纳米氧化锌颗粒通过激活NLRP3炎症小体诱导小胶质细胞炎症反应
Contribution of activating NLRP3 inflammasome to zinc oxide nanoparticles induced inflammation in microglia cells
-
摘要:
背景 随着纳米颗粒的广泛应用，其安全性已引起人们关注。已有文章报道纳米颗粒能够诱导炎症因子分泌水平增高，但纳米颗粒对核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）炎症小体激活的相关研究较少。
目的 探讨纳米氧化锌颗粒（nano-ZnO）暴露对小胶质细胞NLRP3炎症小体激活及炎症因子分泌的影响。
方法 取对数生长期的小胶质细胞BV2暴露于不同浓度的nano-ZnO（0、2.5、5.0、10.0、15.0、20.0 mg·L-1），采用四甲基偶氮唑盐（MTT）比色法检测各组细胞存活率，检测乳酸脱氢酶（LDH）活性判断细胞膜完整性，采用酶联免疫吸附试验（ELISA）检测各组细胞培养液中白介素（IL）-1β、IL-18分泌水平，采用Western blotting法测定各组细胞NLRP3、前半胱天冬酶-1（pro-caspase-1）、凋亡相关斑点样蛋白（ASC）和活化型半胱天冬酶-1（cleaved caspase-1）蛋白表达水平。
结果 随着nano-ZnO暴露浓度的增加，BV2细胞活力降低（
r =-0.814，P < 0.05），且与对照组相比，15.0、20.0 mg·L-1 nano-ZnO暴露组细胞活力下降（P < 0.05）。细胞培养液上清中LDH活性随nano-ZnO暴露浓度的增加而升高（r =0.962，P < 0.05）。与对照组相比，2.5、5.0、10.0、15.0 mg·L-1 nano-ZnO组细胞均出现IL-1β和IL-18分泌水平升高（均P < 0.05），且具有明显的剂量-效应关系（r IL-1β=0.919，P < 0.05；r IL-18=0.994，P < 0.05）。Western blotting检测结果显示，各暴露组NLRP3、pro-caspase-1、ASC以及cleaved caspase-1的蛋白表达水平均随浓度增加而升高（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）；且与对照组相比，5.0、10.0、15.0mg·L-1 nano-ZnO暴露致NLRP3及cleaved caspase-1表达水平升高，10.0、15.0mg·L-1 nano-ZnO暴露致ASC及pro-caspase-1表达水平升高（均P < 0.05）。结论 nano-ZnO暴露可激活小胶质细胞NLRP3炎症小体并进一步诱导炎症因子表达，增强细胞炎症反应。
Abstract: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.
下载:
