| BackgroundPulmonary fibrosis is a chronic,progressive,fibrotic interstitial lung disease.It is characterized by the destruction of the alveolar structure,reduced lung compliance and accumulation of the extracellular matrix,eventually,leading to respiratory failure and death.Its mechanisms are unclear,and the treatment options are limited.Current studies have shown that the fibrotic response is driven by abnormally activated alveolar epithelial cells.Various cytokines produced by these cells induce epithelial-to-fibroblast differentiation and fibroblast-to-myofibroblast activation by inducing resident mesenchymal cell proliferation,circulatory fibroblast attraction,and epithelial-mesenchymal transformation.Fibroblasts and myofibroblasts secrete excessive extracellular matrix,mainly containing collagen,leading to scarring and destruction of lung structures.Lung cancer or primary bronchial carcinoma is a malignant tumor originating in the respiratory epithelium.It is the most common primary malignancy of the lung.The cause is not entirely clear.Lung cancer is the leading cause of cancer-related death worldwide.In recent years,many clinical observations have shown that when patients with pulmonary fibrosis are combined with lung cancer,the progression of pulmonary fibrosis will be significantly accelerated,and some patients even die early from complications of pulmonary fibrosis.There are many common cytobiological behaviors and signaling pathways between pulmonary fibrosis and lung cancer.It is confirmed that pulmonary fibrosis can cause the development of lung cancer,Meanwhile,Although lung cancer can exacerbate pulmonary fibrosis,which has great impacts on disease progression,have not been studied and reported in detail,although it has become a serious problem,clinical interference and prognosis of the patients,the mechanisms behind had not yet been student in detail.According to existing data and previous studies of our team,we found that lung cancer might secrete Wnt/β-catenin signaling pathway and TGF-β/Smad signaling pathway-related activating factors,which play an important role in the occurrence and development of pulmonary fibrosis.Therefore,in this study,the specific mechanism of lung cancer promoting pulmonary fibrosis exacerbation was studied starting from Wnt/β-catenin signaling pathway and TGF-β/Smad signaling pathway,with the same cytobiological behaviors between lung cancer and pulmonary fibrosis.It is of great significance for the treatment of pulmonary fibrosis in patients with pulmonary fibrosis combined with lung cancer and can also provide referenceable basic theoretical support for drug development and clinical treatment.ObjectiveIn this study,normal mouse lung fibroblast cells(MLg-2908 cells)and mouse alveolar epithelial cells(TC-1 cells)were co-cultured in vitro with mouse lung adenocarcinoma cells(LLC cells)to simulate the environment of lung tissue cells co-existing with lung cancer cells.By detecting the alterations of the expression spectrum of two cells,common cytobiological behaviors,and signaling pathways to investigate the related signaling pathways and molecular mechanisms of lung cancer cell microenvironment promoting the transformation of innate lung tissue cells to fibrotic phenotype and the progression of pulmonary fibrosis.Furthermore,we established a mouse model of pulmonary fibrosis with lung cancer to observe the effect of lung cancer on the progression of pulmonary fibrosis by acquiring the lung tissue cells combining the lung cancer microenvironment in vivo.Last,we tested the serum and lung tissue proteins of the mice,then evaluated the impacts lung cancer had on PF and the specific mechanism of lung cancer promoting the progression of pulmonary fibrosis.The results will provide the theoretical basis for the treatment of patients with PF combined lung cancer.Methods1.The morphological changes of TC-1 cells were observed by taking photos after co-culture with LLC cells.2.CCK8 proliferation test and scratch test were adopted to investigate the impact on MLg-2908 and TC-1 cells’ proliferation and migration ability after co-culture with LLC cells.3..Indirect immunofluorescence assay was used to detect the expression of epithelial-mesenchymal transformation-related proteins in TC-1 cells and myofibroblast activation of MLg-2908 cells after co-culture with LLC cells.4.The expression levels of key proteins in Wnt/β-catenin signaling pathway and TGF-β/Smad signaling pathway were detected by WB after co-culture of MLg-2908,TC-1 cells,and LLC cells.5.Gene expression of collagen I and collagen III were detected by RT-PCR after co-culture of MLg-2908,TC-1 cells,and LLC cells.6.ELISA was used to detect the content of secretory cytokines related to pulmonary fibrosis in the microenvironment after co-culture of MLg-2908,TC-1 cells,and LLC cells.7.In vivo experiments demonstrated PF combined with lung cancer can promote the progression of PF and is related to Wnt/β-catenin signaling pathway and TGF-β/Smad signaling pathway.The mouse pulmonary fibrosis model and pulmonary fibrosis combined with lung cancer model were established.The progression of pulmonary fibrosis in mice of each group was detected by H&E staining,Masson staining,immunohistochemical analysis and mouse lung HYP content,and the differences among pulmonary fibrosis-related cytokines in mice of each group were detected by ELISA.Mouse lung tissue proteins were extracted,and the expression of Wnt/β-catenin signaling pathway and TGF-β/Smad signaling pathway-related proteins were detected by WB assay.Results1.Mesenchymal changes were observed in the morphology of TC-1 cells after co-culture 24 h with LLC cells.2.The proliferation and migration ability of MLg-2908,TC-1 cells were significantly increased after co-culture with LLC cells(p< 0.001).3.Indirect immunofluorescence indicates mesenchymal cells markers Vimentin and myofibroblasts markers α-smooth muscle actin(α-SMA)were significantly increased in both cell lines.Meanwhile,epithelial cells marker E-cadherin was decreased in both cell lines(p< 0.001).4.After co-culture of MLg-2908,TC-1 cells and LLC cells,phosphorylation and expression levels of proteins related to Wnt/β-catenin signaling pathway and TGF-β/Smad signaling pathway were increased(p< 0.001).5.Collagen I and Collagen III mRNA levels are increased,indicating an elevated expression.Also,fibrosis-related secretory cytokines are significantly increased(p<0.001).6.In vivo experiment showed that the degree of pulmonary fibrosis in the pulmonary fibrosis combined with lung cancer mice group was more severe than that in the pulmonary fibrosis model group(p< 0.001).5.The expression levels of lung protein Wnt/β-catenin signaling pathway and related proteins of TGF-β/Smad signaling pathway were increased in the pulmonary fibrosis with lung cancer model group compared with the pulmonary fibrosis model group(p< 0.001).Conclusion1.Lung cancer cells can promote the development of EMT in lung epithelial cells and enhance the proliferation and migration of lung epithelial cells and lung fibroblasts.2.LLC cells can induce myofibroblast activation and fibroblast differentiation in MLg-2908 and TC-1 cells respectively,by regulating Wnt/β-catenin signaling pathway and TGF-β/Smad signaling pathway in vitro and promoting the occurrence and development of pulmonary fibrosis.3.When pulmonary fibrosis is combined with lung cancer,the progression of pulmonary fibrosis in mice is more severe.The activation of Wnt/β-catenin signaling pathway and TGF-β/Smad signaling pathway in lung tissues indicates that the lung cancer might accelerate the progression of PF via Wnt/β-catenin signaling pathway and TGF-β/Smad signaling pathway. |
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