Leech Poecilobdella manillensis protein extract ameliorated hyperuricemia by restoring gut microbiota dysregulation and affecting serum metabolites

Figure 1 Effect of leech Poecilobdella manillensis total protein extract treatment on hyperuricemia. A: Serum uric acid levels; B: Urine uric acid levels; C: Liver xanthine oxidase activity; D: Serum creatinine levels; E: Blood urea nitrogen levels; F: Renal tissue hematoxylin and eosin staining pathology under 200 × magnification. Statistical significance determined by ANOVA is indicated as follows: ^aP < 0.05 vs hyperuricemia model group (HUA); ^bP < 0.01 vs HUA; ^cP < 0.001 vs HUA; ^dP < 0.05 vs normal control group (CON); ^eP < 0.01 vs CON; ^fP < 0.001 vs CON. CON: Normal control group; HUA: Hyperuricemia model group; AP: Allopurinol treatment group; LTP: Leech Poecilobdella manillensis total protein extract treatment group; XOD: Xanthine oxidase; BUN: Blood urea nitrogen.

Figure 2 Effect of leech Poecilobdella manillensis total protein extract on facilitating uric acid excretion, evaluated by enzyme-linked immunosorbent assay. A: Renal urate transporter 1 (URAT1) concentration level; B: Renal glucose transporter 9 (GLUT9) concentration level; C: Renal ATP-binding cassette transporter G2 (ABCG2) concentration level; D: Jejunum URAT1 concentration level; E: Jejunum GLUT9 concentration level; F: Jejunum ABCG2 concentration level. Statistical significance determined by ANOVA is indicated as follows: ^aP < 0.05 vs hyperuricemia model group (HUA); ^bP < 0.01 vs HUA; ^cP < 0.001 vs HUA; ^dP < 0.05 vs normal control group (CON); ^eP < 0.01 vs CON; ^fP < 0.001 vs CON. Sample sizes: Renal tissue n = 6 for each group; Jejunum tissue n = 4 for each group. CON: Normal control group; HUA: Hyperuricemia model group; AP: Allopurinol treatment group; LTP: Leech Poecilobdella manillensis total protein extract treatment group; URAT1: Urate transporter 1; GLUT9: Glucose transporter 9; ABCG2: ATP-binding cassette transporter G2.

Figure 3 Effect of leech Poecilobdella manillensis total protein extract on epithelial tight junction proteins, evaluated by enzyme-linked immunosorbent assay. A: Renal zonula occludens-1 (ZO-1) concentration level; B: Renal occludin concentration level; C: Jejunum ZO-1 concentration level; D: Jejunum occludin concentration level. Statistical significance determined by ANOVA is indicated as follows: ^aP < 0.05 vs hyperuricemia model group (HUA); ^bP < 0.01 vs HUA; ^cP < 0.001 vs HUA; ^dP < 0.05 vs normal control group (CON); ^fP < 0.001 vs CON. Sample sizes: Renal tissue n = 6 for each group; Jejunum tissue n = 4 for each group. CON: Normal control group; HUA: Hyperuricemia model group; AP: Allopurinol treatment group; LTP: Leech Poecilobdella manillensis total protein extract treatment group; ZO-1: Zonula occludens-1.

Figure 4 Leech Poecilobdella manillensis total protein extract alters the gut microbiota in potassium oxonate-induced hyperuricemia mice. A: Shannon index across three groups; B: Simpson index across three groups; C: Partial least squares discriminant analysis of three groups; D: Principal coordinate analysis of three groups; E: Distribution plot of relative abundance at the phylum level of bacteria; F: Distribution plot of relative abundance at the genus level of bacteria; G: Cladogram from linear discriminant analysis effect size (LEfSe) analysis identifying highly differentiated taxa from phylum to genus levels; H: Linear discriminant analysis graph from LefSe analysis identifying highly differentiated taxa from phylum to genus levels; I: Predicted functional pathways of gut microbiota in hyperuricemia model group (HUA) vs leech Poecilobdella manillensis total protein extract treatment group; J: Predicted functional pathways of gut microbiota in normal control group vs HUA. Statistical significance is indicated as follows: ^aP < 0.05 vs hyperuricemia model group. Sample size: n = 6 per group. CON: Normal control group; HUA: Hyperuricemia model group; LTP: Leech Poecilobdella manillensis total protein extract treatment group; LDA: Linear discriminant analysis.

Figure 5 Effect of leech Poecilobdella manillensis total protein extract on vital microbial genus. A: The absolute abundance of vital microbial genera in all groups, assessed through the Kruskal-Wallis ANOVA; B and C: Significant differences between each two groups at the genus level of gut microbiota, determined by DESeq2 method [B: normal control group vs hyperuricemia model group (HUA); C: HUA vs leech Poecilobdella manillensis total protein extract treatment group; n = 6 for each group]. Statistical significance is indicated as follows: ^aP < 0.05 vs hyperuricemia model group (HUA); ^bP < 0.01 vs HUA; ^cP < 0.001 vs HUA; ^dP < 0.05 vs normal control group (CON); ^eP < 0.01 vs CON. CON: Normal control group; HUA: Hyperuricemia model group; LTP: Leech Poecilobdella manillensis total protein extract treatment group.

Figure 6 Leech Poecilobdella manillensis total protein extract alters the plasma metabolites in potassium oxonate-induced hyperuricemia mice. A and B: Partial least squares discriminant analysis (PLS-DA) score plots for normal control group (CON), hyperuricemia model group (HUA), and leech Poecilobdella manillensis total protein extract treatment group (LTP) in positive and negative ion mode, respectively; C and D: Score plots and permutation tests of orthogonal PLS-DA (OPLS-DA) between the HUA and CON groups; E and F: Score plots and permutation tests of OPLS-DA between the LTP and HUA groups; G: Volcano plot showing most significant metabolites identified by univariate analysis between the HUA and CON groups; H: Volcano plot showing most significant metabolites identified by univariate analysis between the LTP and HUA groups; I: Summary plot for pathway analysis of CON vs HUA; J: Summary plot for pathway analysis of LTP vs HUA, a pathways coexisted in both the microbiota and metabolome. CON: Normal control group; HUA: Hyperuricemia model group; LTP: Leech Poecilobdella manillensis total protein extract treatment group; FC: Fold change.

Figure 7 Effect of leech Poecilobdella manillensis total protein extract on vital metabolites. A: Heatmaps showing the trends in differential metabolites between groups; B: The concentrations of vital metabolites for all groups, assessed through the Kruskal-Wallis ANOVA. Statistical significance is indicated as follows: ^aP < 0.05 vs hyperuricemia model group (HUA); ^bP < 0.01 vs HUA; ^cP < 0.001 vs HUA; ^dP < 0.05 vs normal control group (CON); ^eP < 0.01 vs CON; ^fP < 0.001 vs CON. CON: Normal control group; HUA: Hyperuricemia model group; LTP: Leech Poecilobdella manillensis total protein extract treatment group.

Figure 8 Correlation analysis between the gut microbiota and metabolites from hyperuricemia and leech Poecilobdella manillensis total protein extract groups. The R values are represented by gradient colors, where purple cells indicate positive correlations and green cells indicate negative correlations, respectively. Statistical significance is denoted as follows: ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 9 Correlation analysis between gut microbiota and hyperuricemia-related parameters. The R values are represented by gradient colors, where purple cells indicate positive correlations and green cells indicate negative correlations, respectively. Statistical significance is denoted as follows: ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. BUN: Blood urea nitrogen; UUA: Urinary uric acid; ZO-1: Zonula occludens-1; URAT1: Urate transporter 1; GLUT9: Glucose transporter 9; ABCG2: ATP-binding cassette transporter G2; XOD: Xanthine oxidase; SCRE: Serum creatinine; SUA: Serum uric acid.

Figure 10  Mechanisms associated with treatment of Poecilobdella manillensis for hyperuricemia. A: decreased of uric acid generation in liver by reducing levels of xanthine oxidase; B: Reduction of uric acid excretion from kidney by regulating the expression of glucose transporter 9, urate transporter 1, and ATP-binding cassette transporter G2; C: Improvement of gut microbiota dysbiosis and regulation of sphingolipid metabolism and galactose metabolism. CON: Normal control group; HUA: Hyperuricemia model group; LTP: Leech Poecilobdella manillensis total protein extract treatment group; ZO-1: Zonula occludens-1; URAT1: Urate transporter 1; GLUT9: Glucose transporter 9; ABCG2: ATP-binding cassette transporter G2; XOD: Xanthine oxidase.