基于16S rRNA基因测序技术分析原发性胆汁性胆管炎伴抑郁小鼠粪便样本中肠道菌群的变化

Changes of Gut Microbiota in Fecal Samples of Mice With Primary Biliary Cholangitis Associated With Depression by Using 16S rRNA Gene Sequencing Technology

背景:肠道菌群失调或紊乱与脑和肝脏疾病之间存在联系,肠道菌群可能通过肠⁃肝⁃脑轴影响原发性胆汁性胆管炎(PBC)和抑郁的发生和发展。目的:应用16S rRNA基因测序技术分析PBC伴抑郁小鼠的肠道菌群情况。方法:将12只雌性小鼠随机分为对照组、胆汁淤积组、胆汁淤积+抑郁组和治疗组。对照组小鼠给予正常饲料和水喂养;胆汁淤积组小鼠采用含0.1%DDC的饲料连续喂养2周构建胆汁淤积模型;胆汁淤积+抑郁组小鼠先采用慢性温和不可预期应激方式刺激2周,再以含0.1%DDC的饲料喂养2周构建胆汁淤积+抑郁模型;治疗组小鼠在构建胆汁淤积+抑郁模型的基础上,腹腔注射盐酸氯米帕明(7.5 mg·kg^(-1)·d^(-1))2周。造模期间观察小鼠的行为学变化。造模结束后各组小鼠称重,行负重游泳试验、强迫游泳试验、悬尾试验和肝组织HE染色;采集新鲜粪便,基于16S rRNA基因测序技术分析小鼠肠道菌群的变化。结果:与对照组相比,胆汁淤积组和胆汁淤积+抑郁组小鼠体质量明显减轻(P<0.05),抑郁样行为明显加重(P<0.05);与胆汁淤积+抑郁组相比,治疗组小鼠体质量明显升高(P<0.05),抑郁样行为明显减轻(P<0.05)。胆汁淤积组、胆汁淤积+抑郁组和治疗组小鼠肝组织出现不同程度的胆汁淤积性损伤;与胆汁淤积+抑郁组相比,治疗组肝脏病理损伤明显减轻。小鼠粪便样本经测序抽平处理后获得个5491个操作分类单元(OTU),4组共有的OTU为162个。对照组与胆汁淤积组、胆汁淤积+抑郁组和治疗组小鼠粪便中微生物多样性和群落组成存在较大差异。在门水平上,胆汁淤积组厚壁菌门(Firmicutes)丰度明显升高;胆汁淤积+抑郁组变形菌门(Proteobacteria)丰度明显升高;治疗组厚壁菌门和变形菌门丰度均明显升高,拟杆菌门(Bacteroidetes)丰度明显下降。对组间差异显著的物种行LEfSe分析发现,LDA值>4的微生物类群有38个。结论:PBC伴抑郁小鼠的肠道菌群结构和多样性均发生显著变化,抗抑郁治疗能改善肠道菌群的丰度和多样性,通过调节肠道菌群治疗PBC伴抑郁有可能成为一种新的治疗策略。

Background:There is a link between dysbiosis or disorder of gut microbiota and brain and liver diseases,and gut microbiota may affect the occurrence and development of primary biliary cholangitis(PBC)and depression through the gut⁃liver⁃brain axis.Aims:To analyze the gut microbiota in PBC mice associated with depression by using 16S rRNA gene sequencing technology.Methods:Twelve female mice were randomly divided into control group,cholestasis group,cholestasis+depression group and treatment group.Mice in the control group were given normal feed and water.Mice in the cholestasis group were fed with a feed containing 0.1%DDC for 2 weeks to establish cholestasis model.Mice in the cholestasis+depression group were stimulated with chronic mild unpredictable stress for 2 weeks,followed by 2 weeks of 0.1%DDC⁃containing feed to establish cholestasis+depression model.Mice in the treatment group were injected intraperitoneally with clomipramine hydrochloride(7.5 mg·kg^(-1)·d^(-1))for 2 weeks based on the construction of cholestasis+depression model.During the modeling period,the behavioral changes of the mice were observed.After the modeling,the body weight was recorded,and the weight⁃bearing swimming test,forced swimming test,tail suspension test and HE staining of liver tissue were performed.The fresh feces were collected,and the changes of gut microbiota were analyzed by 16S rRNA gene sequencing technology.Results:Compared with the control group,the body weight of mice in cholestasis group and cholestasis+depression group was significantly decreased(P<0.05),and the depression⁃like behavior was significantly aggravated(P<0.05);compared with the cholestasis+depression group,the body weight of mice in treatment group was significantly increased(P<0.05),and the depression⁃like behavior was significantly ameliorated(P<0.05).Various degrees of cholestatic injury of the liver tissue were observed in the cholestasis group,cholestasis+depression group and treatment group.Compared with the cholestasis+depression group,the pathological injury of the liver tissue in treatment group was significantly reduced.After sequencing and flattening,5491 operational taxonomic units(OTUs)were obtained in fecal samples.There were 162 common OTUs among the 4 groups.The microbial diversity and community composition of the control group,cholestasis group,cholestasis+depression group and treatment group were significantly different.At the phylum level,the abundance of Firmicutes in the cholestasis group was significantly increased;the abundance of Proteobacteria in the cholestasis+depression group was significantly increased;the abundance of both Firmicutes and Proteobacteria was significantly increased in the treatment group,while the abundance of Bacteroidetes was significantly decreased.LEfSe analysis was performed in species with significant differences among the groups,and the results showed that there were 38 microbial groups with LDA value>4.Conclusions:Significant changes are observed in gut microbiota composition and diversity in mice with PBC associated with depression.Antidepressant treatment can improve the abundance and diversity of gut microbiota,and regulating gut microbiota may become a new treatment strategy for PBC associated with depression.