幽门螺杆菌经ROS通路激活NLRP3炎症复合体诱导THP-1细胞分泌IL-1β和IL-18  

Helicobacter pylori induces cytokines IL-1β and IL-18 production in human monocytic cell line through activation of NLRP3 inflammasome via ROS signaling pathway

作　　者：李翔[1] 何跃平[2] 刘胜[1] 罗晶晶[3] 刘硕[1] 张紫柔 姚雯[4] 张艳[1] 

机构地区：[1]南华大学病原生物学研究所,衡阳421001 [2]遵义医学院第五附属珠海医院耳鼻咽喉科,珠海519110 [3]南华大学附属第一医院麻醉科,衡阳421001 [4]南华大学2013级卓越医生班,衡阳421001

出　　处：《中国免疫学杂志》2015年第3期308-313,共6页Chinese Journal of Immunology

基　　金：湖南省教育厅重点项目(11A103);湖南省科技厅科研条件创新专项(2013TT2021);“重要特殊病原体防控”湖南省普通高等学校重点实验室项目[湘教通(2012)312号];“特殊病原体防控”湖南省科技厅重点实验室[湘科计字(2014)5号]项目

摘　　要：目的:研究幽门螺杆菌(H.pylori)对NLRP3炎症复合体活化的影响及活性氧(ROS)在其中的作用。方法:将THP-1细胞与H.pylori SS1共孵育,于不同时间点收集细胞及上清,ELISA检测细胞上清中IL-1β和IL-18的含量;流式细胞术(FCM)检测胞内ROS的产生;实时荧光定量PCR(Real-time PCR)检测细胞中NLRP3、caspase-1 mRNA的表达;Western blot检测细胞中caspase-1活性亚单位p10的表达;检测ROS清除剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)及NLRP3特异性小干扰RNA(small interfering RNA,siRNA)预处理细胞后相关信号分子的表达。结果:H.pylori SS1能以时间依赖性和剂量依赖性方式诱导THP-1细胞产生IL-1β、IL-18和胞内ROS;H.pylori SS1刺激能使THP-1细胞NLRP3和caspase-1 mRNA转录水平显著升高;NAC及NLRP3-siRNA预处理THP-1细胞能显著降低H.pylori SS1诱导的NLRP3炎症复合体相关成份的表达及细胞因子的分泌。结论:H.pylori SS1株通过ROS途径激活NLRP3炎症复合体诱导THP-1细胞分泌IL-1β和IL-18,这可能与机体的先天免疫防御及细菌的致病作用相关。Objective：To investigate the effects of Helicobacter pylori on NLRP3 inflammasomes activation in THP-1 （human monocytic cell line） -derived macrophages and evaluate the role of ROS. Methods：H. pylori strain SS1 was co-cultured with the THP-1- derived macrophages at a multiplicity of infection （MOI） of 1：100 based on trial results with different MOIs （ratios of THP-1 cells to bacteria ranging from 1：25 to 1：200）. The co-culture supernatants and THP-1 cells were collected at various time points （3 h ,6 h, 12 h ,24 h） and cytokine production was quantitated using ELISA analysis. The generation of intraceHular ROS was detected by FCM, and the mRNA transcript levels of NLRP3 and caspase-1 were measured by Real-time PCR. Western blot was employed to analyze the expression of active caspase-1 subunit （pl0）. Then we observed the inhibitory effects of NAC and siRNA specific for NLRP3 on the ex- pression of NLRP3 inflammasome-related components and the secretion of cytokines induced by H. pylori. Results： We found that H. pylori SS1 induced IL-1 {3 and IL-18 production in human acute monocytic leukemia cell line THP-1 in a time- and dose-dependent manner. We further showed that H. pylori could induce the mRNA expressions of NLRP3 and caspase-1 in THP-I cells. Moreover, release of IL-1 [3 and IL-18 from H. pylori-infected THP-1 cells was suppressed by the ROS scavenger NAC,which was an agent known to inhibit NLRP3 inflammasome formation. NAC administration also resulted in a significant decrease in the level of H. pylori-induced caspase-1 protein expression in THP-1 cells. Additionally, secretion of IL-1β and IL-18 in response to H. pylori infection was remarkably reduced by NLRP3-siRNA. Conclusion： The induction of IL-1 fl and IL-18 secretion by H. pylori strain SS1 in THP-1 cells could be mediated through activation of NLRP3 inflammasome via ROS signaling pathway, which may be involved in the host innate immune defence and the pathogenesis of the bacteria.

关 键 词：幽门螺杆菌 活性氧 NLRP3 炎症复合体 IL-1Β IL-18 

分 类 号：R392.32[医药卫生—免疫学]