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Integrated analysis of gut microbiota structure and metabolic function in patients with cirrhosis and refractory ascites

  • Authors:
    • Dianyan Chen
    • Sandu Liu
    • Ka Zhang
    • Ke Pan
    • Linsu Zhang
    • Deyun Shu
    • Jin Zhang
    • Dingyao Ren
  • View Affiliations / Copyright

    Affiliations: Department of Hepatology, The People's Hospital of Qiannan, Duyun, Guizhou 558000, P.R. China, Department of Hepatology, The Third Affiliated Hospital of Sun Yat‑Sen University, Guangzhou, Guangdong 510630, P.R. China, Office of Scientific Research, Qiannan Medicine College for Nationalities, Duyun, Guizhou 558000, P.R. China, Department of Laboratory, The People's Hospital of Qiannan, Duyun, Guizhou 558000, P.R. China
    Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 28
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    Published online on: January 2, 2026
       https://doi.org/10.3892/br.2026.2101
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Abstract

Refractory ascites is a serious complication of decompensated cirrhosis, yet its intestinal microbial and metabolic characteristics remain incompletely understood. The present study combined 16S rRNA sequencing and gas chromatography‑mass spectrometry‑based untargeted metabolomics to investigate the gut microbial composition and metabolite profiles of 70 patients with refractory ascites due to cirrhosis (OB group) and 70 healthy individuals (NC group). Analyses included microbial diversity evaluation, taxonomic comparison, functional prediction, metabolite screening and disease association mapping. Compared with the NC group, the OB group showed reduced α diversity and distinct β diversity patterns, with enrichment of Proteobacteria and a reduction in anaerobic and Gram‑positive taxa. BugBase analysis indicated increased proportions of potentially pathogenic, biofilm‑forming and oxidative stress‑tolerant phenotypes. LEfSe analysis further revealed group‑specific taxa, such as enrichment of Bacteroidota and Actinobacteriota in the NC group and Firmicutes‑related genera including Clostridium and Lactobacillus in the OB group. Functional predictions suggested group differences in carbohydrate and lipid metabolism, membrane transport and vitamin‑related pathways. Metabolomic analysis identified 2,890 altered metabolites and Kyoto Encyclopedia of Genes and Genomes enrichment highlighted an involvement in serotonergic synapse and steroid hormone biosynthesis. Receiver operator characteristic curve analysis revealed a good discriminatory performance (area under the curve >0.93) for 10 selected metabolites. Several of these metabolites, including L‑tryptophan and hypoxanthine, were further linked to inflammatory, neurodegenerative and neoplastic diseases. In conclusion, patients with refractory ascites exhibited notable structural and functional alterations in gut microbiota and metabolism. These findings provide insights into the gut‑liver axis in cirrhosis and may inform future research on microbial or metabolic biomarkers.
View Figures

Figure 1

OTU comparison and α diversity
profiling of intestinal microbiota between the NC and OB groups.
(A) Venn diagram illustrating the number of shared and unique OTUs
in each group. (B) UpSet plot depicting the distribution and
intersection patterns of OTUs across the two cohorts. (C) Boxplots
of α diversity indices (Ace, Chao1, Sobs, Shannon, Simpson and
Coverage) comparing species richness and microbial community
diversity between the NC and OB groups. OTU, operational taxonomic
unit; NC, normal control; OB, refractory ascites group.

Figure 2

β diversity analysis of intestinal
microbiota between the NC and OB groups. (A) PCoA plots based on
Bray-Curtis distances were generated across six taxonomic ranks:
Phylum, class, order, family, genus and species. (B) Analysis of
Similarities compared intragroup and intergroup microbial distances
at each taxonomic level. NC, normal control; OB, refractory ascites
group; PCoA, principal coordinates analysis.

Figure 3

Functional and phenotypic
characterization of intestinal microbiota in the NC and OB groups.
(A) Kyoto Encyclopedia of Genes and Genomes pathway enrichment
predicted by PICRUSt2 presented relative abundances and statistical
differences between the two groups. (B) BugBase-derived heatmap
illustrated the distribution patterns of microbial phenotypes
across the NC and OB samples. (C) Selected microbial phenotypes
predicted by BugBase were compared between the groups. NC, normal
control; OB, refractory ascites group.

Figure 4

Differential taxonomic features of
intestinal microbiota identified by LEfSe analysis between the NC
and OB groups. (A) A taxonomic cladogram was constructed using
LEfSe to visualize the phylogenetic distribution of taxa differing
in relative abundance between the two groups, spanning from the
phylum to the genus level. (B) Corresponding list of significantly
enriched taxa, with red indicating enrichment in NC and green
indicating enrichment in OB. NC, normal control; OB, refractory
ascites group.

Figure 5

Untargeted fecal metabolomic
profiling using GC-MS of the NC and OB groups. (A) Total ion
chromatograms from representative fecal samples were obtained
through GC-MS (an asterisk at 5 min indicates the internal
alignment marker). (B) PCA based on global metabolite features
revealed distinct clustering patterns between the NC and OB groups,
suggesting underlying differences in metabolic composition. (C) A
volcano plot was used to present the distribution of differential
metabolites. NC, normal control; OB, refractory ascites group;
GC-MS, gas chromatography-mass spectrometry; PCA, principal
component analysis; cps, counts per sec; VIP, variable importance
in projection.

Figure 6

Functional interpretation of
differential metabolites through network construction and KEGG
pathway enrichment. (A) A compound interaction network was
constructed based on chemical similarity and classification, with
node colors representing distinct metabolite categories. (B) KEGG
enrichment analysis was performed on the identified metabolites.
KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 7

ROC curve analysis of 10 candidate
metabolites distinguishing the NC and OB groups. ROC curves between
the NC and OB groups were generated for the following metabolites:
(A) 6-ketoprostaglandin E1, (B) prostaglandin D3, (C)
deoxyartemisinin, (D) biotin-XX-hydrazide, (E)
estra-4,9-diene-3,17-dione, (F) prostaglandin E2 methyl ester, (G)
8-epidermal glycoside, (H) pergolide, (I) cincassiol B, and (J)
ganoderic acid Md. Values in black on the ROC curves indicate the
optimal cut-off value, followed by the corresponding sensitivity
and specificity in parentheses. ROC, receiver operating
characteristic; NC, normal control; OB, refractory ascites group;
AUC, area under the curve.
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Copy and paste a formatted citation
Spandidos Publications style
Chen D, Liu S, Zhang K, Pan K, Zhang L, Shu D, Zhang J and Ren D: Integrated analysis of gut microbiota structure and metabolic function in patients with cirrhosis and refractory ascites. Biomed Rep 24: 28, 2026.
APA
Chen, D., Liu, S., Zhang, K., Pan, K., Zhang, L., Shu, D. ... Ren, D. (2026). Integrated analysis of gut microbiota structure and metabolic function in patients with cirrhosis and refractory ascites. Biomedical Reports, 24, 28. https://doi.org/10.3892/br.2026.2101
MLA
Chen, D., Liu, S., Zhang, K., Pan, K., Zhang, L., Shu, D., Zhang, J., Ren, D."Integrated analysis of gut microbiota structure and metabolic function in patients with cirrhosis and refractory ascites". Biomedical Reports 24.2 (2026): 28.
Chicago
Chen, D., Liu, S., Zhang, K., Pan, K., Zhang, L., Shu, D., Zhang, J., Ren, D."Integrated analysis of gut microbiota structure and metabolic function in patients with cirrhosis and refractory ascites". Biomedical Reports 24, no. 2 (2026): 28. https://doi.org/10.3892/br.2026.2101
Copy and paste a formatted citation
x
Spandidos Publications style
Chen D, Liu S, Zhang K, Pan K, Zhang L, Shu D, Zhang J and Ren D: Integrated analysis of gut microbiota structure and metabolic function in patients with cirrhosis and refractory ascites. Biomed Rep 24: 28, 2026.
APA
Chen, D., Liu, S., Zhang, K., Pan, K., Zhang, L., Shu, D. ... Ren, D. (2026). Integrated analysis of gut microbiota structure and metabolic function in patients with cirrhosis and refractory ascites. Biomedical Reports, 24, 28. https://doi.org/10.3892/br.2026.2101
MLA
Chen, D., Liu, S., Zhang, K., Pan, K., Zhang, L., Shu, D., Zhang, J., Ren, D."Integrated analysis of gut microbiota structure and metabolic function in patients with cirrhosis and refractory ascites". Biomedical Reports 24.2 (2026): 28.
Chicago
Chen, D., Liu, S., Zhang, K., Pan, K., Zhang, L., Shu, D., Zhang, J., Ren, D."Integrated analysis of gut microbiota structure and metabolic function in patients with cirrhosis and refractory ascites". Biomedical Reports 24, no. 2 (2026): 28. https://doi.org/10.3892/br.2026.2101
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