PM2.5 Disrupts Autophagy Flow Through TLR4 And Aggravates The Inflammatory Response Of RAW264.7 Macrophages

Objective: To explore the effect and mechanism of PM2.5 on the autophagy and inflammatory response of Raw264.7 macrophages.Methods: Trial grouping: blank control group,PM2.5 group,rapamycin(Rap,autophagy inducer)group,hydroxychloroquine(HCQ,autophagy inhibitor)group,PM2.5+HCQ group,PM2.5+TAK-242 [ Toll-like receptor 4(TLR4)inhibitor] group.Configure different concentrations(0,25,50,75,100 mg/L)of PM2.5 suspension to act on Raw264.7macrophages for 24 hours,and then take 100 mg/L of PM2.5 suspension to act on Raw264.7 macrophages 6 h,12 h,18 h,24 h,36 h,48 h.Detected cell viability with CCK8 kit.Observed autophagy with transmission electron microscope.After Ad-m Cherry-GFP-LC3 B autophagy doublelabeled adenovirus transfected macrophages,the fluorescence expression of each group was observed under a fluorescence microscope.Western blot method to detect the levels of autophagy-related proteins: microtubuleassociated light chain protein 3 ratio(LC3II/I),p62,cathepsin B(CTSB),lysosomal membrane protein 2(LAMP2)and inflammation-related proteins: TLR4,nucleotide binding Domain-like receptor protein 3(NLRP3);ELISA method to detect the levels of inflammatory factors:interleukin(IL)-1β,IL-18,IL-10.Results: The activity of Raw264.7 macrophages decreased with the increase of PM2.5 concentration and the prolonged action time.Compared with the control group,more autophagic vacuoles and autophagosomes were seen in the PM2.5 group under transmission electron microscopy,but autolysosomes were rare;and the ratio of yellow fluorescent dots to red fluorescent dots is higher than that of the control group and the Rap group under the fluorescence microscope.(P<0.05).Compared with the blank control group,in PM2.5 group,the expressions of NLRP3,LC3Ⅱ/Ⅰ,p62,CTSB,IL-1β and IL-18 were up-regulated,and the expressions of LAMP2 and IL-10 were down-regulated(P<0.05).while hydroxychloroquine inhibited autophagy,it promoted the expression of NLRP3,IL-1β,and IL-18,and inhibited the expression of IL-10(P<0.05).Compared with the PM2.5 group,the up-regulation of NLRP3,IL-1β,IL-18 and the downregulation of IL-10 in the PM2.5+HCQ group were more significant(P<0.05).In addition,the expression of TLR4 was high in the PM2.5 group,compared with PM2.5 group,the expressions of NLRP3,LC3Ⅱ/Ⅰ,p62,CTSB,IL-1β,and IL-18 were down-regulated after TLR4 was inhibited by TAK-242,and the expressions of LAMP2 and IL-10 were up-regulated(P<0.05).Conclusion: PM2.5 reduces macrophage activity in a time-and concentration-dependent manner;PM2.5 induces autophagy in Raw264.7macrophages,but blocks the autophagic flux,which in turn aggravates the inflammatory response of macrophages,and this process may be mediated by TLR4.