Study On Molecular Mechanisms Of Staphylococcus Aureus Inducing Autophagy And Inflammatory Response In Macrophages Through TLR2/PI3K Signaling Pathway

BackgroundAsthma is one of the most common airway diseases characterized by airway inflammation.The prevalence of asthma is increasing year by year,and the number of global asthma patients has exceeded 300 million.SA,which is the most common gram-positive pathogen in nature,is believed to be one of risk factors in chronic airway inflammatory diseases including asthma.Moreover,emerging evidence shows that TLR2 signaling pathway regulates autophagy and the release of inflammatory cytokines and is involved in the pathogenesis of asthma.Therefore,we observed the effect of TLR2 on autophagy and inflammation response in the SA induced alveolar macrophage RAW264.7 cells.ObjectiveTo investigate the potential molecular mechanisms of Toll-like receptor 2(TLR2)taking part in autophagy and inflammatory response in SA-stimulated macrophage.Methods1.Detection of expression of autophagic proteins,proinflammatory cytokines and various signaling pathway proteins in macrophages stimulated by SA at different timesMouse mononuclear macrophage RAW264.7 cells were stimulated by SA at different times,and Western blot and ELISA were used to detect the expression levels of autophagic proteins and proinflammatory cytokines and various signaling pathway proteins in macrophages stimulated by SA at different times;the number of autolysosomes in macrophages was observed by confocal laser scanning microscopy(CLSM).2.Evaluate the effect of RNA interfering TLR2 on expression of autophagic proteins,proinflammatory cytokines and various signaling pathways protein in macrophages stimulated by SAStimulation of TLR2 si RNA interfering RAW264.7 macrophages with SA for 2h,and Western blot and ELISA were used to detect the expression levels of autophagic proteins and pro-inflammatory cytokines and various signaling pathway proteins in macrophages stimulated by SA;the number of autolysosomes in macrophages was observed by confocal laser scanning microscopy(CLSM).3.Evaluate the effect of PI3 K inhibition on expression of autophagic proteins,proinflammatory cytokines and various signaling pathways protein in macrophages stimulated by SAStimulation RAW264.7 cells with SA for 2 h with PI3 K specific inhibitor 3-MA.The expression levels of autophagic proteins and pro-inflammatory cytokines and various signaling pathway proteins was detected by Western blot and ELISA.The number of autolysosomes was analyzed by confocal laser scanning microscope(CLSM).Results1.SA induces the expression of autophagic proteins,pro-inflammatory cytokines,and various signaling pathway proteins in macrophageThe expression of TLR2,My D88,PI3 K,and p-NF-κB p65 signaling pathway proteins increased significantly in a time-dependent manner when mouse mononuclear macrophage RAW264.7 cells were stimulated with SA at different times;meanwhile,the expression of autophagy proteins Beclin-1,LC3-II and pro-inflammatory cytokines TNF-α,IL-6,as well as the number of autolysosomes in macrophages also increased significantly in a time-dependent manner.2.TLR2 plays an important role in the expression of autophagic proteins,pro-inflammatory cytokines,and various signaling pathway proteins in SA-induced RAW264.7 cellsStimulation of TLR2 si RNA interfering RAW264.7 macrophages with SA for 2h,and the expression of TLR2,My D88,PI3 K,and p-NF-κB p65 signaling pathway proteins was significantly inhibited;at the same time,the expression of autophagy proteins Beclin-1,LC3-II,and pro-inflammatory cytokines TNF-α,IL-6 and the number of expression of autolysosomes in macrophages were also significantly inhibited.3.PI3 K signaling pathway is crucial for the expression of autophagic proteins,pro-inflammatory cytokines,and NF-κB signaling pathway protein in SA-induced RAW264.7 cellsStimulation of PI3 K inhibitor interfering RAW264.7 macrophages with SA for 2h,and the expression of PI3 K,and p-NF-κB p65 signaling pathway proteins was significantly inhibited;at the same time,the expression of autophagy proteins Beclin-1,LC3-II,and pro-inflammatory cytokines TNF-α,IL-6 and the number of expression of autolysosomes in macrophages were also substantially inhibited.Conclusions1.Activation of autophagy and inflammation response in SA-stimulated macrophages.2.TLR2 takes part in mediating the activation of autophagy and inflammation response in SA-stimulated macrophages.3.TLR2 mediates the activation of autophagy and inflammation response through PI3 K signaling pathway in SA-stimulated macrophages.