Fig. 5
ZnONPs induced cytotoxicity and innate immune responses through Toll-like receptor (TLR) signaling in THP-1-derived macrophages. THP-1 cells were incubated for 24 h in the presence of 10 ng/ml PMA and then exposed to various concentrations of ZnONPs for different times. A Effects of ZnONPs on THP-1-derived macrophage viability after 24 or 48 h. Cell viability was measured by MTT assays. B ZnONP-induced ROS generation in THP-1-derived macrophages. After being exposed to ZnONPs, the cells were incubated with 10 μM DCFH-DA for 30 min to stain H2O2 and analyzed by flow cytometry. Histograms show the percentages of DCFH-DA fluorescence compared with the control. The data are presented as the mean ± SD from three independent experiments. (*p < 0.05, compared with control) C THP-1-derived macrophages were treated with ZnONPs (10 and 15 μg/ml) for 18 h, and the mRNA expression of TLR1, TLR3 and TLR6 was examined by RT‒qPCR and normalized to GAPDH expression. The data were presented as the mean ± SD from three independent experiments. The results showed that ZnONPs significantly triggered TLR1 and TLR3 activation. D THP-1-derived macrophages were treated with ZnONPs (10 μg/ml) for various times, and NFκB (p-p65) protein expression was analyzed by western blotting. E The mRNA levels of the proinflammatory cytokines TNF-α and IL-1β were determined by RT‒qPCR and normalized to GAPDH expression. The data are presented as the mean ± SD from three independent experiments. ZnONPs increased TNF-α and IL-1β mRNA expression at 18 h. (*p < 0.05, **p < 0.01 compared with control). F THP-1-derived macrophages were treated with ZnONPs (10 μg/ml), and CU-CPT22 or CU-CPT 4a for 16 h, then NFκB (p-p65) protein expression was analyzed by western blotting. CU-CPT22 and CU-CPT 4a are inhibitors specific to TLR1/2 and TLR3, respectively. G Cells were treated with ZnONPs (10 μg/ml), and CU-CPT22 (50 μM) or CU-CPT 4a (30 μM) for 24 h. TNF-α and IL-1β protein 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, #p < 0.05, ##p < 0.01 compared with ZnONPs alone)