Modulation of the gut-liver axis by oxymatrine alleviates metabolic dysfunction-associated steatotic liver disease

Figure 1 Oxymatrine improves liver function, lipid content, and inflammatory parameters in serum and liver in metabolic dysfunction-associated steatotic liver disease rats. A: Study schematic; B: Body weight trajectory post-oxymatrine, n = 6; C: Representative rat images (final day); D: Gross liver morphology; E: The contents of serum alanine aminotransferase, aspartate transaminase, total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol, n = 6; F: Levels of cytokines including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-18, n = 6; G: Hepatic triglyceride and free fatty acid contents, n = 6. ^aP < 0.05, ^bP < 0.01. Statistical analysis was conducted using one-way analysis of variance with Tukey’s post hoc test, and results are presented as mean ± SD. ALT: Alanine aminotransferase; AST: Aspartate transaminase; FFA: Free fatty acid; HDL-C: High-density lipoprotein cholesterol; IL: Interleukin; LDL-C: Low-density lipoprotein cholesterol; MET: Metformin; OMT: Oxymatrine; TC: Total cholesterol; TG: Triglyceride; TNF-α: Tumor necrosis factor-α.

Figure 2 Oxymatrine alleviates hepatic pathological damage and lipid accumulation in metabolic dysfunction-associated steatotic liver disease rats. A: Liver histology (hematoxylin eosin staining, representative images), 100 × (200 μm); 400 × (50 μm); B: Liver histology (oil red O staining, representative images). n = 6, 100 × (200 μm); 400 × (50 μm). ^aP < 0.05, ^bP < 0.01. Statistical analysis was conducted using one-way analysis of variance with Tukey’s post hoc test, and results are presented as mean ± SD. HE: Hematoxylin eosin; MET: Metformin; OMT: Oxymatrine; ORO: Oil red O.

Figure 3 Oxymatrine improves intestinal permeability in metabolic dysfunction-associated steatotic liver disease rats. A: Plasma endotoxin levels of the rats from the portal vein, n = 6; B: Bacterial contents in mesenteric lymph nodes, liver tissues, and spleen tissues, n = 6; C: Ileal histology (hematoxylin eosin staining, representative images), 200 × (100 μm); 400 × (50 μm); D: The concentrations of Evans Blue in liver samples, n = 3; E: Permeability of horseradish peroxidase in segments of ileum incubated in Ussing chambers, n = 6; F: Protein levels of occludin and ZO-1 in ileal tissues, n = 3. ^aP < 0.05, ^bP < 0.01. Statistical analysis was conducted using one-way analysis of variance with Tukey’s post hoc test, and results are presented as mean ± SD. MET: Metformin; OMT: Oxymatrine.

Figure 4 Oxymatrine enhances intestinal microbiota diversity in metabolic dysfunction-associated steatotic liver disease rats. A: Chao1, observed species, and Faith’s phylogenetic diversity indices of the alpha diversity analysis, n = 6; B: Principal coordinate analysis results of the beta diversity analysis; C: Nonmetric multidimensional scaling results of the beta diversity analysis; D: Hierarchical clustering tree of the rats; E: Composition of the top 20 intestinal microbiota (phylum level); F: Composition of the top 20 intestinal microbiota (class level); G: Firmicutes/Bacteroidetes ratio at the phylum level, n = 6; H: The relative abundance of Clostridiales, Streptococcus and S24-7 in each group, n = 6; I: A heatmap of the top 50 different bacterial genera among the three groups (control, model, oxymatrine). ^aP < 0.05, ^bP < 0.01. Statistical analysis was conducted using one-way analysis of variance with Tukey’s post hoc test, and results are presented as mean ± SD. Group A: Control; Group B: Model; Group C: Oxymatrine; OMT: Oxymatrine; PD: Phylogenetic diversity.

Figure 5 Oxymatrine alters intestinal microbiota composition in metabolic dysfunction-associated steatotic liver disease rats. A: Linear discriminant analysis effect size analysis results; B: Cladogram of linear discriminant analysis effect size analysis; C: The relative abundance of Lactobacillus, Bacilli, Lactobacillales, Lactobacillaceae, Streptococcus, Lachnospiraceae, Blautia, Pseudomonadaceae, and Pseudomonas among groups (control, model, oxymatrine). Class A: Control; Class B: Model; Class C: Oxymatrine; LDA: Linear discriminant analysis.

Figure 6 Oxymatrine reverses hepatic metabolite changes in metabolic dysfunction-associated steatotic liver disease rats. A: Principal component analysis scores (positive/negative modes); B: Orthogonal partial least squares discriminant analysis score plots (positive and negative ion modes); C: Orthogonal partial least squares discriminant analysis permutation tests: Both ion modes; D: Top 150 hepatic metabolites: Heatmap visualization; E: The levels of adrenic acid, luteolin, oleoylethanolamide and pomiferin in the hepatic metabolites among the three groups, n = 6; F: Enrichment analysis of differential hepatic metabolites in Kyoto Encyclopedia of Genes and Genomes pathways (control, model, oxymatrine). ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001. Statistical analysis was conducted using one-way analysis of variance with Tukey’s post hoc test, and results are presented as mean ± SD. Group A: Control; Group B: Model; Group C: Oxymatrine; NS: Not significant.

Figure 7 Association between different intestinal microbiota and different liver metabolites. ^aP < 0.05, ^bP < 0.01 (color-coded: Red = positive, blue = negative). Data = mean ± SD. Groups were compared by one-way analysis of variance (Tukey’s test).

Figure 8 Improvement of serum parameters and cytokines by fecal microbiota transplantation in metabolic dysfunction-associated steatotic liver disease rats. A: Study schematic; B: The contents of serum alanine aminotransferase, aspartate transaminase, total cholesterol, triglyceride, n = 6; C: The concentrations of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-18, n = 6. ^aP < 0.05, ^bP < 0.01. Statistical analysis was conducted using one-way analysis of variance with Tukey’s post hoc test, and results are presented as mean ± SD. ALT: Alanine aminotransferase; AST: Aspartate transaminase; FMT: Fecal microbiota transplantation; IL: Interleukin; MET: Metformin; SFF: Sterile fecal filtrate; TC: Total cholesterol; TG: Triglyceride; TNF-α: Tumor necrosis factor-α.

Figure 9 Fecal microbiota transplantation improved histopathological liver damage in metabolic dysfunction-associated steatotic liver disease rats. A: Liver histology (hematoxylin eosin staining, representative images), 100 × (200 μm); 400 × (50 μm); B: Liver histology (oil red O staining, representative images). n = 6, 100 × (200 μm); 400 × (50 μm); C: Quantitative oil red O staining of rat liver tissue, n = 3. ^bP < 0.01. Statistical analysis was conducted using one-way analysis of variance with Tukey’s post hoc test, and results are presented as mean ± SD. Abx: Antibiotics; FMT: Fecal microbiota transplantation; HE: Hematoxylin eosin; ORO: Oil red O; SFF: Sterile fecal filtrate.