NOD8对LPS诱导巨噬细胞释放NO、TNF-α和IL-1β的影响

Effect of NOD8 on production of nitric oxide,IL-1β and TNF-α in LPS-treated macrophages

目的:探讨NOD8对脂多糖(LPS)诱导巨噬细胞释放一氧化氮(NO)、肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)的影响。方法:pEGFP-C2及pEGFP-NOD8重组质粒分别转染小鼠巨噬细胞RAW264.7,以LPS刺激RAW264.7细胞0、6、12、24 h后,采用Griess reagent法测定观察细胞分泌的NO水平;ELISA法检测IL-1β和TNF-α的含量;荧光法测定活化的caspase-1水平;Western blotting检测NOD8蛋白表达及NF-κB p65亚基的核转位情况。结果:(1)与转染pEGFP-C2空质粒组比较,转染pEGFP-NOD8质粒组NOD8蛋白表达明显增加。(2)LPS刺激6、12、24 h后,RAW264.7细胞释放NO、IL-1β及TNF-α均明显增加;而在pEGFP-NOD8+LPS组RAW264.7细胞,NO于12、24 h的释放显著降低,IL-1β于6、12、24 h的释放也明显降低,TNF-α的释放则无明显变化。(3)在LPS刺激6、12、24 h后,RAW264.7细胞caspase-1活化水平均明显升高,胞浆NF-κB p65亚基表达明显减少,表明p65核转位增加;而pEGFP-NOD8+LPS组可显著抑制caspase-1的活化以及NF-κB p65亚基的核转位,差异有统计学意义。结论:NOD8可抑制LPS诱导的巨噬细胞NO与IL-1β释放,其作用机制可能与NOD8抑制caspase-1及NF-κB的活化有关。

AIM： To investigate the effect of NOD8 on lipopolysaccharide （LPS）-induced releases of nitric oxide （ NO）, tumor necrosis factor a （TNF-a） and interleukin-1β （IL-1β） in RAW264.7 cells. METHODS： The plas- mids of pEGFP-C2 and pEGFP-NOD8 were transfected into RAW264.7 cells respectively. The transfected and non-trans- fected cells were stimulated by LPS for 0, 6, 12 and 24 h. NO production was evaluated by Griess reagent assay, and the levels of IL-1β and TNF-a were measured by ELISA. The protein expression of NOD8 and the nuclear translocation of nu- clear factor KB （NF-KB） p65 subunit were detected by Western blotting. The level of activated caspase-1 was determined by fluorimetric method. RESULTS ： Compared with pEGFP-C2 group, the protein expression of NOD8 was significantly el- evated in pEGFP-NOD8 ＋ LPS group. The releases of NO, IL-1β and TNF-a were obviously increased after RAW264.7 cells were treated with LPS for 6 h, 12 h and 24 h, and while the secretion of NO was significantly reduced in the ceils transfected with pEGFP-NOD8 and induced by LPS for 12 h and 24 h, and the release of IL-1β was also significantly re- duced at 6 h, 12 h and 24 h. However, no significant difference of TNF-ct release was observed between pEGFP-C2 ＋ LPS group and pEGFP-NOD8 ＋ LPS group. The activation of caspase-1 in RAW264.7 cells stimulated with LPS for 6 h, 12 h and 24 h was markedly increased, and the expression of NF-κB p65 subunit in the cytoplasm was significantly decreased,indicating that p65 nuclear translocation was increased. In addition, the activation of caspase-1 and the nuclear transloca- tion of p65 were significantly inhibited in pEGFP-NOD8 ＋ LPS group. CONCLUSION ： NOD8 suppresses the releases of LPS-induced NO and IL-113 in RAW264.7 cells by inhibiting the activation of caspase-1 and NF-κB.