内毒素耐受对核苷酸结合寡聚化结构域2信号通路的影响

Effect of endotoxin tolerance on nucleotide-binding oligomerization domain containing 2 signaling pathway

为研究内毒素耐受对核苷酸结合寡聚化结构域2(nucleotide-binding oligomerization domain containing 2,NOD2)信号通路的影响,将小鼠单核-巨噬细胞RAW264.7分为两组,分别给予小剂量脂多糖(lipopolysaccharide,LPS)(100ng/mL)或磷酸盐缓冲液(phosphate buffered saline,PBS)预处理20h,建立内毒素耐受组和对照组。每组细胞分别给予大剂量LPS(1 000ng/mL)或热灭活烟曲霉孢子刺激,于刺激后0、2、6、12、24h采用定量聚合酶链反应(polymerase chain reaction,PCR)检测细胞NOD2、受体相互作用蛋白2(receptor-interacting protein 2,RIP2)和肿瘤坏死因子α(tumor necrosis factorα,TNF-α)mRNA表达;采用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测细胞上清液中白细胞介素8(interleukin 8,IL-8)和TNF-α浓度。结果显示,内毒素耐受组无论是大剂量LPS还是热灭活烟曲霉孢子刺激均不能增加NOD2、RIP2和TNF-αmRNA表达及细胞上清液中IL-8、TNF-α浓度;而对照组大剂量LPS和热灭活烟曲霉孢子刺激均可提高NOD2、RIP2和TNF-αmRNA表达及细胞上清液中IL-8、TNF-α浓度,尤以刺激后12h增加显著,与刺激前(0h)比较,差异有统计学意义(P<0.05)。结果提示,内毒素耐受可能对NOD2信号通路有抑制作用。

To investigate the potential effect of endotoxin tolerance on nucleotide-binding oligomerization domain containing 2（NOD2）signaling pathway,murine macrophage RAW264.7cells were pretreated with lipopolysaccharide（LPS,100ng/mL）or phosphate buffered saline（PBS）for 20 hto establish endotoxin tolerance group and control group respectively.Both groups were subjected to the treatment with LPS（1 000 ng/mL）or heat-killed Aspergillus fumigatus（HAf）.The mRNA levels of NOD2,receptorinteracting protein 2（RIP2）and tumor necrosis factorα（TNF-α）were measured at 0,2,6,12,24 hafter stimulation by real-time quantitative polymerase chain reaction（PCR）.The concentrations of secreted TNF-αand interleukin 8（IL-8）were also quantified by enzyme-linked immunosorbent assay（ELISA）.The collective results showed that neither LPS nor HAf had any significant effect on NOD2,RIP2,and TNF-αtranscription or TNF-αand IL-8secretion in the endotoxin tolerance group.On the contrary,a clear upregulation was detected in the control group upon either LPS or HAf treatment,concomitant with a peak at 12 hafter stimulation.These data indicate that the NOD2 signaling pathway might be suppressed under endotoxin tolerance conditions.