Distinct common signatures of gut microbiota associated with damp-heat syndrome in patients with different chronic liver diseases

Abstract

Background: Chronic hepatitis B (CHB) and non-alcoholic fatty liver disease (NAFLD) are prevalent in China. According to traditional Chinese medicine (TCM) theory, damp-heat (DH) syndrome is common in chronic liver disease. However, the biological characteristics related to quantitative diagnosis remain to be determined. This study aimed to identify the consistent alterations in the gut microbiota associated with DH syndrome in patients with CHB or NAFLD. Methods: A total of 405 individuals were recruited, of which 146 were participants who met the consistent TCM diagnosis by three senior TCM physicians and were typical syndromes. All participants were required to provide fresh stool and serum samples. The gut microbiota was assessed by fecal 16S rRNA gene sequencing, and the serum metabolite profiles of participants were quantified by an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system. DH syndrome-related bacteria taxa were identified based on the 146 individuals with typical syndromes and validated in all 405 volunteers. Results: The results showed that CHB and NAFLD patients with typical TCM DH syndrome had consistently elevated serum total bile acid (TBA) levels. Significant alterations in microbial community were observed according to TCM syndromes identification. A total of 870 microbial operational taxonomic units and 21 serum metabolites showed the same variation trends in both the CHB and NAFLD DH syndrome groups. The functional analysis predicts consistent dysregulation of bile acid metabolism. Five genera (Agathobacter, Dorea, Lachnospiraceae_NC2004_group, Subdoligranulum, and unclassified_c__Clostridia) significantly decreased in abundance in patients with DH syndrome. We utilize these five genera combined with TBA to construct a random forest classifier model to predict TCM diagnosis. The diagnostic receiver-operator characteristic (ROC) areas for DH syndrome were 0.818 and 0.791 in internal tenfold cross-validation and the test set based on all 405 individuals, respectively. Conclusion: There are common signatures of gut microbiota associated with DH syndrome in patients with different chronic liver diseases. Serum TBA combined with DH-related genera provides a good diagnostic potential for DH syndrome in chronic liver disease.

Keywords: chronic hepatitis B; damp-heat syndrome; gut microbiota; non-alcoholic fatty liver disease; traditional Chinese medicine.

FIGURE 1

Analysis of TCM information for 146 individuals. (A) Flowchart of this study; (B) PCA analysis based on TCM diagnostic parameters; (C) DH syndrome score comparison.

FIGURE 2

Comparison of α diversity and β diversity of gut microbial communities in patients with and without DH syndrome. (A) Sobs index; (B) Ace index; (C) Shannon index; PCoA with Bray-Curtis distance at OTU level based on (D) CHB_DH and CHB_nonDH individuals and (E) NAFLD_DH and NAFLD_nonDH individuals; (F) Score plot of PLS_DA analysis based on gut microbiota OTUs in patients with and without DH syndrome (the R²Y and Q² values for the model were 0.988 and 0.469, respectively); (G) Score plot of PLS_DA analysis based on TCM symptoms in patients with and without DH syndrome (the R²Y and Q² values for the model were 0.448 and 0.275, respectively); Finite Gaussian mixture models of parameters of (H) COMP1 in Figures 2F, (I) COMP1 in Figure 2G; (J) Spearman’s correlation analysis of both COMP1 parameters.

FIGURE 3

Patients with DH syndrome are associated with aberrant BA metabolism. (A) Functional pathways with the same trend predicted based on 870 OTUs, *p < 0.05, **p < 0.01, ***p < 0.001; (B) heatmap of 21 metabolites with the same expression trends in DH syndrome groups; (C) functional enrichment analysis based on the 21 metabolites.

FIGURE 4

Comparison of the gut microbiome in patients with and without DH syndrome. (A) Phylum-level gut microbial composition; (B) the relative abundance of 30 different bacterial genera between CHB_DH and CHB_nonDH patients; (C) the relative abundance of 16 different bacterial genera between NAFLD_DH and NAFLD_nonDH patients; LEfSe analysis between (D) CHB_DH and CHB_nonDH groups; (E) NAFLD_DH and NAFLD_nonDH groups.

FIGURE 5

Five genera have diagnostic potential for DH syndrome. Relative expression of six bacterial genera in (A) 146 individuals with and without typical DH syndrome and (B) all chronic liver disease individuals with and without DH syndrome adjusted for age, sex, and BMI; (C) Correlation analysis of DH syndrome-associated gut genera and clinical indices; (D) correlation analysis of DH syndrome co-altered trends metabolites and clinical indices; ROC curves were plotted for the diagnosis of DH syndrome in 10-fold cross-validation and test set validation (310 patients with CHB or NAFLD) using the combination of (E) five genera, and (F) five genera combined with serum TBA. The areas under the ROC curves (AUCs) were calculated.