Study On The Role And Molecular Mechanism Of NLRP3 Inflammasome Involved In The Pathogenesis Of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis(IPF)is a chronic and progressive respiratory disease with unknown etiology,and currently there is no effective treatment in clinical practice.Studies have shown that the overactivation and proliferation of fibroblasts is the direct cause of pulmonary fibrosis,and lung mesenchymal stem cells are the main source of fibroblasts in fibrotic lung tissue.The inflammatory microenvironment of lung mesenchymal stem cells plays an important role in the differentiation of lung mesenchymal stem cells into fibroblasts.The inflammatory microenvironment is formed by a variety of inflammatory factors.It has been found that the activation of NLRP3 inflammasome can participate in the formation of inflammatory microenvironment by promoting the maturation and secretion of various inflammatory factors.Therefore,this study aims to investigate whether the activation of NLRP3 inflammasome is involved in the occurrence of pulmonary fibrosis by regulating the differentiation of pulmonary mesenchymal stem cells,and to clarify the relevant molecular mechanisms.Objectives:1.To explore the relationship between NLRP3 inflammasome and the occurrence of pulmonary fibrosis,and clarify the cellular localization of NLRP3 inflammasome in the fibrotic lung tissue.2.To determine whether activation of NLRP3 inflammasome promotes differentiation of lung mesenchymal stem cells into fibroblasts.3.To explore the specific molecular mechanism of NLRP3 inflammasome activation promoting differentiation of lung mesenchymal stem cells into fibroblasts.Methods:1.Lung tissue specimens of IPF patients and pulmonary fibrosis mice were collected,and H&E staining,Masson staining,immunohistochemistry,Western blot and immunofluorescence techniques were used to test the expression and activation of NLRP3 inflammasomes in the fibrotic lung tissues and the cellular localization of NLRP3 inflammasomes.2.Bleomycin-induced alveolar epithelial cell injury model was established,and Real-time PCR,Western blot and immunofluorescence were used to determine the expression and activation of NLRP3 inflammasomes in the injured alveolar epithelial cells.3.Establish damaged alveolar epithelial cells – lung mesenchymal stem cells conditioned medium system,using MCC950 to inhibit the activation of NLRP3 inflammasome in alveolar epithelial cells,Western blot and ELISA and immunofluorescence technique were used to explore the influence of the NLRP3 inflammasome activated epithelial cells to the differentiation of lung mesenchymal stem cells and its molecular mechanism.4.Bleomycin-induced mouse pulmonary fibrosis model was established,and MCC950 was used to inhibit the activation of NLRP3 inflammasome in fibrotic mice.H&E staining,Masson staining,immunohistochemistry,Western blot and immunofluorescence were used to determine the effects of inhibition of NLRP3 inflammasome activation on pulmonary fibrosis in mice,and verify the results in vitro experiments.Results:1.NLRP3 inflammasomes were activated in the lung tissues of IPF patients and fibrotic mice,and the activated NLRP3 inflammasomes were mainly located in alveolar epithelial cells.2.After bleomycin stimulation of alveolar epithelial cells,the expression of NLRP3 inflammasome components in alveolar epithelial cells was up-regulated and activated.3.NLRP3 inflammasome activated alveolar epithelial cells can induce differentiation of lung mesenchymal stem cells into fibroblasts;activated NLRP3 inflammasomes in alveolar epithelial cells inhibit the expression of secretory protein DKK1 through IL-1β,thereby activating the Wnt/β-catenin signaling pathway in lung mesenchymal stem cells.4.In the bleomycin-induced mouse model of pulmonary fibrosis,MCC950 was used to inhibit the activation of NLRP3 inflammasome in fibrotic mice.Compared with bleomycin group,the expression of DKK1 was upregulated,the Wnt/β-catenin signaling pathway was inhibited,the differentiation of lung mesenchymal stem cells into fibroblasts was inhibited,and the degree of pulmonary fibrosis was reduced in mice.Conclusion:Activation of NLRP3 inflammasome in alveolar epithelial cells can inhibit the expression of DKK1 by secreting IL-1β,thus activating the Wnt/β-catenin signaling pathway in lung mesenchymal stem cells,thereby promoting differentiation of lung mesenchymal stem cells into fibroblasts and accelerating the occurrence of pulmonary fibrosis.