Abstract
Up to 50–70% of patients with liver cirrhosis develop hepatic encephalopathy (HE), which is closely related to gut microbiota dysbiosis, with an unclear mechanism. Here, by constructing gut–brain modules to assess bacterial neurotoxins from metagenomic datasets, we found that phenylalanine decarboxylase (PDC) genes, mainly from Ruminococcus gnavus, increased approximately tenfold in patients with cirrhosis and higher in patients with HE. Cirrhotic, not healthy, mice colonized with R. gnavus showed brain phenylethylamine (PEA) accumulation, along with memory impairment, symmetrical tremors and cortex-specific neuron loss, typically found in patients with HE. This accumulation of PEA was primarily driven by decreased monoamine oxidase-B activity in both the liver and serum due to cirrhosis. Targeting PDC or PEA reversed the neurological symptoms induced by R. gnavus. Furthermore, fecal microbiota transplantation from patients with HE to germ-free cirrhotic mice replicated these symptoms and further corroborated the efficacy of targeting PDC or PEA. Clinically, high baseline PEA levels were linked to a sevenfold increased risk of HE after intrahepatic portosystemic shunt procedures. Our findings expand the understanding of the gut–liver–brain axis and identify a promising therapeutic and predictive target for HE.
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Data availability
The metagenomic datasets used in this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB65440. The 16S rRNA sequence datasets in this study have been deposited in the ENA at EMBL-EBI under accession number PRJEB66346. All relevant sample-level patient data used in this study are provided in Supplementary Table 17. The six previously published studies used are available under accession numbers PRJEB6337 (ref. 22) and PRJEB14215 (ref. 23) (Supplementary Table 19), and GSE84044 (ref. 31), GSE48452 (ref. 32), GSE49541 (ref. 32) and GSE41919 (ref. 36). Associated sample-level clinical metadata for external datasets were collected from their relevant publications. Additional public databases used in this study include MetaCyc (https://biocyc.org/download.shtml), UniRef90 database (https://ftp.uniprot.org/pub/databases/uniprot/current_release/), GRCm38, M11 (https://www.gencodegenes.org/mouse/release_M11.html) and NCBI37 human genome (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001405.25/). Source data are provided with this paper.
Code availability
All code is available via the lab’s GitHub page at https://github.com/ZJJY-Bioinformatics/Cirrhosis-AADC.
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Acknowledgements
The authors thanks D. C. Rockey for the useful discussions. This work was supported by the following fundings: National Key Research and Development Program of China (2022YFA0806400, H.Z.), National Science Fund for Distinguished Young Scholars of China (81925026, H.Z.), National Natural Science Foundation of China (82272387, J.G.; 31900101, J.G.; 82130068, H.Z.; 32170111, X.H.; 82372305, X.H.), Guangzhou Key Research Program on Brain Science (202206060001, H.Z.), Natural Science Foundation of Guangdong Province of China (2022A1515010402, X.H.) and Tianjin Science and Technology Plan Project (19ZXDBSY00030, X. Qi).
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Authors and Affiliations
Contributions
H.Z., J.G., X.H., X. Qi, J.C., Y.H. and E.E.-O. conceived the study and interpreted the data. J.G., H.Z., X. Qi, J.C., X.H., M.H. and Y.X. wrote and revised the paper. H.Z., J.G., X.H. and X. Qi were involved in the funding acquisition. J.G., W.L., Y.X. and Y. Wang performed and interpreted the metagenomic analysis, GBM construction, 16S rRNA sequencing and transcriptome analysis. X. Qi, J.C., H.X., H.W., M.S., S.L., Y.P., R.S., J.Y., X. Quan, Y. Wei, J.G. and C.H. contributed to participant recruitment, sample collection and biobank management. X.H., M.H., Y.X., Y. Wang, Q.X., S.X., L.W. and Z.H. were involved in the construction of the cirrhotic animal model, R. gnavus colonization, FMT experiment, and PDCI and PEA antibody treatment. X.H., M.H., Y.X., Y. Wang, L.W. and Y. Wan performed and interpreted the behavioral experiment, MAO-B activity detection and immunofluorescence analysis. Y.T., F.L., X.H., M.H. and Y.X. were involved in the genetic manipulation. F.X., P.C., M.H., Y.X., J.G. and L.W. performed the HPLC–MS/MS analysis. T.J., M.H., Y.X. and X.H. performed the screening of the PDCI. E.E.-O. and Y.H. provided critical comments on the paper. All authors discussed and approved the paper.
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Competing interests
J.G., X.H. and H.Z. are listed as inventors on two patents concerning the use of R. gnavus as a therapeutic or diagnostic target for hepatic encephalopathy.
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Nature Medicine thanks Patricia Bloom and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Alison Farrell and Joao Monteiro, in collaboration with the Nature Medicine team.
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Extended data
Extended Data Fig. 1 Systematic screening for possible AADCs.
(a) The detailed process for systematic identification of AADCs. (b) Sequence similarity network (SSN) clusters of 329 sequences from IGC with pyridoxal_deC domains in combination with 107 known decarboxylases. Sequences in clusters 2, 5, 6, 7, and 14 (highlighted in red) were chosen for further analysis. (c) Phylogenetic tree analysis of 97 sequences from clusters 2, 5, 6, 7 and 14 in b. Tips colors indicate enzyme types inferred from known sequences. The text numbers represent the CD-hit cluster number of each sequence, with the 39 sequences picked for validation marked by an asterisk; the inner circle shows the SSN cluster of the corresponding sequence; the middle circle indicates the taxonomy of each sequence at the phylum level; the outermost circle shows the conserved domain organization of each sequence.
Extended Data Fig. 2 Colonization of R. gnavus has no significant effect on normal GF mice.
(a) The change of body weight in mice. n = 8 mice per group. Data are presented as mean ± SD. (b-f) Representative image of H&E staining in colon (b), lung (c), liver (d), spleen (e) and kidney (f); Scale bar, 100 μm. (g) Representative image of H&E staining in six brain regions: cortex, hippocampus, thalamus, striatum, amygdala and hypothalamus. The scale bars in the cortex, thalamus, striatum, amygdala and hypothalamus were 100 μm; in the hippocampus it was 400 μm; in the half brain it was 1000 μm.
Extended Data Fig. 3 The distribution of astrocytes and microglia in 6 brain regions of R. gnavus-colonized cirrhotic mice.
(a) Left panel: representative images of astrocyte (GFAP, red) in brain coronal slice. Middle panel: each white dot in brain section represents one GFAP-positive astrocyte. Right panel: brain section shown the distribution of GFAP-positive astrocytes in 6 brain regions, cyan: cortex; white: hippocampus; green: thalamus; blue: hypothalamus; red: striatum; yellow: amygdala. Scale bars in whole brain section were 1000 µm, in local region were 100 µm. Bar graph showing the density of astrocytes in 6 brain regions. The six brain regions were illustrated below, cortex (CTX); hippocampus (HIP); thalamus (THA); hypothalamus (HYP); striatum (ST); amygdala (AMY). (b) Left panel: representative images of microglia (Iba-1, green) in brain coronal section. Middle panel: each white dot in brain section represents one Iba-1-positive microglial cell. Right panel: the distribution of Iba-1-positive microglial cells in 6 brain regions, cyan: cortex; white: hippocampus; green: thalamus; blue: hypothalamus; red: striatum; yellow: amygdala. Scale bars in whole brain section were 1000 µm, in local region were 100 µm. Bar graph showing the density of microglia in 6 brain regions. Each dot represents a single individual, n = 3 in Con and n = 4 in Rg group. Data are presented as mean ± SD and analyzed by two-sided Mann-Whitney U test (a, b).
Extended Data Fig. 4 PDC plays a critical role in mediating neurotoxic effects of R. gnavus in cirrhotic mice.
(a) Experimental scheme. (b) The colonization rate of wild-type R. gnavus (Rg-WT) and a RGna_RS09085 insert mutant of R. gnavus (Rg-Mu). (c) Quantification of PEA in the brain. (d) Left panel: schematic of the open field apparatus and central region. Right panel: heatmap of overall activity comparison between the two groups. (e) The ratio of distance traveled in the center to total distance. (f) Left panel: schematic of the water maze apparatus and platform position, the red dot depicts the location of the mouse at the start of the recording. Right panel: heatmap of overall activity comparison between the two groups. (g) Time spent in the platform quadrant in the probe test. (h) The percentage of mice without coma. (i) Left panel: representative images of immunofluorescent labelling of GFAP (astrocyte, red) in the cerebral cortex of each treatment, scale bars, 20 µm. Representative astrocytes were 3D reconstructed (scale bars, 8 µm) and color-coded to indicate the spectrum of volumes (scale bars, 20 µm). Right panel: astrocyte morphological parameters of relative GFAP fluorescence intensity, volume, and number of branch points. (j) Left panel: representative images of immunofluorescent labelling of Iba-1 (microglia, green) in the cerebral cortex of each treatment, scale bars, 20 µm. Representative microglia were 3D reconstructed and their convex hulls (grey shadow) were illustrated, scale bars, 8 µm. Somas and branches were color-coded to indicate the spectrum of soma volumes and branches length, scale bars, 20 µm. Right panel: microglia morphological parameters of the ratio of soma volume/convex hull volume, total branch length, and number of intersections. Each dot represents a single individual, n = 11 mice per group (a-j). Data are presented as mean ± SD. Statistical analysis: two-sided Student’s t-test (b); one-way ANOVA followed by two-sided Dunnett's post hoc test (c, e, g, i, j); two-sided Log-rank (Mantel-Cox) test (h). Panel a created with BioRender.com.
Extended Data Fig. 5 High-throughput screening and in vitro evaluating of PDCI.
(a) Schematic diagram illustrating the study design for the high-throughput screening and evaluation of PDCI. (b) The OD600 of R. gnavus (Rg) growth in the PYG broth supplemented with 100 μM of candidate inhibitors. n = 3 biological replicates per group. (c) The relative PEA levels in the culture supernatant of Rg supplemented with 10 μM of candidate inhibitors. n = 3 biological replicates per group. (d) The relative PEA levels in the culture supernatant of Rg supplement with 100 μM of candidate inhibitors. n = 3 biological replicates per group. Data are presented as mean ± SD and analyzed by one-way ANOVA followed by two-sided Dunnett's post hoc test (c, d). Panel a created with BioRender.com.
Extended Data Fig. 6 PEA is required for the neurotoxic effects of R. gnavus.
(a) Experimental scheme. PEA-mAB: PEA monoclonal antibody. (b) Quantification of PEA in the brain. (c) Left panel: schematic of the open field apparatus and central region. Right panel: heatmap of overall activity comparison between the three groups. (d) The ratio of distance traveled in the center to total distance. (e) Left panel: schematic of the water maze apparatus and platform position, the red dot depicts the location of the mouse at the start of the recording. Right panel: heatmap of overall activity comparison between the three groups. (f) Time spent in the platform quadrant in the probe test. (g) The percentage of mice without coma. (h) Left panel: representative images of immunofluorescent labelling of GFAP (astrocyte, red) in the cerebral cortex of each treatment, scale bars, 20 µm. Representative astrocytes were 3D reconstructed (scale bars, 8 µm) and color-coded to indicate the spectrum of volumes (scale bars, 20 µm). Right panel: astrocyte morphological parameters of relative GFAP fluorescence intensity, volume, and number of branch points. (i) Left panel: representative images of immunofluorescent labelling of Iba-1 (microglia, green) in the cerebral cortex of each treatment, scale bars, 20 µm. Representative microglia were 3D reconstructed and their convex hulls (grey shadow) were illustrated, scale bars, 8 µm. Somas and branches were color-coded to indicate the spectrum of soma volumes and branches length, scale bars, 20 µm. Right panel: microglia morphological parameters of the ratio of soma volume/convex hull volume, total branch length, and number of intersections. Each dot represents a single individual, n = 12 mice per group (b-i). Data represent 2 independent experiments and are presented as mean ± SD. Statistical analysis: one-way ANOVA followed by two-sided Dunnett's post hoc test (b, d, f, h, i); two-sided Log-rank (Mantel-Cox) test (g). Panel a created with BioRender.com.
Extended Data Fig. 7 PEA triggers a HE-like syndromes in cirrhotic mice.
(a) Experimental scheme. (b) Quantification of PEA in the brain of healthy or cirrhotic mice receiving the indicated dose of PEA (n = 5 in cirrhosis control, non-cirrhosis control and non-cirrhosis + 20 μg/g PEA groups; n = 6 in cirrhosis + 1, 10, 20 μg/g PEA groups). (c) Left panel: schematic of the open field apparatus and central region. Right panel: heatmap of overall activity in the open field apparatus. (d) The ratio of distance traveled in the center to total distance. (e) Left panel: schematic of the water maze apparatus and platform position, the red dot depicts the location of the mouse at the start of the recording. Right panel: heatmap of overall activity. (f) Time spent in the platform quadrant in the probe test. (g) All the mice were intraperitoneally injected with 40% CCl4 to induced severe liver damage, line graph shows the percentage of mice without coma. (h) Upper panel: representative images of immunofluorescent labelling of GFAP (astrocyte, red) in the cerebral cortex of each treatment, scale bars, 20 µm. Representative astrocytes were 3D reconstructed (scale bars, 8 µm) and color-coded to indicate the spectrum of volumes (scale bars, 20 µm). Lower panel: astrocyte morphological parameters of relative GFAP fluorescence intensity, volume, and number of branch points. (i) Upper panel: representative images of immunofluorescent labelling of Iba-1 (microglia, green) in the cerebral cortex of each treatment, scale bars, 20 µm. Representative microglia were 3D reconstructed and their convex hulls (grey shadow) were illustrated, scale bars, 8 µm. Somas and branches were color-coded to indicate the spectrum of soma volumes and branches length, scale bars, 20 µm. Lower panel: microglia morphological parameters of the ratio of soma volume/convex hull volume, total branch length, and number of intersections. Each dot represents a single individual, n = 12 mice per group (c-i). Data represent 2 independent experiments and are presented as mean ± SD. Statistical analysis: two-sided Student’s t-test (d, f, h, i); two-sided Log-rank (Mantel-Cox) test (g). Panel a created with BioRender.com.
Extended Data Fig. 8 Fecal microbiota from HE patient induces neurobehavioral and neuropathological changes in cirrhotic mice.
(a) Germ-free (GF) mice were colonized with fecal microbiota from 3 healthy control (HC) and 3 HE patients (HE). Each donor was transferred to 3-5 recipient mice. GF mice gavaged with saline were used as pure controls. (b) Relative PDC levels in fecal samples from both donors and recipient mice. (c) Quantification of PEA levels in the brains of recipient mice (n = 8 mice per group). (d) Heatmap of overall activity comparison between three groups in open field test. (e) The ratio of distance traveled in the center to total distance. (f) Heatmap of overall activity comparison between three groups in water maze test. (g) Time spent in the platform quadrant in the probe test. (h) Mediation analysis suggested that HE fecal microbiota induce memory deficits through R. gnavus. (i) Left panel: representative images of immunofluorescent labelling of GFAP (astrocyte, red) in the cerebral cortex of each treatment, scale bars, 20 µm. Representative astrocytes were 3D reconstructed (scale bars, 8 µm) and color-coded to indicate the spectrum of volumes (scale bars, 20 µm). Right panel: astrocyte morphological parameters of relative GFAP fluorescence intensity, volume, and number of branch points. (j) Left panel: representative images of immunofluorescent labelling of Iba-1 (microglia, green) in the cerebral cortex of each treatment, scale bars, 20 µm. Representative microglia were 3D reconstructed and their convex hulls (grey shadow) were illustrated, scale bars, 8 µm. Somas and branches were color-coded to indicate the spectrum of soma volumes and branch length, scale bars, 20 µm. Right panel: microglia morphological parameters of the ratio of soma volume/convex hull volume, total branch length, and number of intersections. Each dot represents a single individual, n = 12 in GF and HC, n = 14 in HE (b, d-j). Data are presented as mean ± SD. Statistical analysis: two-sides Student’s t test (b); Mediation analysis using R package ‘mediation’ (h); one-way ANOVA followed by two-sides Tukey’s post hoc test (c, e, g, i, j). Panel a created with BioRender.com.
Extended Data Fig. 9 Engraftment fidelity for colonization of mice with human microbiota.
(a-b) α-diversity as measured by the Shannon index (a) or Chao index (b). (c-d) Taxonomic profile in donors and recipients (HC or HE) at the phylum level. (e-f) Taxa engraftment in mice at the genus level from 16S rRNA gene sequencing. The cumulative relative abundance of shared taxa in human donors (e), as well as the fraction of donor-derived taxa (genus level) engrafted in the mice relative to taxa in human donor (f). (g) The PCoA of weighted UniFrac distances. (h-i) GraPhlAn plot of LEfSe linear discriminant analysis of microbiome profiles up to the genus level from 16S rRNA gene sequencing of HC or HE (h, mice; i, human donors). n = 3, 3, 12, 12 for DonorsHC, DonorsHE, MiceHC, MiceHE separately, data are presented as mean ± SD (a-f).
Extended Data Fig. 10 Targeting PEA alleviates neurotoxicity induced by fecal microbiota from HE patients.
(a) Schematic outline of the fecal microbiota transplantation experiments. (b) The relative PDC levels in each treatment. (c) Quantification of PEA in the brain of the mouse. (d) Left panel: heatmap of overall activity comparison between three groups in the open field apparatus, white frame indicated the central region. Right panel: the ratio of distance traveled in the center to total distance. (e) Left panel: heatmap of overall activity comparison between three groups in the water maze apparatus, black dashed circle indicated the platform. Right panel: time spent in the platform quadrant in the probe test. (f) All the mice were intraperitoneally injected with 40% CCl4 to induce severe liver damage, line graph shows the percentage of mice without coma. (g) Left panel: representative images of immunofluorescent labelling of GFAP (astrocyte, red) in cerebral cortex of each treatment, scale bars, 20 µm. Representative astrocytes were 3D reconstructed (scale bars, 8 µm) and color-coded to indicate the spectrum of volumes (scale bars, 20 µm). Right panel: astrocyte morphological parameters of relative GFAP fluorescence intensity, volume, and number of branch points. (h) Left panel: representative images of immunofluorescent labelling of Iba-1 (microglia, green) in the cerebral cortex of each treatment, scale bars, 20 µm. Representative microglia were 3D reconstructed and their convex hulls (grey shadow) were illustrated, scale bars, 8 µm. Somas and branches were color-coded to indicate the spectrum of soma volumes and branches length, scale bars, 20 µm. Right panel: microglia morphological parameters of the ratio of soma volume/convex hull volume, total branch length, and number of intersections. Each dot represents a single individual, n = 11 mice per group (a-h). Data are presented as mean ± SD. Statistical analysis: one-way ANOVA followed by two-sides Dunnett's post hoc test (b, c, d, e, g, h); two-sides Log-rank (Mantel-Cox) test (f). Panel a created with BioRender.com.
Supplementary information
Supplementary Tables
Supplementary Tables 1–20.
Supplementary Video 1
R. gnavus induces symmetrical tremor of the large joint in cirrhotic mice.
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He, X., Hu, M., Xu, Y. et al. The gut–brain axis underlying hepatic encephalopathy in liver cirrhosis. Nat Med 31, 627–638 (2025). https://doi.org/10.1038/s41591-024-03405-9
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DOI: https://doi.org/10.1038/s41591-024-03405-9
