脂多糖联合三磷酸腺苷对肺上皮细胞来源A549细胞IL-1β分泌的影响及其机制研究

Effect of LPS combined with ATP on the expression of IL-1β of human A549 cells regulated by NLRP3 inflammasome and its mechanism

目的探讨白细胞介素1β(IL-1β)在脂多糖(LPS)联合三磷酸腺苷(ATP)诱导的人肺上皮细胞来源A549细胞中的表达变化及其机制。方法以肺上皮细胞来源的A549细胞作为研究对象,采用Western blotting法确认A549细胞中是否存在NLRP3炎症体各组件蛋白(NLRP3、ASC、caspase-1)的表达。检测不同浓度LPS(0、1、5、10、50、100μg/ml)对A549细胞NLRP3炎症体通路蛋白表达(Western blotting检测)及IL-1β分泌(ELISA法)的影响,选择最佳LPS浓度。采用LPS联合ATP(LPS+ATP)刺激A549细胞,并检测其对NLRP3炎症体通路蛋白表达和IL-1β分泌的影响。采用siRNA干扰A549细胞NLRP3基因,观察LPS+ATP诱导的A549细胞中IL-1β的分泌情况。结果 Western blotting检测结果显示,A549细胞中存在NLRP3炎症体组件蛋白NLRP3、ASC、caspase-1的表达。与对照组比较,LPS单独处理细胞后,NLRP3、pro-IL-1β、caspase-1p45蛋白表达增加(P<0.05),且呈剂量依赖关系,但未检测到活化形式caspase-1p20蛋白的表达及细胞因子IL-1β的分泌。与LPS单独刺激组相比,LPS+ATP刺激组可检测到活化形式的caspase-1p20蛋白表达及细胞因子IL-1β分泌(P<0.05)。采用siRNA干扰NLRP3基因表达可显著抑制LPS+ATP诱导的IL-1β表达(P<0.05)。结论 A549细胞中存在NLRP3炎症体通路,LPS+ATP诱导A549细胞分泌的细胞因子IL-1β在下调NLRP3基因后显著减少,初步提示IL-1β的释放受NLRP3炎症体途径调控。

Objective To investigate the expression and the mechanism of interleukin-1 beta（IL-1β） in human A549 cells regulated by lipopolysaccharide（LPS） combined with adenosine triphosphate（ATP）. Methods The protein expressions of NLRP3, ASC and caspase-1 in A549 cell lines were firstly assessed by Western blotting, and then the effects of different concentrations（0, 1, 5, 10, 50 and 100μg/ml） of LPS on the protein expression of NLRP3 inflammasome pathway and the secretion of IL-1β in A549 cell lines were determined by Western blotting and ELISA, respectively. Secondly, the effects of co-stimulation of LPS and ATP on the activation of inflammasome and the secretion of IL-1β in A549 cell lines were detected. Finally, the effects were observed of siRNA interference to the NLRP3 gene expression in A549 cell lines on the secretion of proinflammatory cytokines-IL-1β induced by LPS＋ATP. Results NLRP3, ASC and caspase-1 proteins were observed in A549 cell lines. The expressions of NLRP3, caspase-1p45 and pro-IL-1β in A549 cell lines increased by treatment of LPS alone compared with that of control group（P〈0.05） in a dosedependent manner, but no expression of caspase-1p20 protein and no secretion of cytokine IL-1β were detected. While the expression of activated caspase-1p20 protein and the secretion of cytokine IL-1β were found by co-stimulation of LPS＋ATP compared with that of LPS alone stimulation group（P〈0.05）. After siRNA interference of the expression of NLRP3 gene, the LPS＋ATP-induced release of proinflammatory cytokines-IL-1β was inhibited significantly（P〈0.05）. Conclusions The NLRP3 inflammasome pathway is present in A549 cell lines. The release of cytokines-IL-1β in LPS＋ATP-induced A549 cell lines decreases after down-regulation of NLRP3 genes, implying the release of cytokines-IL-1β may be regulated by the NLRP3 inflammasome pathway.