Early Inhibition Of Alveolar Macrophage PRDX4 Expression Reverses Fibrosis

Aim:Silicosis is a lethal occupational disease that leads to the formation of silica nodules and extensive fibrosis in the lungs,for which there is still no effective therapeutic drug.Alveolar macrophages(AMs)are key cells mediating chronic lung inflammation and fibrosis in silicosis,and PRDX4 is one of the six members of the peroxisome family(PRDXs)whose main function is to maintain intracellular environmental homeostasis by scavenging reactive oxygen species(ROS)produced by cellular stress.Recent studies have reported that high PRDX4-specific expression in chronic obstructive pulmonary disease(COPD)promotes further progression of inflammatory and fibrotic responses in the lung,but the exact mechanism of action is unclear.Meanwhile,clinical studies have shown that PRDX4 is highly expressed in AMs of patients with early silicosis.Therefore,this study focused on the role of PRDX4 in the regulation of early inflammation and fibrosis in silicosis and whether PRDX4 can modulate the activity of early AMs to exert therapeutic effects.Methods:1)Exploration of the correlation between PRDX4 and inflammation and fibrosis process in silicosis tissue.a)Search for inflammation and fibrosis genes related to silicosis occurrence using GSEA database with silicosis fibrosis and inflammation as keywords.b)Download of RNA-seq data of silicosis lung tissue from the internet to analyze the correlation between PRDX4 and inflammation and fibrosis process in silicosis.c)Use of C57BL/6 mice to drip nasal CS to construct a self-repair model of early silicosis mice,and H&E,Masson staining and bias light techniques to verify the distribution of lung inflammation,fibrotic lesions and CS particles.d)Real-time fluorescence quantitative PCR technique(RT-qPCR)to detect key inflammatory genes(TNF-α,TGF-β,IL6,IL1α and IL1β)and fibrosis genes(α-SMA,COL1A1 and COL3A1)expression levels in mouse lung tissues after different repair times.e)Real-time fluorescence quantitative PCR technique to detect gene expression levels of six members of the PRDXs family.f)Pearson correlation analysis of the correlation between PRDXs and key inflammatory and fibrosis gene expression.2)Exploration of cellular expression,localization and preliminary function of PRDX4 in silicosis tissue.a)Tissue immunofluorescence staining technique to detect the expression levels of PRDX4 on T cells,macrophages and neutrophils in mouse and human silicosis tissues.b)CS stimulation of mouse(Raw264.7)and human macrophages(THP-1 and PBMC-m)for different times followed by RT-qPCR to detect the expression levels of PRDXs and key inflammatory and fibrotic genes.c)The effect of knockdown of PRDX4 expression on inflammatory and fibrotic gene expression in mouse macrophages after knockdown of PRDX4 expression using si RNA technology.d)Detection of inflammatory and fibrotic gene expression after inhibition of PRDX4 protein activity in human macrophages using PRDX4 protease inhibitor(Conoidin A).3)Investigation of the mechanism of action of PRDX4 in regulating macrophage activity.a)Screening of key transcription factors(TFs)regulating inflammatory and fibrotic genes using transcription factor database(TRRUST).b)Cellular immunofluorescence staining to verify nuclear translocation of key TFs after Conoidin A treatment of CS-stimulated macrophages.c)Protein immunoblotting(Western Blot)to verify the protein expression levels of PRDX4,related kinases(AKT and TAK1)and key TFs after Conoidin A treatment of CS-stimulated macrophages.d)Detection of key TFs expression after treatment of CS-stimulated macrophages using kinase inhibitors and detection of expression of inflammatory and fibrotic genes.4)Exploration of CS-stimulated macrophages to induce epithelial cell differentiation to myofibroblasts in vitro.a)Macrophage/epithelial cell transformation model was constructed using CS-stimulated mouse macrophages(Raw264.7)/mouse lung epithelial cells(MLE12).b)Macrophage/epithelial cell transformation model was constructed using CS-stimulated human-derived macrophages(THP-1 and PBMC-m)/human-derived lung epithelial cells(A549)to construct a macrophage/epithelial cell transformation model.c)CS-stimulated human-derived macrophages(THP-1 and PBMC-m)/human-derived lung fibroblasts(WI-38)to construct a macrophage/myofibroblast transformation model.d)Cell scratch assay to detect the migratory differentiation ability of A549 and WI-38 cells.e)Giemsa staining to detect the proliferation ability of A549 and WI-38 cells.f)RT-qPCR to verify the expression levels of marker genes during epithelial to myofibroblast differentiation.5)Exploration of PRDX4 systemic inhibition(Conoidin A)and AAV adeno-associated virus targeted knockdown of PRDX4 expression in AMs cells of mice with early silicosis(AAV＿Psp146_sh PRDX4)for the treatment of pulmonary fibrosis in silicosis.a)Establishment of PRDX4 systemic inhibition and targeted knockdown of PRDX4 expression in AMs cells of mice with early silicosis,respectively.b)Body weights of mice were recorded daily,and body weights and H&E and Masson staining were compared to verify the therapeutic effects of PRDX4 inhibitors and AAV adeno-associated virus-targeted knockdown of PRDX4 in silicosis.c)RT-qPCR was performed to detect the expression levels of PRDXs and inflammatory and fibrosis-related factors.d)Tissue immunofluorescence staining was performed to detect AAV＿Psp146_sh PRDX4 specific knockdown of the effectiveness of PRDX4 within AMs.e)Tissue immunofluorescence staining technique to detect the expression levels of pulmonary function markers(FAM13A)and compare the effects of PRDX4 inhibitors and AAV adeno-associated viruses on lung function.Results: 1)RNA-seq data from silicosis lung tissues showed that the process of silicosis development includes an early inflammatory phase dominated by AMs and a late fibrosis phase dominated by fibroblasts;PRDX4 was significantly and positively correlated with the process of inflammation and fibrosis;the lungs of mice in the early inflammatory phase had a self-repair function.2)PRDX4 is selectively highly expressed on silicosis lung macrophages;PRDX4 induces macrophage expression of inflammatory factors and promotes myofibroblast activation via the AKT/NF-κB pathway.3)Systemic administration of an early PRDX4 inhibitor(Conoidin A)significantly increased lung self-repair in silicosis mice;AAV＿Psp146_sh PRDX4 viral vector targeted to knock down PRDX4 in AMs not only significantly inhibited lung fibrosis in mice but also had lower side effects.Conclusion: This project elucidates the molecular mechanism by which PRDX4 promotes the silicosis process by activating the AKT/NF-κB pathway to upregulate macrophage inflammatory and fibrotic factor secretion.Inhibition of PRDX4 in AMs of mice with early silicosis can accelerate the repair of lung injury,and targeted inhibition of PRDX4 in AMs may become a novel therapeutic approach for silicosis patients.Figure [8] Table [2] Reference [43]...