Effects of alcohol on the symptoms of gouty arthritis and taxonomic structure of gut microbiota in C57BL/6 mice

Figures

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  Figure 1. Effect of alcohol on body weight and foot joint gout symptoms in mice. (A) Schematic diagram of the experimental treatments. MSU, monosodium urate. (B) Schematic representation of the appearance of foot joints in six groups of mice. CTL, control. AGA, acute gouty arthritis. (C) Comparison of changes in footpad swelling in six groups of mice. (D) Comparison of changes in footpad pain thresholds in six groups of mice. (E) Comparison of changes in serum uric acid (UA) levels in six groups of mice. (F) Food intake of the six groups of mice. (G) Comparison of changes in alcohol intake in mice of the alcohol group. (H) Comparison of body weight changes in six groups of mice after 6 weeks of feeding. Data are presented as mean ± standard deviation. Statistical differences are determined by p < 0.05 (*) and p < 0.01 (**), respectively; “ns” represents no statistical significance.

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  Figure 2. Gout-associated inflammatory markers aggravated by alcohol in mice. (A) HE analysis of the right hindfoot pads of six groups of mice. (B) Comparison of changes in the levels of cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), and the enzymatic activity of myeloperoxidase (MPO), in foot joint tissues of six groups of mice. (C) Comparison of changes in serum cytokine (i.e., IL-1β, IL-6, and TNF-α) levels in six groups of mice (based on ELISA). Data are presented as mean ± standard deviation. Statistical differences are determined by p < 0.05 (*) and p < 0.01 (**), respectively; “ns” represents no statistical significance.

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  Figure 3. Comparison of changes in gut microbiota in six groups of mice. (A) Alpha diversity analysis of the intestinal microbiota in six groups of mice. (B) Principal coordinate analysis (PCoA) of intestinal microbiota in six groups of mice. (C) Variations in the relative proportions of Firmicutes and Bacteroidota in the intestines of six groups of mice. (D) Comparison of changes in the composition of the intestinal microflora at the phylum level in six groups of mice. (E) Comparison of changes in the composition of the gut microbiota at the genus level in six groups of mice. Data are presented as mean ± standard deviation. The statistical difference is determined by p < 0.05 (*).

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  Figure 4. Comparative analysis of changes in the genus level of key bacteria in mice in high alcohol concentration groups. (A) Volcano plot analysis of changes in the intestinal microbiota of mice in the 10% Alcohol group compared to the AGA group. (B) Volcano plot analysis of changes in the intestinal microbiota of mice in the 20% Alcohol group compared to the AGA group. The significant difference is determined by t-test. Bacteria with relative abundance >1% are identified as key bacteria. (C) Heatmap showing the relative abundance of the key bacteria co-occurring in both (A,B).

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  Figure 5. Correlation of microbiota among the top 50 taxa at the genus level in the intestines of four groups of mice, including the CTL group (A), the Model group (B), the 10% Alcohol group (C), and the 20% Alcohol group (D). The size of the circle represents the relative abundance, the line represents the correlation between the two taxa at both ends of the line, the thickness of the line represents the level of the correlation, the orange line represents positive correlation, and the green line represents negative correlation.

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  Figure 6. Comparison of the relative abundances of functional profiles of the intestinal microbiota of mice in high alcohol concentration groups based on Picrust. (A) Volcano plot analysis of altered KEGG pathways in mice of the 10% Alcohol group vs. Model group, with mean relative abundance >0.1%. (B) Volcano plot analysis of altered KEGG pathways in mice of the 20% Alcohol group vs. Model group, with mean relative abundance >0.1%. The significant difference is determined by t-test. (C) Relative abundance of five key metabolic pathways identified in both (A) and (B) showing the significant difference in the four groups of mice. Data are presented as mean ± standard deviation.

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  Figure 7. Correlation analyses between microorganisms, pro-inflammatory cytokines, enzymes, and metabolic pathways in mice. (A) Correlation between key intestinal microorganisms (i.e., Parasutterella and Alistipes) and pro-inflammatory cytokines, enzymes, and metabolic pathways. (B) Correlation between metabolic pathways and both pro-inflammatory cytokines and enzymes. The relative R values range from −1 to 1 (i.e., blue to red). B, blood; H, hepatic tissue; F, foot joint tissue. Statistical differences are determined by p < 0.05 (*) and p < 0.01 (**), respectively.

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  Figure 8. Intestinal bacterial disorders and abnormal metabolic pathways as well as increased levels of pro-inflammatory factors and uric acid caused by alcohol and monosodium urate (MSU) crystals to exacerbate gout symptoms.