| Background Zinc Oxide Nano-particles(ZnO-NPs) is a new type of high-functional inorganic product, which has the characteristic that the macroscopic objects do not have, such as the surface effect, quantum size effect, macroscopic tunnel effect and high dispersability. It is widely used in many fields about optics,chemicals, electronics, ceramics, biology, medicine, national defense and others. At the same time, the toxic effects of ZnO-NPs have also been widespread concerned. ZnO-NPs could enter the body mainly through the respiratory system and induce the inflammatory response in respiratory epithelial cells. NLRP3 inflammasome is a protein complex which is composed by NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC) and caspase-1. The activated NLRP3 inflammasome induced by ROS could cut the pro-IL-1β and pro-IL-18 and promote the maturation and secretation of them, which induce the secretion of other inflammatory factors and promote the occurrence and development of inflammatory reactions. Studies have confirmed that the activation of nuclear factor Kappa B(NF-κB) could regulate the activation of NLRP3 inflammasome and promote the secretion of inflammatory factor. However, there is no research explain that whether the NLRP3 inflammatory could be activated in the inflammatory reactions which induced by ZnO-NPs. We use the ZnO-NPs stimulate A549 cells to study the effect and mechanism of the activation of NLRP3 inflammasome in the inflammatory induced by ZnO-NPs.Method 1. The oxidative damage effects in A549 cells induced by ZnO-NPs. A549 cells were stimulated by ZnO-NPs with different concentrations(0、10、20、40 μg/m L) for 24 hours. Cells were collected after exposure. The intracellular ROS levels were detected by flow cytometry. The contents of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) in the cells were respectively measured by the detection assay kit of MDA and SOD. 2. The levels of activation of NF-κB in the A549 cells induced by ZnO-NPs.A549 cells were treated by ZnO-NPs with different concentrations for 24 hours. The phosphorylation p65 in the cells was detected by Western Blot in order to reflect the level of activation of NF-κB. 3. The levels of the activation of NLRP3 inflammasome in the A549 cells. A549 cells were respectively dealwith by ZnO-NPs with different concentrations for 12 hours,the cells were collected after exposure and detected the expression levels of NLRP3 m RNA by Real-time fluorescent quantitative polymerase chain reaction(Real-time PCR). The A549 cells were induced by ZnO-NPs with different concentrations for 12 hours and collected after the exposure. The proteins expression levels of NLRP3 and activated subunit p10 of caspase-1 were detected by Western Blot. The expression levels of inflammatory cytokines(IL-1β, IL-18) in A549 cell culture supernatants were measured by Enzyme Linked Immuno-sorbent Assay(ELISA). 4. The influences of inhibitor in the pathway. 4.1 The activation of NF-κB and NLRP3 inflammasome in A549 cells were inhibited by the N-acetyl-L-cysteine(NAC) of ROS inhibitor. The A549 cells were pretreated with NAC, and then exposed to ZnO-NPs for 24 hours. The cells and cultural supernatant were collected after the exposure. The expression levels of phosphorylation p65, NLRP3 and caspase-1 activated subunit p10 were detected by Western Blot. The expression levels of IL-1β and IL-18 in supernatants were detected by ELISA. 4.2 The levels of ROS and NLRP3 inflammasome in the A549 cells were inhibited by the BAY11-7082 of NF-κB inhibitor. A549 cells were pretreated with BAY11-7082 and then exposed to ZnO-NPs with different concentrations for 8 hours. The cells were collected after the exposure and the level of ROS were measured by low cytometry. A549 cells were pretreated with BAY11-7082 and then exposed to ZnO-NPs with different concentrations for 24 hours. The cells and cultural supernatant were collected after the exposure and the expression levels of NLRP3 and caspase-1 activated subunit p10 were detected by Western Blot. The expression levels of inflammatory cytokines IL-1β and IL-18 in supernatants were detected by ELISA. 4.3 The expression levels of inflammatory cytokines of IL-1β and IL-18 in the A549 cells were inhibited by the Glibenclamide of NLRP3 inhibitor. A549 cells were pretreated with Glibenclamide then exposed to ZnO-NPs with different concentrations for 24 hours. The expression levels of inflammatory cytokines of IL-1β and IL-18 in the supernatants were detected by ELISA.Results 1. The oxidative damage effects in A549 cells induced by ZnO-NPs. The results showed that in the same dose the expression levels of ROS and MDA were increased accompanied to the exposure times, the differences were statistical significance(P <0.05). In the same time, the contents of ROS and MDA were increased accompanied to the increasing of exposure dose, the differences were statistical significance(P <0.05). The activity of SOD were decreased with the increasing of exposure time and dose, the differences were statistical significance(P <0.05). 2. The levels of the activation of NF-κB in the A549 cells induced by ZnO-NPs. The results showed that the levels of phosphorylation p65 in A549 cells of 0, 10, 20, 40 μg/m L respectively were 0.23, 0.45, 0.96, and 1.12. It indicated that the activition of NF-κB were related to the increased of the exposure dose. 3. The levels of the activation of NLRP3 inflammasome in the A549 cells induced by ZnO-NPs. 3.1 The levels of the activation of NLRP3 in the A549 cells induced by ZnO-NPs. The results of Real Time-PCR showed that accompany with the elevation of exposure dose, the expression levels of NLRP3 m RNA were increased, the differences were statistical significance(P <0.05). The result of Western Blot indicated that accompanied with the increased of exposure dose the levels of the activation of NLRP3 inflammasome and p10 were raised. 3.2 The expression levels of inflammatory cytokines IL-1β, IL-18 in A549 cells induced by ZnO-NPs. The results indicated that with the increasing of exposure dose, the expression levels of inflammatory cytokines IL-1β and IL-18 were elevated. Compare to 0 μg/m L, the the higher expression levels of inflammatory cytokines IL-1β and IL-18 in 20 μg/m L and 40μg/m L were statistical significance(P <0.05). The difference between 40 μg/m L and 10 μg/m L were statistical significance(P <0.05). 4. The effect of inhibitor 4.1 The effect of NAC of ROS inhibitor on the expression of NF-κB and NLRP3 inflammasome. The expression levels of phosphorylation p65, NLRP3 and p10 of the exposure groups were higher than the control groups, so were the levels of cytokines IL-1β and IL-18. Compare to the exposure group, the levels of cytokines IL-1β and IL-18 in the NAC inhibitor group were downregulated, the difference were statistical significance(P <0.05). 4.2 The effect of BAY11-7082 of NF-κB inhibitor on the expression of ROS and NLRP3 inflammasome. The difference of expression levels of ROS between the exposure(9543.44±272.81) and the BAY11-7082 inhibitor group(9042.65±314.19) were no statistical significance(P =0.07). The result of WB indicated the BAY11-7082 could downgrade the expression levels of phosphorylation p65, NLRP3 and p10 in the exposure group. The results of ELISA show that the levels of cytokines IL-1β and IL-18 in the exposure group were(1.60±0.09) μg/m L and(739.02±29.40) pg/m L and the inhibitor group were(1.08±0.12) μg/m L and(331.27±70.65) pg/m L, The difference were statistical significance(P<0.05). 4.3 The effect of Glibenclamide of NLRP3 inhibitor on the expression of cytokines IL-1β and IL-18. The levels of cytokines IL-1β and IL-18 in the exposure group were(1.60±0.09)μg/m L and(739.02±29.40)pg/m L, they are higher than the control group. After the application of Glibenclamide, the levels of cytokines IL-1β and IL-18 in the inhibitor were( 0.96±0.10) μg/m L and( 173.02±20.44) pg/m L. Glibenclamide could down regulate the levels of cytokines IL-1β and IL-18, the differences were statistical significance(P <0.05).Conclusion ZnO-NPs could induce the activation of the NLRP3 inflammasome in A549 cells, and the activation of NLRP3 inflammasome may be through the ROS-NF-κB-NLRP3 signaling pathway. |
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