摘要
目的探究H.pylori空泡毒素VacA对胃上皮细胞NLRP3炎性小体激活的影响。方法采用WT H.pylori26695和△VacA H.pylori26695感染AGS细胞,Western blotting、qRT-PCR、ELISA检测NLRP3、Pro-Caspase-1、Caspase-1、pro-IL-1β和IL-1β的表达。siRNA-NC、siRNA-NLRP3转染AGS后,分别用PBS、WT H.pylori26695和△VacA H.pylori26695刺激,qRT-PCR和细胞因子ELISA检测试剂盒分别检测pro-IL-1β的mRNA表达水平和IL-1β的蛋白表达水平。收集慢性胃炎患者标本,鉴别出H.pylori阳性患者的VacA基因型(s1m1型和s1m2型),通过qRT-PCR检测NLRP3、caspase-1、pro-IL-1β的mRNA表达水平及采用细胞因子ELISA试剂盒检测IL-1β蛋白表达水平。C57BL/6小鼠腹腔注射DMSO或者DMSO+Mcc950,H.pylori菌液灌胃,qRT-PCR和ELISA检测胃组织IL-1β的表达。结果H.pylori感染AGS细胞,与对照组相比,WT H.pylori26695组IL-1β的mRNA和蛋白表达水平均明显升高(P<0.01);△VacA H.pylori26695组与WT H.pylori26695组相比,IL-1β的mRNA和蛋白表达水平均明显下降(P<0.01)。与siRNA-NC组相比,采用WT H.pylori26695和△VacA H.pylori 26695刺激转染siRNA-NLRP3的AGS细胞pro-IL-1β的mRNA水平和IL-1β蛋白表达水平均明显下调(P<0.01)。H.pylori阳性患者较阴性患者NLRP3、Caspase-1和IL-1β的mRNA表达水平明显升高(P<0.01);与VacA s1m2型患者相比,VacA s1m1型患者NLRP3、Caspase-1和IL-1β的mRNA表达水平显著升高(P<0.01),同时IL-1β成熟分泌增加。H.pylori慢性胃炎小鼠动物模型中,与对照组相比,Mcc950组中在WT H.pylori26695和△VacA H.pylori26695灌胃后,pro-IL-1β的mRNA水平和IL-1β蛋白水平表达均明显下调(P<0.01)。结论H.pylori空泡毒素VacA能够激活胃上皮细胞NLRP3炎性小体,促进IL-1β的成熟分泌。
This study aimed to explore the effects of H.pylori VacA on NLRP3 inflammasome activation in gastric epithelial cells.Human gastric adenocarcinoma cell line AGS were stimulated with wild type(WT)H.pylori26695 strain or△VacA H.pylori26695 strain for 6 h,and then the expressions of NLRP3,Caspase-1,pro-Caspase-1,pro-IL-1βand IL-1βwere detected by qRT-PCR,Western blotting and ELISA,respectively.AGS cells were transfected with siRNA-NC or siRNA-NLRP3,and then infected with PBS,WT H.pylori26695 orΔVacA H.pylori26695.The mRNA and protein levels of pro-IL-1βwere detected by ELISA and qET-PCR,respectively.The specimens of patients with chronic gastritis were collected,and the VacA genotypes(s1m1 and s1m2)were identified.The chronic gastritis model of C57BL/6 mice was established by intragastric administration of H.pylori.Compared with VacA s1m2 patients group,the levels of NLRP3,Caspase-1 and pro-IL-1βmRNA in VacA s1m1 patients group were significantly increased(P<0.01).In vitro,the levels of pro-IL-1βmRNA and protein levels in the WT H.pylori26695 group were significantly higher than those of control group(P<0.01).Compared with the WT H.pylori26695 group,the mRNA and protein expression levels of IL-1βwere significantly decreased in theΔvacA H.pylori26695 group(P<0.01).In vitro transfection experiment,pro-IL-1βmRNA were significantly decreased in the siRNA-NLRP3 group compared with the siRNA-NC group.In vivo,the mRNA levels and protein levels of IL-1βwere significantly down-regulated in the Mcc950 group,as compared with the control group(P<0.01).Taken together,H.pylori VacA can activate NLRP3 inflammatory corpuscles in gastric epithelial cells and promote the secretion of IL-1β.
作者
王廷义
左钱飞
张意
朱建儒
赵喆
赵靖涛
兰春慧
WANG Tingyi;ZOU Qianfei;ZHANG Yi;ZHU Jianru;ZHAO Zhe;ZHAO Jingtao;LAN Chunhui(DepartmentofGastroenterology,DapingHospital,ArmyMedicalUniversity,Chongqing400010,China;Department of Microbiology and Biochemical Pharmacy,Army Military Medical University,Chongqing 400010,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2020年第1期58-63,68,共7页
Immunological Journal
基金
国家自然科学基金（81472006）
