产耶尔森杆菌素大肠杆菌诱导小鼠十二指肠炎症的研究

Study on the Induction of Duodenum Inflammation in Mice by Yersiniabactin Producing Escherichia coli

【目的】探究耶尔森杆菌素(yersiniabactin,Ybt)在大肠杆菌(Escherichia coli,E.coli)致小鼠十二指肠炎症中的作用,为猪源大肠杆菌诱导肠道炎症的机制提供新的理论依据。【方法】使用R语言功能包挖掘大肠杆菌感染细胞的基因表达综合(gene expression omnibus,GEO)数据库,进行差异表达基因火山图、热图富集和KEGG通路分析;使用分子对接软件预测Ybt与Toll样受体4(Toll-like receptor 4,TLR4)的结合;以注射脂多糖(lipopolysaccharide,LPS)的小鼠为阳性对照,用大肠杆菌ZB-1及前期构建的产Ybt缺陷菌株ZB-1ΔHPI感染小鼠,HE染色观察感染后十二指肠损伤,实时荧光定量PCR检测TLR4、核因子κB(nuclear factor kappa-B,NF-κB)、白细胞介素-18(interleukin 18,IL-18)和IL-1β的mRNA水平;ELISA检测pro-IL-18、pro-IL-1β、IL-18和IL-1β在十二指肠中的分泌水平;免疫组织化学(immunohistochemistry,IHC)染色观察炎性因子IL-18和IL-1β在十二指肠中的表达定位。【结果】GEO数据库火山图及热图分析显示,大肠杆菌感染显著上调了TLR4、NF-κB、IL-1β和趋化因子(CXCL1、CXCL3、CXCL5等)的表达(P<0.05),KEGG富集发现TLR4/NF-κB通路参与了大肠杆菌感染;此外,分子对接显示,Ybt能与TLR4结合;小鼠十二指肠组织HE染色显示,产Ybt大肠杆菌感染诱导了小鼠严重的十二指肠炎症及损伤;实时荧光定量PCR结果显示,ZB-1组的TLR4、NF-κB、IL-18和IL-1βmRNA水平显著高于ZB-1ΔHPI组(P<0.05)。ELISA检测结果显示,与ZB-1ΔHPI组相比,ZB-1组显著促进了pro-IL-Iβ、IL-Iβ、pro-IL-18和IL-18的释放(P<0.05)。同时,IHC结果显示,大量IL-18和IL-1β表达于肠绒毛上皮细胞,产Ybt大肠杆菌感染显著促进了这一结果(P<0.05)。【结论】Ybt具有较强的促炎作用,结合TLR4通过TLR4/NF-κB通路促进大肠杆菌诱导的小鼠十二指肠炎症。

【Objective】The aim of this study was to investigate the effect of yersiniabactin(Ybt)on duodenum inflammation induced by Escherichia coli(E.coli)in mice,and provide new theoretical basis for the mechanism of intestinal inflammation induced by E.coli from pigs.【Method】The R package was used to mine the gene expression omnibus(GEO)datasets of E.coli infection cells for differential gene volcano plot,heatmap enrichment,and KEGG pathway analysis.Molecular docking software was used to predict the binding of Ybt with Toll-like receptor 4(TLR4).Mice injected with lipopolysaccharide(LPS)were used as positive control,and mice were infected with E.coli ZB-1 and previously constructed Ybt-deficient strain ZB-1ΔHPI.Duodenum damage after infection was observed by HE staining.Real-time quantitative PCR was used to detect mRNA levels of TLR4,nuclear factor kappa-B(NF-κB),interleukin 18(IL-18),and IL-1β.ELISA was performed to measure the secretion levels of pro-IL-18,pro-IL-1β,IL-18 and IL-1βin the intestines.Immunohistochemistry(IHC)staining was used to observe the expression and localization of inflammatory factors IL-18 and IL-1βin the intestines.【Result】Volcano plot and heatmap analysis of public databases showed significant upregulation of TLR4,NF-κB,IL-1βand chemokines(CXCL1,CXCL3,CXCL5,and so on)in response to E.coli infection(P<0.05).KEGG enrichment analysis revealed the involvement of TLR4/NF-κB pathway in E.coli infection.Additionally,molecular docking showed that Ybt could bind to TLR4.HE staining demonstrated that E.coli infection with Ybt induction resulted in more severe duodenum inflammation and damage in mice.Real-time quantitative PCR results showed that mRNA levels of TLR4,NF-κB,IL-18,and IL-1βwere significantly higher in ZB-1 group compared to ZB-1ΔHPI group(P<0.05).ELISA results showed that ZB-1 group significantly promoted the release of pro-IL-Iβ,IL-Iβ,pro-IL-18 and IL-18 compared to ZB-1ΔHPI group(P<0.05).Furthermore,IHC results showed abundant expression of IL-18 and IL-1βin duodenum villous epithelial cells,and Ybt producing E.coli infection significantly enhanced this result(P<0.05).【Conclusion】Ybt exhibited a strong pro-inflammatory effect and promotes E.coli-induced duodenum inflammation in mice through the TLR4/NF-κB pathway.These finding providde new theoretical evidence for the mechanism of duodenum inflammation induced by porcine-derived E.coli.