摘要
目的探讨细胞活性氧(reactive oxygen species, ROS)在幽门螺杆菌(Helicobacter pylori,Hp)诱导胃上皮细胞NLRP3炎症小体激活中的作用。方法培养Hp国际标准株NCTC11639和胃黏膜上皮细胞株GES-1。用Hp感染细胞GES-1 6 h,采用活性氧检测试剂盒检测细胞内ROS;用25 mmol/L的活性氧ROS清除剂N-乙酰半胱氨酸(N-acetylcysteine, NAC)预处理GES-1细胞30 min,再按照MOI为100∶1、200∶1加入Hp与细胞共孵育6 h和12 h,通过Western blot检测细胞内NLRP3、caspase-1 p10、ASC蛋白水平,采用ELISA检测上清液中IL-1β含量;实验设未感染对照组。结果与未感染组比较,Hp感染组细胞内ROS生成增多;无论是感染6 h还是感染12 h, NLRP3、caspase-1及ASC蛋白的表达及细胞培养上清液中IL-1β的浓度均随着感染复数的增加而增加(均P<0.05);NAC预处理后再用Hp感染,细胞NLRP3、caspase-1、ASC蛋白表达降低,IL-1β的浓度也降低(均P<0.05);Hp感染6 h和12 h结果一致。结论 ROS途径可能参与Hp对GES-1细胞NLRP3炎症小体的激活。
Objective To explore the role of ROS in Helicobacter pylori-induced NLRP3 inflammasome activation of gastric epithelial cells. Methods The NCTC11639 international standard strain of H. pylori and the GES-1 gastric mucosal epithelial cell line were cultured. After H. pylori infected GES-1 cells for 6 h, an ROS detection kit was used to detect intracellular ROS. GES-1 cells were pretreated with 25 mmol/L ROS scavenger N-acetylcysteine(NAC) for 30 min, and then H. pylori was added to the cells at an MOI of 100:1 and 200:1. After incubation for 6 h and 12 h, the levels of NLRP3, caspase-1 p10, and ASC protein in the cells were determined using Western blotting, and IL-1β content in the supernatant was measured using an enzyme-linked immunosorbent assay. Results Compared to the uninfected cells, ROS production increased in cells infected with H. pylori. The expression of NLRP3, caspase-1 and ASC protein and the concentration of IL-1β in cell culture supernatant increased with an increase in the MOI, and differences in levels were significant(P<0.05). After NAC pretreatment and H. pylori infection, the expression of NLRP3, caspase-1, and ASC protein and the concentration of IL-1β decreased significantly(P<0.05);The results were consistent between 6 h and 12 h after H. pylori infection(P<0.05). Conclusion The ROS pathway may be involved in the activation of NLRP3 inflammasomes in GES-1 cells as a result of H. pylori infection.
作者
谢渊
刘正美
周建奖
赵艳
鲍利雅
王琴容
XIE Yuan;LIU Zheng-mei;ZHOU Jian-jiang;ZHAO Yan;BAO Li-ya;WANG Qin-rong;无(Key Laboratory of Endemic and Ethnic Diseases,Ministry of Education&Key Laboratory of Medical Molecular Biology,Guizhou Medical University,Guiyang,Guizhou,550004,China;Affiliated Hospital,Guiyang Medical University;Maternity and Child Health Care Hospital)
出处
《中国病原生物学杂志》
CSCD
北大核心
2021年第4期379-383,共5页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.31960028,31660031)
贵阳市科技计划项目(筑科合同[2017]30-4)
贵州省科技基金项目(黔科合基础[2020]1Z010)
贵州省科技厅中央引导地方科技发展专项(黔科中引地[2019]4008号)。