猪源E.coli HPI通过NLRP3/ASC/Caspase-1诱导巨噬细胞焦亡的机制  

Mechanism of macrophage pyroptosis induced by porcine-derived E.coli HPI via NLRP3/ASC/Caspase-1 pathway

作　　者：沈珏 邓静 王浩 肖金龙 万全 张博 赵维薇 赵汝[1] 肖鹏 高洪[1] SHEN Jue;DENG Jing;WANG Hao;XIAO Jinlong;WAN Quan;ZHANG Bo;ZHAO Weiwei;ZHAO Ru;XIAO Peng;GAO Hong(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China;College of Food Science and Technology,Yunnan Agricultural University,Kunming 650201,China;Faculty of Animal Science and Technology,Yunnan Agricultural University,Kunming 65020l,China)

机构地区：[1]云南农业大学动物医学学院,云南昆明650201 [2]云南农业大学食品科学技术学院,云南昆明650201 [3]云南农业大学动物科学技术学院,云南昆明650201

出　　处：《中国兽医学报》2024年第3期450-457,共8页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31960692,31660704);2021年度云南省研究生优质课程建设资助项目(2021YJSYZKC05)。

摘　　要：为探讨猪源E.coli高致病性毒力岛(high pathogenicity island,HPI)对巨噬细胞焦亡的影响,以内毒素(LPS)联合三磷酸腺苷(ATP)建立焦亡阳性对照,使用E.coli WT株(E.coli HPI^(+))和前期构建的E.coli HPI敲除株(E.coliΔHPI)感染巨噬细胞,在感染后不同时间点收集细胞,运用PI染色观察细胞膜损伤情况;RT-qPCR测定NLRP3/ASC/Caspase-1焦亡通路中关键因子的mRNA水平;共聚焦观察NLRP3和Caspase-1的炎性复合体的组装;Western blot技术测定GSDMD和cleaved-GSDMD的表达。结果显示,与E.coliΔHPI组相比,E.coli HPI^(+)焦亡通路关键因子NLRP3、ASC、Caspase-1、GSDMD及炎症因子IL-18、IL-1β的mRNA水平均升高;NLRP3和Caspase-1蛋白的表达量升高,共聚焦观察到共定位;E.coli HPI^(+)组的GSDMD及其活性形式cleaved-GSDMD的表达水平高于E.coliΔHPI组。E.coli HPI感染巨噬细胞后,通过上调NLRP3/ASC/Caspase-1通路中关键因子mRNA的水平,诱导NLRP3和Caspase-1炎性复合体的组装,促进cleaved-GSDMD的表达,从而加剧细胞膜损伤,最终诱导巨噬细胞发生焦亡。In order to investigate the effect of porcine-derived E.coli high pathogenicity island(HPI)on pyroptosis of macrophages,endotoxin(LPS)combined with adenosine triphosphate(ATP)was used to establish a positive control of pyroptosis,and macrophages were infected with E.coli WT strain(E.coli HPI)and the previously constructed E.coli HPI knockout strain(E.coliΔHPI).Cells were collected at different time points after infection,and cell membrane damage was determined by PI staining.RT-qPCR was used to determine the mRNA levels of key factors in the pyrosis pathway of NLRP3/ASC/Caspase-1.The assembly of inflammatory complexes of NLRP3 and Caspase-1 was observed through confocal microscopy.Western blot was used to determine the expression of GSDMD and cleaved-GSDMD.The results showed that the mRNA levels of the key factors NLRP3,ASC,Caspase-1,GSDMD and inflammatory factors IL-18 and IL-1βof E.coli HPI pyrosis pathway were increased compared with the E.coli AHPI group.The expression of NLRP3 and Caspase-1 proteins increased,and colocalization was observed under confocal microscopy.The expression levels of GSDMD and its active form,cleaved-GSDMD,were higher in the E.coli HPI group than in the E.coliΔHPI group.The above results suggest that E.coli HPI infection of macrophages induces the assembly of NLRP3 and Caspase-1 inflammatory complexes and promotes the expression of cleaved-GSDMD by upregulating the level of key factor mRNA in the NLRP3/ASC/Caspase-1 pathway,thereby exacerbating cell membrane damage and ultimately inducing pyroptosis in macrophages.

关 键 词：大肠杆菌 高致病性毒力岛 巨噬细胞 细胞焦亡 NLRP3/ASC/Caspase-1通路 

分 类 号：S852.33[农业科学—基础兽医学]