SPLUNC1负向调控肺炎克雷伯菌夹膜多糖诱导细胞因子分泌

SPLUNC1 negatively regulates Klebsiella pneumoniae capsular polysaccharide-induced secretion of cytokines in human lung epithelial cells

目的:观察肺炎克雷伯菌夹膜多糖(CPS)对人肺上皮细胞表达短腭、肺及鼻咽上皮克隆1(SPLUNC1)的影响,并探讨其在介导细胞因子分泌中的作用。方法:培养肺炎克雷伯菌并提取其CPS,将其与体外培养人肺上皮细胞系NCI-H292孵育。实时定量PCR和ELISA检测SPLUNC1蛋白和mRNA表达的影响。提取细胞核蛋白,ELISA测定CPS处理前后NF-κB活性的变化,采用Western blot检测IκB的表达,并采用NF-κB抑制剂PDTC预处理,观察其在SPLUNC1表达中的作用。ELISA检测CPS处理前后培养上清TNF-α和IL-8分泌的变化;并采用siRNA沉默NCI-H292细胞SPLUNC1,观察其对TNF-α和IL-8分泌的影响。结果:实时定量PCR结果显示,0.5~3μg/ml CPS处理后,SPLUNC1 mRNA水平明显增高,并呈一定的剂量依赖性,ELISA也得到了类似的结果。0.5~3μg/ml CPS也能激活NF-κB,主要表现在NF-κB的DNA结合活性增高、IκB表达减少。同时,CPS也能诱导肺上皮细胞分泌TNF-α和IL-8,采用NF-κB抑制剂PDTC预处理细胞后,细胞因子分泌明显减少。此外,采用siRNA沉默SPLUNC1表达后,TNF-α和IL-8的分泌有所增多。结论:肺炎克雷伯菌夹膜多糖可经激活NF-κB诱导人肺上皮细胞系表达SPLUNC1,后者能在一定程度上负向调控TNF-α和IL-8分泌。

Objective:To observe the effect of Klebsiella pneumoniae capsular polysaccharide(CPS)on the expression of short palate,lung and nasal epithelial clone 1(SPLUNC1)in human pulmonary epithelial cells,and to explore its role in mediating cytokine secretion.Methods:Klebsiella pneumoniae was cultured and its CPS was extracted.The CPS was incubated with human lung epithelial cell line NCI-H292 in vitro.Real-time quantitative PCR and ELISA were used to detect the expression of SPLUNC1 protein and mRNA.Nucleoprotein was extracted and the activity of NF-κB was measured by ELISA before and after CPS treatment.The expression of IκB was detected by Western blot and pretreated by PDTC,an inhibitor of NF-κB,to observe its role in the expression of SPLUNC1.The changes of TNF-αand IL-8 secretion in culture supernatants before and after CPS treatment were detected by ELISA,and the effects of siRNA on TNF-αand IL-8 secretion were observed by silencing SPLUNC1 of NCI-H292 cells.Results:Real-time quantitative PCR showed that after 0.5-3μg/ml CPS treatment,the level of SPLUNC1 mRNA increased significantly,and showed a dose-dependent manner.ELISA also obtained similar results.0.5-3μg/ml CPS could also activate NF-κB,mainly in the increased DNA binding activity of NF-κB and the decreased expression of IκB.At the same time,CPS could also induce lung epithelial cells to secrete TNF-αand IL-8.After pretreatment with PDTC,the secretion of cytokines decreased significantly.In addition,the secretion of TNF-αand IL-8 increased after SPLUNC1 was silenced by siRNA.Conclusion:Klebsiella pneumoniae entrapment polysaccharide can induce the expression of SPLUNC1 in human lung epithelial cell lines by activating NF-κB,which can negatively regulate the secretion of TNF-αand IL-8.