PM2.5通过TLR4破坏自噬流加重Raw264.7巨噬细胞 炎症反应.

Objective To explore the effect and mechanism of PM2.5 on the autophagy and inflammatory response of Raw264.7 macrophages. Methods CCK-8 was used to detect the effect of PM2.5 on the viability of Raw264.7 cells, and transmission electron microscopy was used to observe the autophagy structure. The autophagic flux was observed by fluorescence microscope after Ad-mCherry-GFP-LC3B transfection to explore the effect of PM2.5 on macrophage activity and autophagy. Then the blank control group, the PM2.5 group, the rapamycin (Rap, autophagy inducer) group, the hydroxychloroquine (HCQ, autophagy inhibitor) group, the PM2.5+HCQ group and PM2.5+TAK-242 [Toll-like receptor 4 (TLR4) inhibitor] group were set. Western blot method was used to detect levels of autophagy-related proteins, including microtubule-associated light chain protein 3 ratio (LC3Ⅱ/Ⅰ), p62, cathepsin B (CTSB), lysosomal membrane protein 2 (LAMP2) and inflammation-related proteins TLR4 and nucleotide binding Domain-like receptor protein 3 (NLRP3)]. ELISA method was used to detect the levels of inflammatory factors, including interleukin (IL)-1β, IL-18 and IL-10. Results The activity of Raw264.7 macrophages decreased with the increase of PM2.5 concentration and the prolonged action time. Transmission electron microscopy showed that there were more autophagic vacuoles and double-membrane autophagosomes in the PM2.5 group, while autophagic lysosomes were rare, and under the fluorescence microscope, the ratio of yellow fluorescent dots to red fluorescent dots was higher in the PM2.5 group than that in the control group and the Rap group (P＜ 0.05). Compared with the blank control group, the expressions levels of NLRP3, LC3Ⅱ/Ⅰ, p62, CTSB, IL-1β and IL-18 were up-regulated in the PM2.5 group, and the expressions of LAMP2 and IL-10 were down-regulated (P＜0.05). While HCQ 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 blank control group, the PM2.5 group and the HCQ group, NLRP3, IL-1β and IL-18 were significantly up-regulated and IL-10 was significantly down-regulated in the PM2.5+HCQ group (P＜0.05). In addition, the expression of TLR4 was high in the PM2.5 group. Expressions of NLRP3, LC3Ⅱ/Ⅰ, p62, CTSB, IL-1β and IL-18 were down-regulated after TLR4 was inhibited by TAK-242, and 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 Raw264.7 macrophages, but blocks autophagic flow and aggravates the inflammatory response of macrophages, which may be mediated by TLR4. [ABSTRACT FROM AUTHOR]