Fig. 6

ZnONPs obstructed the fusion of autophagosome with lysosome and caused NLRP-3 inflammasome activation leading to IL-1β expression and prolonged skin inflammation. THP-1 cells were differentiated with PMA for 24 h and then exposed to 10 μg/ml ZnONPs for various times. A After being exposed to ZnONPs, THP-1- derived macrophage proteins were extracted, and LC3 II protein expression was assessed by western blotting. GAPDH was used as a loading control. Histograms show the quantification of LC3 II normalized to GAPDH in three independent experiments. The results were expressed as the mean ± SD. B Immunofluorescence assays with LC3 (green) and LAMP-1 (red) antibodies were performed to identify the location of autophagosomes and lysosomes. Scale bars: 50 μm. The fusion of autophagosomes with lysosomes was blocked when cells were exposed to ZnONPs for 24 h. C NLRP3, ASC and Caspase-1 protein expression was analyzed by western blotting, and GAPDH was used as a loading control. Histograms representing the quantification of NLRP3, ASC, caspase-1, and caspase-1 (p20) normalized to GAPDH in three independent experiments. The results were expressed as the mean ± SD. D Analysis of NLRP3 and ASC protein interactions by immunoprecipitation at various time points. E Cells were treated with various concentrations of ZnONPs for 24 h. IL-1β levels in the culture medium were measured by ELISA. The data were presented as the mean ± SD from three independent experiments. (*p < 0.05, **p < 0.01 compared with control). F Mouse skin proteins were collected from different experimental groups after treatment for 72 h. NLRP3 and LC3 protein expression was analyzed by western blotting, and GAPDH was used as a loading control. G Frozen skin sections were collected from the different experimental groups after treatment for 72 h and stained with an antibody against IL-1β. The expression of IL-1β was determined by immunohistochemistry. Scale bars: 200 μm