摘要
目的以佛波酯(PMA)诱导分化的THP-1巨噬细胞为模型,探讨NLRP3炎性小体在幽门螺杆菌Tipα蛋白诱导炎性细胞因子分泌中的作用。方法采用重组纯化Tipα蛋白刺激巨噬细胞,ELISA检测其促炎细胞因子TNF-α、IL-1β和IL-18的分泌水平。分别用NF-κB通路阻断剂PDTC和ROS清除剂NAC预处理细胞,ELISA检测预处理前后Tipα诱导细胞分泌促炎细胞因子水平差异,Western Blot检测NLRP3和Caspase-1分子的表达水平差异。结果 Tipα蛋白可显著诱导巨噬细胞产生TNF-α、IL-1β和IL-18;Tipα蛋白浓度为40μg/mL时,刺激细胞6h后,TNF-α、IL-18和IL-1β表达水平接近峰值(P<0.05)。特异性阻断NF-κB信号通路后,巨噬细胞分泌的促炎细胞因子及Caspase-1和NLRP3分子的表达水平均较阻断前明显降低,ROS清除剂NAC预处理细胞后,巨噬细胞分泌的IL-18和IL-1β较处理前有明显降低(P<0.05),TNF-α无明显变化;Western Blot结果显示Caspase-1和NLRP3分子的表达水平较阻断前均有明显降低。结论 Tipα能够促进巨噬细胞产生促炎细胞因子IL-1β和IL-18;NLRP3/Caspase-1途径可能参与了Tipα诱导的IL-1β和IL-18分泌。
We preliminarily investigated the role of NLRP3 inflammasome in Helicobacter pylori(H.pylori)Tipα-induced pro-inflammatory cytokines secretion in PMA-differentiated human acute monocytic leukemia cell line THP-1.PMA-differentiated THP-1 cells were treated with pure recombinant Tipαprotein.The secretion levels of TNF-α,IL-1βand IL-18 in supernatant of culture medium were detected by ELISA.Then we blocked MyD88/NF-κB and NLRP3/Caspase-1 pathways by PDTC or the general ROS scavenger,NAC,respectively,and determined the secretion levels of proinflammatory cytokines and the expression levels of NLRP3 and Caspase-1.The results showed that Tipαprotein can significantly induced the secretion of TNF-α,IL-1β and IL-18 in THP-1 cells in a time and dose-dependent manner.Levels of TNF-α,IL-1β and IL-18 approached their peaks at 6 hpost-treatment by 40μg/mL of Tipαprotein(P<0.05).Moreover,the blockade of NF-κB signaling pathway by PDTC can inhibit the secretion of proinflammatory cytokines and the expression of NLRP3 and Caspase-1.When THP-1 cells were pre-treated with ROS scavenger NAC,the Tipα-induced increased IL-1β and IL-18 secretion was obviously eliminated(P<0.05),while TNF-αlevel had no significant difference,and the expression levels of Caspase-1 and NLRP3 also have a significant decrease.Our results demonstrated that Tipαcan promote THP-1-drived macrophages to secrete proinflammatory cytokines TNF-α,IL-1β and IL-18,and the NLRP3/Caspase-1 pathway may be involved in the Tipαproteininduced IL-1β and IL-18 secretion.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2017年第12期1071-1076,共6页
Chinese Journal of Zoonoses
基金
湖南省高校创新平台开放基金项目(No.16K078)
湖南省科教联合基金项目(No.2017JJ5049)
“特殊病原体防控”湖南省科技厅重点实验室资助项目[No.湘科计字(2014)5号]
湖南省分子靶标新药研究协同创新中心资助项目[湘教通(2014)405号
(2015)351号]联合资助~~
