Secretome Study Of Silica Induced RAW264.7 Macrophages Polarization

BackgroundSilicosis,as a kind of the most prevalent and serious occupational pneumoconiosis diseases,caused by long-term inhalation of crystalline silica,which mainly characterizes silicotic nodule and pulmonary fibrosis.Recently,the morbidity and mortality of silicosis were significantly increased.SiO₂ as a carcinogen is considered as an important role in the pathogenesis of lung cancer.Even if the patient is no longer exposed to silica particles,pulmonary fibrosis cannot be reversed and lack of effective treatment.Many studies explored the relationship between macrophages and SiO₂ by establishing in vitro and in vivo models.However,detailed mechanism of activation of macrophages for exposure by SiO₂was not still clear.ObjectiveTo clarify the role of macrophages in early inflammatory response of silicosis,we used proteomic methods to study the change of secretome in RAW264.7 macrophages.It provides a new theoretical basis for determining the mechanism of SiO₂ induced macrophage inflammatory reaction.MethodsIn this study,we used iTRAQ coupled 2D LC-MS/MS to study the change of secretome in RAW264.7 macrophages.Signaling pathways and the altering expressed secretory proteins were screened in RAW264.7 cells exposure to silica particles using quantitative secretome,and map the signaling network to clarify the mechanism of activated macrophages.Results1.Compared to the control group,the viability rates of cells treated with 25,50,100,150,200μg/ml SiO₂ for 24 hours were significantly decreased（P<0.05）.Fluorescence staining showed that the cells presented obvious morphological changes,such as nuclear condensation,fragmentation,small apoptotic bodies,showing typical apoptotic features.The cells cycle results confirmed that SiO₂ could generate the S phase arresting in cells compared with that of control groups and induce apoptosis（P<0.05）.2.The confocal results showed that the intensity red fluorescence in cells treated with 50μg/ml SiO2 for 24 h was significantly increased compared with that of corresponding to control group.Comparing to control group,the proportion of CD11b⁺CD86⁺cells was obviously increased from 3.28%to 20.3%（P<0.05）.3.A total of 3522 and 3413 proteins were successfully identified and quantified in MS analysis.By prediction of secreted proteins in 3413 quantified proteins,2321 proteins were regarded as secretory proteins and applied to the subsequent analysis,which accounted for68.00%of all quantified proteins in MS data.By setting a 2-fold change as the cutoff value for significant up-or down-regulation of the proteins in this study,we screened 330differentially expressed proteins;of those,210 proteins presented down-regulated expression and 120 proteins presented up-regulated expression.4.By GO enrichment analysis,altering expressed proteins were involved in many vital biological processes,including oxidative stress,mitochondrial damage,cell apoptosis and acute inflammatory response.5.Based on the MS results,the expression of differentially expressed proteins in cell lysates and culture supernatant were further validated by Western blot and ELISA.The levels of mitochondrial apoptosis-related proteins p-AKT and Bax were increased compared with the control group,respectively（P<0.05）.The expression of iNOS as a marker of M1 type in macrophage polarization was remarkably raised,however,the expression of Arg1 as a marker of M2 type was not change.In the SiO₂ treatment groups,the expression levels of TNF-α,RIP2,NF-κB were substantially elevated compared with the control group（P<0.05）,while the expression of IκB was significantly decreased.The levels TNF-αin cellular supernatants were measured by ELISA,and treatment with SiO₂ led to an obviously increase in macrophages（P<0.05）.Conclusion1.By GO enrichment analysis,altering expressed proteins were involved in many vital biological processes,including oxidative stress,mitochondrial damage,cell apoptosis and acute inflammatory response.2.SiO₂ could induce cells apoptosis by oxidative stress and activation of dependent-mitochondrial pathway.3.SiO₂ could induce macrophage polarization by activation of NOD-RIP2-NF-κB signaling pathway in RAW264.7 macrophages.