基于16S rRNA基因测序技术分析急性肝内胆汁淤积大鼠的肠道菌群

Analysis of intestinal flora in rats with acute intrahepatic cholestasis based on 16S rRNA gene sequencing

目的 探讨ANIT诱导的大鼠急性肝内胆汁淤积(acute intrahepatic cholestasis,AIC)与肠道菌群的关系。方法 以SPF级的SD大鼠为实验对象,随机分为正常对照组、模型组,每组24只,依据取材时间不同又将各组随机分为24 h、48 h、72 h 3个亚组,每组8只。模型组通过一次性予2%ANIT按100 mg·kg^(-1)·d^(-1)灌胃造模诱发AIC模型,正常对照组予等容积色拉油灌胃。通过高通量测序法对肠道粪便细菌16S rRNA的V_(3)~V_(4)区进行基因测序及生物信息学分析。结果 AIC造模后,随着时间增加,α多样性指数逐步升高,造模72 h组相比造模24 h组,Shannon指数有显著性差异;β多样性发现,利用基于加权Unifrac距离PcoA分析或NMDS分析均显示:正常对照组、造模24 h组和造模48 h组的肠道菌群显示比较明显的聚集效应;在门水平上,厚壁菌门(Firmicutes)丰度在造模48 h后显著下降,变形菌门(Proteobacteria)的丰度显著上升,拟杆菌门(Bacteroidetes)的丰度显著上升;LEfSe对组间差异显著的物种分析结果显示,LDA值大于4的微生物类群有35个。结论 模型组干预后大鼠肠道菌群结构和多样性均发生显著变化。

Objective To investigate the correlation between rat gut microbiota and ANIT induced acute intrahepatic cholestatic(AIC).Methods SPF SD rats were randomly divided into normal control group and model group.According to the time of sampling,each group was divided into 24 h,48 h,and 72 h subgroups,with 8 rats in each subgroup.Rats of model group were given a single dose of 2% ANIT(100 mg·kg~(-1)·d~(-1)) by gavage to establish the model.Changes in the gut microbiota composition were analyzed using 16S rRNA sequencing.Results After AIC modeling,α diversity index gradually increased with the increase of time.The Shannon index had significant difference after 72 h modeling compared with 24 h modeling,β diversity found that PcoA analysis or NMDS analysis based on weighted Unifrac distance showed that:the intestinal flora in the control group,24 h and 48 h after modeling showed obvious aggregation effect.At the phyla level,the abundance of Firmicutes decreased significantly 48 h after modeling,the abundance of proteobacteria increased significantly,and the abundance of Bacteroides increased significantly.The results of species analysis with significant differences between groups by LEfSe showed that there were 35 microbial groups with LDA values greater than 4.Conclusion The structure and diversities of gut microbiota of model group rats were significantly changed compared with normal control group.