Contribution of activating NLRP3 inflammasome to zinc oxide nanoparticles induced inflammation in microglia cells

Background The wide application of nanoparticles has raised public concerns about their safety. Studies have shown that nanoparticles could increase the secretion of pro-inflammatory factors, but the influence of nanoparticles on activation of nucleotide-binding oligomerization domainlike receptor protein 3 (NLRP3) inflammasome is rarely reported.

Objective This experiment investigates the effect of zinc oxide nanoparticle (nano-ZnO) exposure on the activation of NLRP3 inflammasome and the expression of inflammatory cytokines in microglia cells.

Methods BV2 cells in logarithmic growth phase were exposed to nano-ZnO at concentrations of 0, 2.5, 5.0, 10.0, 15.0, and 20.0 mg·L^-1, respectively. Cell viability was determined by methyl thiazolyl tetrazolium (MTT) assay. Integrity of cell membrane was evaluated using lactate dehydrogenase (LDH) activity. Secretion levels of interleukin (IL)-1β and IL-18 in the medium of each group were detected by enzyme linked immunosorbent assay (ELISA). Expression levels of NLRP3, pro-caspase-1, apoptosis-associated speck-like protein containing CARD (ASC), and cleaved caspase-1 in each group were determined by Western blotting.

Results With the increase of nano-ZnO doses, the cell viability of BV2 cells was decreased (r=-0.814, P < 0.05). Compared with the control group, the cell viabilities in the 15.0 and 20.0 mg·L^-1 nano-ZnO groups were reduced (P < 0.05). The LDH activity in supernatant was enhanced along with the increasing of nano-ZnO concentrations (r=0.962, P < 0.05). Compared with the control group, the secretion levels of IL-1β and IL-18 were increased in the 2.5, 5.0, 10.0, and 15.0 mg·L^-1 nano-ZnO groups, and there were significant dose-response relationships (r_IL-1β=0.919, P < 0.05; r_IL-18=0.994, P < 0.05). The Western blotting results showed that the expression levels of NLRP3, pro-caspase-1, ASC, and cleaved caspase-1 were increased with higher nano-ZnO doses (r_NLRP3=0.897, P < 0.05; r_{pro-caspase-1}=0.758, P < 0.05; r_ASC=0.751, P < 0.05; r_{cleaved caspase-1}=0.723, P < 0.05). Compared with the control group, the expression levels of NLRP3 and cleaved caspase-1 were increased in the 5.0, 10.0, and 15.0mg·L^-1 nano-ZnO groups, and the expression levels of ASC and pro-caspase-1 were increased in the 10.0 and 15.0 mg·L^-1 nano-ZnO groups (P < 0.05).

Conclusion Exposure to nano-ZnO could induce the activation of NLRP3 inflammasome, further increase the expression of inflammatory cytokines, and enhance cell inflammation response.