E.coli HPI通过NLRP3/caspase-1信号通路诱导细胞焦亡而促进肠道炎症  

E.coli HPI promotes enteritis by inducing pyroptosis through NLRP3/caspase-1 signaling pathway

作　　者：张靖松 单春兰 王浩 潘天灵 沈珏 肖金龙 赵汝 肖鹏 高洪 ZHANG Jingsong;SHAN Chunlan;WANG Hao;PAN Tianling;SHEN Jue;XIAO Jinlong;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;College of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学动物医学院,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：《中国病理生理杂志》2024年第10期1777-1787,共11页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.31960692,No.31660704);2021年度云南省研究生优质课程建设项目(No.2021YJSYZKC05);云南省教育厅科学研究基金项目(No.2024Y335)。

摘　　要：目的:探究大肠埃希菌(Escherichia coli,E.coli)强毒力岛(high-pathogenicity island,HPI)对细胞焦亡及肠道炎症的影响。方法:用含HPI的E.coli株(HPI+)、HPI缺失的E.coli株(△HPI)和脂多糖(lipopolysaccharide,LPS)分别处理昆明小鼠和IPEC-J2细胞(猪小肠上皮细胞)。测定小鼠肠道乳酸脱氢酶(lactate dehydrogenase,LDH)活性、超氧化物歧化酶(superoxide dismutase,SOD)活性、IgA表达和分泌性IgA(secretory IgA,SIgA)含量;HE和TUNEL染色观察肠道损伤;RT-qPCR、免疫组织化学染色和Western blot检测小鼠肠组织和IPEC-J2细胞中核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)/caspase-1信号通路关键调控点的表达;ELISA检测小鼠血清和IPEC-J2细胞培养上清液中白细胞介素1β(interleukin-1β,IL-1β)和IL-18含量;共聚焦显微镜观察NLRP3与caspase-1的共定位。siRNA沉默IPEC-J2细胞的NLRP3,验证NLRP3在E.coli HPI感染中的关键调控功能。结果:相对于△HPI感染,E.coli HPI显著降低小鼠肠道SOD活性,增加IgA+B细胞,并促进LDH的释放和SIgA的分泌;ELISA、HE染色和TUNEL染色结果显示,E.coli HPI促进了小鼠肠道上皮细胞DNA断裂、组织损伤和炎症;Western blot结果显示,相对于△HPI,E.coli HPI感染使得小鼠肠道消皮素D的N端片段(gasdermin D N-terminal fragment,GSDMD-N)蛋白水平显著升高;RT-qPCR和免疫组织化学染色结果显示,E.coli HPI显著促进了小鼠肠道和IPEC-J2细胞中NLRP3、含caspase募集结构域的凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a caspase recruitment domain,ASC)、caspase-1、GSDMD、IL-1β和IL-18的mRNA和蛋白表达,IL-1β和IL-18分泌增加;共聚焦显微镜观察发现,相对于△HPI感染,E.coli HPI显著促进了NLRP3炎症小体的组装,使得NLRP3与caspase-1发生共定位;此外,在NLRP3沉默的IPEC-J2细胞中观察到E.coli HPI诱导的细胞炎症、细胞损伤及NLRP3/caspase-1信�AIM:This study aims to explore the impact of Escherichia coli(E.coli)high-pathogenicity island(HPI)on pyroptosis and intestinal inflammation.METHODS:Kunming mice and IPEC-J2 cells(porcine small intestinal epithelial cells)were treated with HPI-containing E.coli strain(HPI+),HPI-deleting E.coli strain(ΔHPI),or lipopolysaccharide(LPS).The intestinal lactate dehydrogenase(LDH)activity,superoxide dismutase(SOD)activity,IgA expression and secretory IgA(SIgA)content were assessed in mice.The expression of key regulatory factors in the nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)/caspase-1 signaling pathway-related proteins in mouse intestinal tract and IPEC-J2 cells was analyzed by RT-qPCR,immunohistochemical staining and Western blot.The levels of interleukin-1β(IL-1β)and IL-18 in mouse serum and IPEC-J2 cell culture supernatants were measured by ELISA.The pivotal role of NLRP3 in HPI+infection was confirmed by silencing NLRP3 in IPEC-J2 cells using siRNA.RESULTS:The HPI+infection markedly decreased SOD activity,increased IgA+B cell count,and induced the LDH release and SIgA secretion in the mouse intestine compared withΔHPI infection.The results of ELISA,HE staining and TUNEL staining indicated that E.coli HPI triggered DNA damage,tissue injury and inflammation in mouse intestinal epithelial cells.Western blot revealed an increase in intestinal gasdermin D N-terminal fragment(GSDMD-N)protein level with HPI+infection compared withΔHPI infection.E.coli HPI significantly up-regulated mRNA and protein expression of NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),caspase-1,GSDMD,IL-1βand IL-18 in mouse intestinal tissues and IPEC-J2 cells,accompanied by the elevated secretion of IL-1βand IL-18.The confocal microscopy demonstrated an enhanced assembly of the NLRP3 inflammasome with HPI+infection compared withΔHPI infection,leading to colocalization of NLRP3 and caspase-1.Furthermore,NLRP3 silencing in IPEC-J2 cells attenuated E.coli HPI-induced

关 键 词：大肠埃希菌 强毒力岛 细胞焦亡 NLRP3/caspase-1信号通路 

分 类 号：R378.21[医药卫生—病原生物学] R574[医药卫生—基础医学] Q255[生物学—细胞生物学]