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

[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、15.0 mg·L^-1 nano-ZnO暴露致NLRP3及cleaved caspase-1表达水平升高,10.0、15.0 mg·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。

[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(rIL-1β=0.919, P < 0.05;rIL-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(rNLRP3=0.897, P < 0.05;rpro-caspase-1=0.758, P < 0.05;rASC=0.751, P < 0.05;rcleaved 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 15.0 mg·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.