抗生素对流感病毒感染小鼠肠道菌群、微生物代谢及屏障结构的影响

Effects of antibiotics on gut microbes,metabolites and barrier structure in mice infected with influenza virus

[目的]本文旨在研究流感感染小鼠使用抗生素后肠道微生物、代谢物及肠道紧密连接蛋白的变化。[方法]30只雄性BALB/c小鼠随机分为3组:对照组、感染组和抗生素组。感染组和抗生素组同时感染流感病毒,抗生素组感染1 d后饮水中添加组合抗生素,3组小鼠感染9 d后取样。16S rRNA基因测序后进行微生物组学分析;用气相色谱-飞行时间质谱(GC-TOF/MS)法进行代谢组学分析;用免疫组化法和免疫印迹法进行结肠紧密连接蛋白的分析。[结果]与对照组相比,感染组微生物多样性指数(Chao1、Shannon、Simpson)、细菌丰度和代谢物主成分分析(PCA)均无显著差异。与感染组相比,抗生素组微生物多样性指数均极显著下降(P<0.01)。拟杆菌门和变形菌门相对丰度极显著增加(P<0.01),厚壁菌门相对丰度极显著降低(P<0.001)。抗生素组与对照组在PCA中完全分离,有机酸代谢与筛选出的差异微生物具有较强相关性。感染组和抗生素组的紧密连接蛋白(Claudin-1、Occludin)的表达量均极显著降低(P<0.01),抗生素组Claudin-1的表达量与感染组相比极显著降低(P<0.001)。[结论]抗生素降低肠道微生物的多样性,增加致病菌的侵入,减少有益菌的繁殖,加剧流感诱导的肠道菌群失调,减少有机酸的产生,导致更严重的肠道屏障功能损伤。

[Objectives]The paper aimed to study the changes of intestinal microorganisms,metabolites and tight junction proteins in mice infected with influenza after antibiotics was given.[Methods]Thirty BALB/c male mice were randomly divided into three groups:the control group,the virus-infected group and the antibiotic group.Virus-infected group and antibiotic group were infected with influenza virus simultaneously.One day after infection,mice of antibiotic group were treated with combinative antibiotics by drinking water,and samples were collected at 9 days post infection.Using 16S rRNA gene sequencing and gas chromatography-time of flight mass spectrometry(GC-TOF/MS)was to analyze microbiome and metabonomics of microbiota.The expression level of tight junction proteins was detected by immunohistochemistry and western blot.[Results]Compared with control group,there were no significant differences in microbial diversity indexes,phylum level and principle components analysis(PCA)of virus-infected group.However,microbial diversity indexes(Chao1,Shannon,Simpson)were significantly reduced(P<0.01),and the abundance of Bacteroidetes and Proteobacteria showed a significant increase(P<0.01)together with a remarkable decline(P<0.001)in Firmicutes of antibiotic group when compared with the virus-infected group.The antibiotic group was completely separated from the control group in PCA,metabolites related to organic acids had strong correlations with intestine microorganisms that were screened from antibiotic group.The level of tight junction proteins(Claudin-1,Occludin)expression significantly decreased(P<0.01)in virus-infected group and antibiotic group,and the expression of Claudin-1 in antibiotic group was significantly reduced(P<0.001)compared with virus-infected group.[Conclusions]Antibiotics treatment depleted the diversity of intestinal microbial flora,increased the opportunity for pathogenic bacteria invasion and reduced the colonization of probiotics,which exacerbated the microbial communities dysbiosis,decreased the production of organic acids and caused more serious impairment of intestinal barrier function.