乙型肝炎病毒感染性疾病患者肠道菌群结构和多样性分析

摘要/Abstract

摘要： 目的探究乙型肝炎病毒(HBV)感染性疾病患者肠道菌群间的差异性。方法收集2020年2—9月新疆医科大学第一附属医院肝病专科收治的133例肝病患者粪便,包括HBV携带者(HBV组)23例、乙型肝炎肝硬化(LC组)25例、肝细胞癌(HCC组)38例、慢加急性肝衰竭(ACLF组)16例、自身免疫性肝炎(AIH组)13例和原发性胆汁性肝硬化(PBC组)18例。提取粪便样本基因组DNA,特定性引物扩增DNA样本中16S rRNA基因的V3~V4可变区。Usparse软件分析扩增序列,并依据分析结果(相似度≥97%)划分OTU,Silva数据库对OTU进行分类信息标注;QIIME软件进行肠道菌群物种多样性和组间菌群差异性分析。结果 6组样本中门水平上丰度前4的菌门依次为Firmicutes、Proteobacteria、Bacteroidota、Actinobacteria,HBV组、LC组、HCC组、ACLF组、AIH组和PBC组样本中4大菌门总占比分布为88.76%、97.22%、96.15%、92.88%、95.91%和93.36%。HBV组和HCC组Actinobacteria丰度显著高于LC组,HBV组Actinobacteriota和其他菌门丰度均显著高于LC组、HCC组和ACLF组,LC组和ACLF组的Actinobacteriota及ACLF组的其他菌门丰度均显著低于PBC组;LC组Bacteroidota丰度显著高于HCC组;HCC组的Firmicutes丰度显著高于HBV组。6组样本肠道菌群丰度依次为HBV组、PBC组、LC组、HCC组、ACLF组、AIH组。HBV组、LC组、HCC组和PBC组组内菌群差异性较大,组间肠道菌群群落结构差异较大,ACLF组和AIH组中肠道菌群群落比较集中,群落结构相似。HBV组物种数明显多于LC组、HCC组和ACLF组,LC组少于低于AIH组和PBC组,HCC组和ACLF组均显著低于PBC组(P<0.05);HBV组Shannon指数明显高于LC组和ACLF组,LC组、HCC组、ACLF组Shannon指数均明显低于PBC组(P<0.05)。结论 HBV感染者随着肝损伤病情加重肠道菌群丰度和多样性下降,且HBV感染者肠道菌群丰度和多样性与AIH和PBC患者均均存在明显差异。

关键词: 乙型肝炎病毒, 肝损伤, 肠道菌群, 16sRNA

Abstract: Objective To examine the variations in gut microbiota among patients infected with hepatitis B virus (HBV).Methods Stool samples were collected from 133 patients with liver disease admitted to the department of liver disease. This cohort comprised 23 HBV carriers (HBV group), 25 with hepatitis B cirrhosis (LC group), 38 with hepatocellular carcinoma (HCC group), 16 with hepatitis B-induced liver failure (ACLF group), 13 with autoimmune hepatitis (AIH group) and 18 with primary biliary cirrhosis (PBC group). Genomic DNA was isolated from these fecal samples, folloed by amplification of the V3~V4 variable region of the 16S rRNA gene using specific primers. Amplified sequences were then analyzed using UPARSE to delineate the OTUs with a set similarity threshold of ≥97%. These OTUs were annotated with taxonomic data from the Silva database. Subsequently, the QIIME software was employed to assess both the intra-and inter-group diversity of the gut microbiota.Results The predominant bacteria across the 6 groups were Firmicutes, Proteobacteria, Bacteroidota and Actinobacteria. Their combind proportions in the HBV, LC HCC, ACLF, AIH, and PBC group was 88.76%, 97.22%, 96.15%, 92.88%, 95.91% and 93.36%, respectively. Notably, the abundance of Actinobacteria was significantly greater in the HBV and HCC groups compared to the LC group. Furthermore, the abundance of Actinobacteriota, as well as other bacteria, in the HBV group surpassed that in the LC, HCC, and ACLF group. The abundance of Actinobacteriota, as well as other bacteria, was notably lower in the LCand ACLF groups compared to the PBC group. In the LC group, the presence of Bacteroidota was significantly greater than in the HCC group. While the HCC group had a higher abundance of Firmicutes compared to the HBV group. When ranking the groups by their intestinal flora anundance, the order is HBV, PBC, LC, HCC group, ACLF, and AIH.The microbial composition in the HBV, LC, HCC, and PBC groups showed significantly variances, with distinct differences in the community structures of their intestinal microflora. On the other hand, the ACLF and AIH groups displayed a more cohesive intestinal microflora community, sharing similar structural characteristics. The number of species in the HBV group was significantly higher than in the LC, HCC, and ACLF group. The LC group had fewer species than both the AIH and PBC group. Both the HCC and ACLF groups had significantly fewer species compared to the PBC group (P<0.05). As for the Shannon index, the HBV group′s score was significantly higher than the LC and ACLF groups, Additionally, the scores for the LC, HCC, and ACLF groups were all significantly lower than that of the PBC group (P<0.05).Conclusion As liver injury in HBV patients worsened, there was a noticeable decrease in the abundance and diversity of their gut microbiota. Furthermore, the abundance and diversity of gut microbiota in HBV patients were markedly different from those in AIH and PBC patients.

Key words: Hepatitis B virus, Liver injury, gut microbiota, 16sRNA

赵成燕, , 周欣怡, 高冉冉, 韩丹, 郑嵘炅, 鲁晓擘. 乙型肝炎病毒感染性疾病患者肠道菌群结构和多样性分析[J]. 肝脏, 2023, 28(11): 1339-1345.

ZHAO Cheng-yan, ZHOU Xin-yi, GAO Ran-ran, HAN Dan, ZHENG Rong-jiong, LU Xiao-bo. Analysis of gut microbiota structure and diversity in patients with hepatitis B virus infection[J]. Chinese Hepatolgy, 2023, 28(11): 1339-1345.

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