Study On The Role And Mechanism Of RAB6 In The Pathogenesis Of PM2.5-Induced Pulmonary Fibrosis

Objective:Pulmonary fibrosis is a interstitial lung disease characterized by chronic non-specific inflammation and a large amount of collagen fiber deposition leading to destruction of lung function.The pathogenesis of IPF is poorly understood,and there is currently no effective treatment for this fatal disease,which seriously threatens human health.Ras-related protein(RAB6)can regulate cell signal transduction and protein transport.Whether RAB6 is involved in the development of pulmonary fibrosis is unknown.The purpose of this study is to clarify the effect of RAB6 on the self-renewal ability of alveolar stem cells,reveal the role and mechanism of RAB6-regulated alveolar stem cells in PM2.5-induced pulmonary fibrosis,and provide new strategies for the diagnosis and treatment of pulmonary fibrosis.Method:1.The clinical IPF and its control lung tissue samples were collected,and the expression of RAB6 gene in the samples was detected by Q-PCR.The lung injury mice model were established by intratracheal injection of PM2.5.The effects of PM2.5 exposure on lung inflammation and fibrosis in mice were evaluated by MASSON staining,hydroxyproline test,ELISA test,and TUNEL staining.The COLOIA and ACTA2 gene expression in lung tissues were detected by Q-PCR,and RAB6 gene and protein expression were detected by Q-PCR,immunohistochemistry and Western blot2.The RAB6 knockout mouse model was constructed,and the lung injury mouse model was constructed by intratracheal injection of PM2.5.The effects of RAB6 knockout on lung inflammation and fibrosis were assessed by MASSON staining,hydroxyproline detection,and ELISA;the effects of RAB6 knockout on oxidative stress in PM2.5-exposed mice were detected by flow cytometry and western blotting;The effect of RAB6 knockout on the death of mouse alveolar epithelial cells was detected by TUNEL,immunofluorescence,transmission electron microscopy and Western blot3.The mouse type 2 alveolar epithelial(AEC2)cells were sorted by flow cytometry,and identified by immunofluorescence,Q-PCR and western blotting.The effect of RAB6 knockout on the proliferation and self-renewal of PM2.5-exposed AEC2 cells was detected by flow cytometry,clone formation and stem cell sphere formation.The effect of RAB6 knockout onthe stem gene expression and wnt pathway of PM2.5-exposed AEC2 cells was detected by flow cytometry,clone formation and stem cell sphere formation.4.The interaction between RAB6 protein and DKK1 protein was detected by Co-immunoprecipitation(COIP)and immunofluorescence co-localization experiment.The effects of DKK1 protein on the proliferation,self-renewal and wnt signal of RAB6 knock-out AEC2 cells were detected by flow cytometry,clone formation,Q-PCR and Western blot AEC2 cells overexpressing RAB6 were constructed by transfection,and the effects of DKK1 inhibitor on the proliferation,self-renewal and wnt signal of AEC2 cells overexpressing RAB6 were detected by flow cytometry,clone formation,Q-PCR and Western blot.The effects of DKK1 inhibitors on lung inflammation and fibrosis in PM2.5-exposed mice were evaluated by MASSON staining,hydroxyproline test,and ELISA testResult:1.Compared with control lung tissue,the expression of RAB6 gene in IPF lung tissue is up-regulated.Compared with control mice,the Ashcroft score of PM2.5-exposed mice was significantly increased,the expression of COL1A2 and ACTA2 genes were up-regulated,the degree of apoptosis was increased,the expression of α-SMA protein was up-regulated,the levels of IL-1β and TNFa in BALF increased,and the levels of RAB6 gene and protein are up-regulated2.Compared with the WT-PM2.5 group of mice,the Ashcroft score,IL-1β and TNFα content,COL1A2,ACTA2 gene and α-SMA protein expression in RAB6-/--PM2.5 group of mice were downregulated.The oxidative stress test results showed that compared with the WT-PM2.5 group,the ROS level in the lung tissue of the RAB6-/--PM2.5 group was significantly reduced,the SOD activity and GSH/GSSG were significantly increased,and the MDA content was significantly reduced,PRDX5 protein expression increased significantly.3.Compared with AEC2 cells in the WT-PM2.5 group,the apoptosis rate of AEC2 cells in the RAB6-/--PM2.5 group was reduced,the cell self-renewal ability,SOX2,OCT4 and NANOG gene expression,β-catenin and c-Myc protein expression were all significantly increased.4.There is an interaction and co-localization between RAB6 and DKK1 protein.Compared with the AEC2 cells in the RAB6-/--PBS group,the apoptosis rate of AEC2 cells in the RAB6-/--DKK1 protein group was significantly increased,the number of clones,SOX2,OCT4 and NANOG gene expression,β-catenin and c-Myc protein expression were significantly increased.Compared with the AEC2 cells in the NC-Gallocyanine group,the proliferation and self-renewal ability of AEC2 cells in the RAB6-Gallocyanine group,β-catenin and SOX2 protein expression,and SOX2,OCT4 and NANOG gene expression were significantly increased.The results of in vivo experiments in mice showed that compared with the PM2.5 group mice,the Ashcroft score,ACTA2 gene expression,α-SMA protein expression,TNFα and IL-1β contents of the PM2.5+Gallocyanine group mice were significantly reduced.Conclusion:1.RAB6 knockout inhibits PM2.5-induced oxidative stress,apoptosis and fibrosis in mouse lung tissue.2.RAB6 regulates wnt signal through DKK1 and participates in the regulation of AEC2 cell proliferation and self-renewal.3.DKK1 inhibitor inhibits pulmonary fibrosis in PM2.5-exposed mice in vivo.