PM2.5 aggravates the inflammatory response of Raw264.7 macrophages by disrupting autophagic flow through TLR4

doi:10.11958/20220086

Abstract

Abstract:

Objective To explore the effect and mechanism of PM_2.5 on the autophagy and inflammatory response of Raw264.7 macrophages. Methods CCK-8 was used to detect the effect of PM_2.5 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 PM_2.5 on macrophage activity and autophagy. Then the blank control group, the PM_2.5 group, the rapamycin (Rap, autophagy inducer) group, the hydroxychloroquine (HCQ, autophagy inhibitor) group, the PM_2.5+HCQ group and PM_2.5+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 PM_2.5 concentration and the prolonged action time. Transmission electron microscopy showed that there were more autophagic vacuoles and double-membrane autophagosomes in the PM_2.5 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 PM_2.5 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 PM_2.5 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 PM_2.5 group and the HCQ group, NLRP3, IL-1β and IL-18 were significantly up-regulated and IL-10 was significantly down-regulated in the PM_2.5+HCQ group (P<0.05). In addition, the expression of TLR4 was high in the PM_2.5 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 PM_2.5 reduces macrophage activity in a time- and concentration-dependent manner. PM_2.5 induces autophagy in Raw264.7 macrophages, but blocks autophagic flow and aggravates the inflammatory response of macrophages, which may be mediated by TLR4.

Key words: particulate matter, autophagy, inflammation, Toll-like receptor 4, NLR family, pyrin domain-containing 3 protein, atherosclerosis, PM_2.5

CLC Number:

R364.5

Figures/Tables 8

Tab.1 Comparison of the effect of PM2.5 on macrophage activity at different times between the two groups

组别	0 h	6 h	12 h	18 h	24 h	48 h
对照组	100.0±1.1	99.0±1.2	101.0±1.1	100.0±1.4	100.0±1.3	100.0±1.2
PM_2.5组	100.0±1.6	91.0±2.0	82.3±3.2	67.0±2.4	52.0±2.3	41.0±2.8
t	0.292	1.345^＊	3.363^＊	4.178^＊	4.765^＊	5.193^＊

Fig.1 Observation of autophagy structure under transmission electron microscope (×8 000)

Fig.2 Changes of autophagy in Raw264.7 cells transfected with Ad-mCherry-GFP-LC3B

Fig.3 Western blot detection of LAMP2, LC3, CTSB, p62 protein expression in each group

Tab.2 Comparison of expressions of inflammatory factor levels between the four groups

组别	IL-1β	IL-18	IL-10
对照组	25.47±8.70	25.32±5.79	45.59±7.55
PM_2.5组	156.79±13.77^a	129.71±9.75^a	24.31±3.82^a
HCQ组	124.09±11.46^a	106.57±9.13^a	19.32±3.40^a
PM_2.5+HCQ组	205.55±29.13^abc	218.54±11.56^abc	7.24±2.52^abc
F	55.819^＊＊	217.096^＊＊	61.207^＊＊

Fig.4 Western blot detection of expression levels of autophagy and NLRP3 protein

Tab.3 Comparison of inflammatory factor levels after inhibition of TLR4 between the three groups

组别	IL-1β	IL-18	IL-10
对照组	25.54±8.70	24.99±5.81	46.11±7.56
PM_2.5组	157.19±11.84^a	130.21±9.65^a	23.10±4.12^a
PM_2.5+TAK-242组	74.54±6.47^ab	72.56±7.53^ab	86.92±10.13^ab
F	129.037^＊＊	132.806^＊＊	52.373^＊＊

Fig.5 After inhibiting TLR4, the expression of autophagy and inflammation-related proteins detected by Western blot assay

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