【摘要】 目的探讨乙型肝炎肝硬化(HBLC)伴有或不伴有腹水患者肠道菌群的差异。方法收集2016年10月-12月于上海中医药大学附属曙光医院肝硬化科就诊的HBLC患者57例,其中无腹水30例(HBLC-WOA),有腹水但无SBP 27例(HBLCWA),并纳入28例健康志愿者(HC),采用16S rRNA测序技术,比较两组肝硬化患者间肠道菌群的分布差异。计数资料2组间比较采用Wilcoxon秩和检验;计量资料2组间比较采用Mann-Whitney U检验,3组比较采用Kruskal-Wallis H检验。组间各物种相对丰度差异的比较采用多重假设检验,采用Benjamini和Hochberg错误发现率(fdr)进行调整,Pfdr<0. 05为差异有统计学意义。相关分析采用Spearman秩相关检验。不同条件下组间的差异比较采用相似性分析(ANOSIM)和非参数多元方差分析(Adonis)。结果伴随腹水的出现,HBLC患者粪便微生物群的多样性降低(P=0. 042)。PCo A结果显示,HBLC-WOA组、HBLC-WA组与HC组间比较差异有统计学意义(HBLC-WOA vs HC,ANOSIM:R=0. 159,P=0. 001,Adonis:R2=0. 067,P=0. 001; HBLC-WA vs HC,ANOSIM:R=0. 323,P=0. 001,Adonis:R2=0. 107,P=0. 001)。在属水平上,与HC组比较,HBLC-WA组罕见小球菌属和假丁弧菌属的丰度显著降低(P值分别为<0. 01、<0. 001),肠杆菌属、大肠埃希菌属和韦荣球菌属的丰度显著增加(P值分别为<0. 05、<0. 001、<0. 01)。大肠埃希菌属和韦荣球菌属均与CTP评分、凝血酶原时间和国际标准化比率(INR)均呈正相关,与血清Alb水平均呈负相关(P值均<0. 05)。假丁弧菌属、norank_f_毛螺菌科、unclassified_f_毛螺菌科、布劳特氏菌属与Alb水平均呈正相关,与CTP评分和C反应蛋白水平均呈负相关(P值均<0. 05)。KEGG通路分析显示,伴随腹水的出现,与转录相关蛋白、α-亚油酸代谢、金黄色葡萄球菌感染、细菌侵袭上皮细胞和胆汁分泌通路相关的菌群丰度逐渐上升(Pfdr值均<0. 05),而与黄酮类生物合成通路相关的菌群丰度逐渐下降(Pfdr<0. 05)。且细菌侵袭上皮细胞通路与大肠埃希菌属的丰度呈正相关(P <0. 001),肠杆菌属丰度与胆汁分泌通路呈正相关(P <0. 001)。结论 HBLC-WA患者肠道菌群紊乱以硬壁菌门的罕见小球菌属和假丁弧菌属丰度降低,变形菌门的肠杆菌属和大肠埃希菌属丰度增加为特征,肠杆菌可能参与胆汁分泌通路,大肠埃希菌可能参与上皮细胞的细菌侵袭通路,提示对于无SBP的HBLC-WA患者,调节肠道菌群的同时,可能也要考虑SBP的预防性治疗。更多还原

【Abstract】 Objective To investigate the difference in intestinal flora between hepatitis B liver cirrhosis( HBLC) patients with or without ascites. Methods A total of 57 patients with HBLC who visited Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from October to December 2016 were enrolled,among whom 30 had no ascites( HBLC-WOA group) and 27 had ascites without spontaneous peritonitis( SBP)( HBLC-WA group),and 28 healthy volunteers were enrolled as healthy controls( HC group). Intestinal flora was compared between the two groups of HBLC patients using 16 S rRNA sequencing. The Wilcoxon rank sum test was used for comparison of categorical data between two groups,and the Mann-Whitney U test was used for comparison of continuous data between two groups;the Kruskal-Wallis H test was used for comparison of continuous variables between more than two groups. Multiple hypothesis tests were used for comparison of relative abundance between species and was adjusted by Benjamini and Hochberg false discovery rate( fdr),and Pfdr< 0. 05 was considered statistically significant. The Spearman rank correlation test was used for correlation analysis. An analysis of similarity( ANOSIM) and a non-parametric multivariate analysis of variance( Adonis) were used for comparison between groups under different conditions. Results The abundance of fecal microbiota gradually decreased with the appearance of ascites in HBLC patients( P = 0. 042).There were significant differences between the HBLC-WOA group and the HC group( ANOSIM: R = 0. 159,P = 0. 001; Adonis: R2=0. 067,P = 0. 001) and between the HBLC-WA group and the HC group( ANOSIM: R = 0. 323,P = 0. 001; Adonis: R2= 0. 107,P =0. 001). At the genus level,compared with the HC group,the HBLC-WA group had significant reductions in the abundance of Subdoligranulum and Pseudobutyrivibrio( P < 0. 01 and P < 0. 001) and significant increases in the abundance of Enterobacter,Escherichia,and Veillonella( P < 0. 05,P < 0. 001,and P < 0. 01). Escherichia and Veillonella were positively correlated with Child-Turcotte-Pugh( CTP) score,prothrombin time,and international normalized ratio and were negatively correlated with serum albumin( Alb) level( all P <0. 05). Pseudobutyrivibrio,norank _f _ Lachnospiraceae,unclassified _ f _ Lachnospiraceae,and Blautia were positively correlated with Alb level and were negatively correlated with CTP score and C-reactive protein level( all P < 0. 05). The KEGG pathway analysis showed that with the appearance of ascites,there were gradual increases in the abundance of the pathways associated with transcription-related proteins,alpha-linolenic acid metabolism,Staphylococcus aureus infection,bacterial invasion of epithelial cells,and bile secretion( all Pfdr< 0. 05),as well as a gradual reduction in the abundance of the pathway associated with the biosynthesis of flavonoids( Pfdr< 0. 05). The pathway associated with bacterial invasion of epithelial cells was positively correlated with the abundance of Escherichia( P < 0. 001),and the abundance of Enterobacter was positively correlated with the pathway of bile secretion( P < 0. 001). Conclusion Intestinal flora disturbance is observed in HBLC-WA patients,featuring the reductions in the abundance of Subdoligranulum and Pseudobutyrivibrio belonging to Firmicutes and the increases in the abundance of Enterobacter and Escherichia belonging to Proteobacteria. Enterobacter may be involved in the pathway of bile secretion,and Escherichia may be involved in the pathway associated with bacterial invasion of epithelial cells. It is suggested that regulation of intestinal flora,as well as the prophylactic treatment of SBP,should be considered for HBLC-WA patients without SBP.更多还原