Figures and data
Gout susceptibility increases with age, related to gut microbiota
(a) Mice of different ages.
(b-d) The foot thickness ratios (b), foot tissue’s IL-1β concentrations (c) and serum concentrations of uric acid of three different age ranges groups (d) were tested after MSU administration (n=6).
(e-f) The three different age ranges groups’ ASVs (Amplicon Sequence Variants)
(e) and PcoA analysis (using Bray-Curtis dissimilarity and Jaccard distance) (f). (g-i) The foot thickness ratios (g), foot tissue’s IL-1β concentrations (h) and serum concentrations of uric acid of three different age ranges groups (i) (treated with antibiotics (ABX) cocktail) were tested after MSU administration (n=6).
Values are presented as the mean ± SEM. Differences were assessed by One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
Aged-to-young FMT worsens acute gout, whereas young-to-aged FMT reduced this disease.
(a) Overall experimental design and timeline for experiments.
(b) Representative H&E-stained images of left foot tissues. Scale bars 1000 μm and 3x magnification.
(c-e) Foot tissue inflammatory parameters, including IL-1β (c), IL-6 (d) and TNF-α (e) concentrations, from the indicated mice are shown (n=6).
(f-h) The peritoneal fluid concentrations of IL-1β (f), IL-6 (g) and TNF-α (h) inflammatory parameters were measured in the indicated mice(n=6).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
“Younger” gut microbiota suppresses NLRP3 inflammasome pathway, “aging” gut microbiota promotes
(a-b) Representative western blot images and band density (Young+PBS, Young+Old and Young+Aged) of foot tissue NLRP3 pathways proteins (n=3).
(c-d) Representative western blot images and band density (Old+PBS and Old+Young) of foot tissue NLRP3 pathways proteins (n=3).
(e-f) Representative western blot images and band density (Aged+PBS and Aged+Young) of foot tissue NLRP3 pathways proteins (n=3).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
Beneficial effects of FMT from young to aged mice on uric acid metabolism.
(a) All groups’ serum concentrations of uric acid (n=6).
(b-d) The activity of uric acid-producing enzymes of liver in the cross-age fecal microbiota transplantation group and its control group (n=6), including ADA (b), GDA (c) and XOD (d).
(e) The activity of XOD of kidney in the cross-age fecal microbiota transplantation group and its control group (n=6).
(f) Relative kidney injury molecule-1 (KIM-1) expression in the indicated groups by qPCR (n = 6).
(g and h) Relative renal genes for uric acid reabsorption expression in the indicated groups by qPCR (n = 6), including URAT1 (g) and GLUT9 (h).
(i-k) Relative renal genes for uric acid excretion expression in the indicated groups by qPCR (n = 6), including OAT1 (i), OAT3 (j) and ABCG2 (k).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
Modifications in the gut microbiota composition following cross-age FMT.
(a) Bacterial composition at the phylum levels (top 10) of the indicated groups (n=6).
(b-c) Alpha diversity indices including Chao1 (b) and observed_otus (c) index in the indicated groups (n=6).
(d) β-diversity difference among the seven groups analyzed by the PcoA using Bray-Curtis dissimilarity and Jaccard distance.
(e) Volcano Plot of inter-group significance analysis using metastat (t-test, p < 0.05).
(f) The ternary plot of three different groups among the seven groups at genus levels (top 10).
Changes in fecal microbiota metabolism and pathways after cross-age FMT.
(a) Comparison of differential KEGG enrichment bubble plots between Young+PBS and Young+Old.
(b) Comparison of differential KEGG enrichment bubble plots between Young+PBS and Young+Aged.
(c) Comparison of differential KEGG enrichment bubble plots between Old+PBS and Old+Young.
(d) Comparison of differential KEGG enrichment bubble plots between Aged+PBS and Aged+Young.
The enrichment analysis was performed at the KEGG Pathway level using a hypergeometric test, as shown in the figure below. The pathways that were significantly enriched in the differential metabolites compared to the background of all identified metabolites. Pathway enrichment analysis enables us to determine the major biochemical metabolic pathways and signaling transduction pathways that are implicated by the differential metabolites.
Butyrate supplementation inhibits gout and MSU-induced peritonitis.
(a) The concentration of primary short-chain fatty acids (SCFAs) in fecal samples from both young (3 months), aged (24 months) mice and Aged+Young (FMT from young to aged). Graphs were generated to illustrate the changes in individual SCFAs, with a sample size of 6 in the Young+PBS, Aged+PBS and Aged+Young group(n=6).
(b-e) The foot thickness ratios (b) were tested after MSU administration, foot tissue inflammatory parameters, including IL-1β (c), IL-6 (d) and TNF-α (e) concentrations, from the indicated mice are shown(n=6).
(f) Representative H&E-stained images of left foot tissues. Scale bars 1000 μm and 3x magnification.
(g-i) The peritoneal fluid concentrations of IL-1β (g), IL-6 (h) and TNF-α (i) inflammatory parameters were measured in the indicated mice (n=6).
(j) Representative western blot images of foot tissue NLRP3 pathways proteins and band density (n=3).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
Serum uric acid-lowering effect of butyrate in old or aged mice.
(a) The serum uric acid concentrations in Old+PBS and Old+Butyrate, Aged+PBS and Aged+Butyrate (n=6).
(b-d) The activity of uric acid-producing enzymes of liver in the Old or Aged control group and the old or aged group supplemented with butyrate (n=6), including ADA (b), GDA (c) and XOD (d).
(e) The activity of XOD of kidney in the Old or Aged control group and the old or aged group supplemented with butyrate (n=6).
(f) Relative kidney injury molecule-1 (KIM-1) expression in the indicated groups by qPCR (n = 6).
(g and h) Relative renal genes for uric acid reabsorption expression in the indicated groups by qPCR (n = 6), including URAT1 (g) and GLUT9 (h).
(i-k) Relative renal genes for uric acid excretion expression in the indicated groups by qPCR (n = 6), including OAT1 (i), OAT3 (j) and ABCG2 (k).
(l) Relative colonic genes for uric acid expression in the indicated groups by qPCR, including ABCG2 (n = 6).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
Oligonucleotides
Changes in fecal microbiota metabolism and pathways after cross-age FMT.
(a) All groups’ foot thickness ratios were tested after MSU administration (n=6). (b-d) The serum concentrations of IL-1β (b), IL-6 (c) and TNF-α (d) inflammatory parameters were measured in the indicated mice(n=6).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
(a-b) Representative western blot images and band density (Young+PBS, Young+Old and Young+Aged) of peritoneal cells NLRP3 pathways proteins (n=3).
(c-d) Representative western blot images and band density (Old+PBS and Old+Young) of peritoneal cells NLRP3 pathways proteins (n=3).
(e-f) Representative western blot images and band density (Aged+PBS and Aged+Young) of peritoneal cells NLRP3 pathways proteins (n=3).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
(a and b) Serum AST (a) and ALT (b) concentrations in the cross-age fecal microbiota transplantation group and its control group (n=6).
(c and d) Serum Crea (c) and BUN (d) concentrations in the cross-age fecal microbiota transplantation group and its control group (n=6).
(e-h) Relative colonic gene expression in the indicated groups by qPCR (n = 6), including ABCG2 (e), Occludin (f), ZO-1 (g) and JAMA (h).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
(a) Bacteroidetes relative abundance of the indicated groups (n=6).
(b) Firmicutes relative abundance of the indicated groups (n=6).
(c) The Firm/Bac ratio in the different groups (n=6).
(d-e) Alpha diversity indices including Shannon (d) and Simpson (e) index in the indicated groups (n=6).
(f) Bacterial composition at the genus levels (top 15) of the indicated groups(n=6).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
(a) Metabolite classification of various groups.
(b) Volcano plots of differential metabolites in each group.
The horizontal axis of volcano plot represents the log2 (Fold Change) in metabolite abundance between different groups, while the vertical axis of volcano plot represents the significance level (-log10 (P-value)) of the differences. Each point on the volcano plot represents a metabolite, with significantly upregulated metabolites depicted as red points and significantly downregulated metabolites depicted as green points. The size of the circles corresponds to the VIP (Variable Importance in Projection) value.
(a) Comparison of differential metabolite volcano plots and KEGG enrichment bubble plots between Young+PBS and Old+PBS.
(b) Comparison of differential metabolite volcano plots and KEGG enrichment bubble plots between Young+PBS and Aged+PBS.
To visualize the differential metabolites resulting from pairwise comparisons, we generated volcano plots that provide a clear representation of the upregulation and downregulation of metabolites, especially those with significant fold change differences. The enrichment analysis was performed at the KEGG Pathway level using a hypergeometric test, as shown in the figure below. The pathways that were significantly enriched in the differential metabolites compared to the background of all identified metabolites. Pathway enrichment analysis enables us to determine the major biochemical metabolic pathways and signaling transduction pathways that are implicated by the differential metabolites.
(a-h) The difference analysis of the detected short chain fatty acids, including acetic acid (a), propionic acid (b), isobutyric acid (c), butyric acid (d), 2-merhylbutyrate (e), isovaleric acid (f), valeric acid (g), hexanoic acid (h).
(i-k) The serum concentrations of IL-1β (i), IL-6 (j) and TNF-α (k) inflammatory parameters were measured in the indicated mice (n=6).
(l) Representative western blot images and band density of peritoneal cells NLRP3 pathways proteins (n=3)
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
(a-c) Relative colonic gene expression in the indicated groups by qPCR (n = 6), including Occludin (a), ZO-1 (b) and JAMA (c).
Values are presented as the mean ± SEM. Differences were assessed by t-test or One-Way ANOVA and denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, “ns” indicates no significant difference between groups.
