乳铁蛋白负向调控TLR2激动剂诱导的人肺上皮细胞细胞因子分泌  

Lactoferrin negatively regulates TLR2 agonist-induced secretion of cytokines in human lung epithelial cells

作　　者：曹二龙 谭潇 刘选梅 肖非 马婷 贺印旎 CAO Erlong;TAN Xiao;LIU Xuanmei;XIAO Fei;MA Ting;HE Yinni(Department of Medical Laboratory Technology,College of Medical Laboratory,Shaoyang University,Shaoyang 422000,China)

机构地区：[1]邵阳学院医学检验学院医学检验技术教研室

出　　处：《免疫学杂志》2019年第9期780-784,共5页Immunological Journal

基　　金：邵阳市科技计划(2017ZD08);2016年度校级大学生研究性学习和创新性实验计划(校发[2016]11号)

摘　　要：目的观察乳铁蛋白对Toll样受体2(TLR2)激动剂Pam3CSK4刺激人肺上皮细胞分泌促炎细胞因子的影响,并探索其潜在的机制。方法体外培养人肺上皮细胞系NCI-H292,采用100ng/ml Pam3CK4联合100、200和400μg/ml乳铁蛋白孵育细胞。ELISA检测乳铁蛋白处理前后培养上清TNF-α和IL-8分泌的变化;实时定量PCR和ELISA检测短腭、肺及鼻咽上皮克隆1(SPLUNC1)蛋白和mRNA表达的影响。提取细胞核蛋白,ELISA测定乳铁蛋白处理前后NF-κB活性的变化,采用Western blot检测IκB的表达;并用siRNA沉默NCI-H292细胞SPLUNC1,观察其对TNF-α和IL-8分泌的影响。结果100、200和400μg/ml乳铁蛋白预处理肺上皮细胞后,可下调Pam3CK4诱导分泌TNF-α和IL-8。实时定量PCR结果显示,100、200和400μg/ml乳铁蛋白处理后,SPLUNC1mRNA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。采用转录和翻译抑制剂放线菌素D(ActD)和放线菌酮(CHX)预处理,可显著降低乳铁蛋白诱导SPLUNC1的表达。此外,乳铁蛋白处理可抑制Pam3CK4激活NF-κB,主要表现为NF-κB的DNA结合活性降低、IκB降解减少。siRNA沉默SPLUNC1表达后,可明显削弱乳铁蛋白对TNF-α和IL-8的抑制作用。结论乳铁蛋白对Pam3CK4诱导的细胞因子分泌具有抑制作用,其机制与上调SPLUNC1表达、抑制NF-κB的活性有关。This study was performed to investigate the effect of lactoferrin (LF) on the secretion of proinflammatory cytokines by human lung epithelial cells stimulated by Toll-like receptor 2 (TLR2) agonist Pam3CSK4,and to explore its potential mechanism.Human lung epithelial cell line NCI-H292 was cultured in vitro,and then incubated with 100 ng/ml Pam3CK4 combining with 100 μg/ml,200 μg/ml or 400 μg/ml LF.The changes of TNF-α and IL-8 secretion in culture supernatant before and after LF treatment were detected by ELISA, and the changes in the protein and gene expression of short palate,lung and nasopharyngeal epithelial clone 1 (SPLUNC1) were detected by real-time quantitative PCR and ELISA.Nucleoprotein was extracted and the activity of NF-κB was measured by ELISA before and after LF treatment.The expression of IκB was detected by Western blot;the effect of siRNA on the secretion of TNF-α and IL-8 was observed by silencing SPLUNC1 in NCIH292 cells.Data showed that pretreatment with 100, 200 and 400 μg / ml LF could down-regulate the Pam3CK4-induced TNF-α and IL-8 secretion.Real- time quantitative PCR results showed that after treatment with 100,200 and 400 μg/ml LF,the level of SPLUNC1 gene was significantly increased in a dose-dependent manner,and similar results were obtained by ELISA. Pretreatment with transcription,translation inhibitors actinomycin D (ActD) and actinomycin ketone (CHX) significantly reduced the expression of SPLUNC1 induced by LF.In addition,LF treatment inhibited the activation of NF-κB by Pam3CK4,which was mainly manifested in the decrease of DNA binding activity of NF-κB and the increase of IκB expression.Furthermore,silencing SPLUNC1 expression with siRNA could significantly weaken the inhibitory effect of LF on TNF-α and IL-8.In conclusion,LF can inhibit Pam3CK4-induced cytokines secretion by up-regulating the expression of SPLUNC1 and inhibiting the activity of NF-κB.

关 键 词：乳铁蛋白 TOLL样受体2 细胞因子 

分 类 号：R392.9[医药卫生—免疫学]