine, health > Internal Medicine > Respiratory system and chest diseases > Pulmonary disease > Other
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