Modulation of Gut Microbiota by Magnesium Isoglycyrrhizinate Mediates Enhancement of Intestinal Barrier Function and Amelioration of Methotrexate-Induced Liver Injury

Abstract

Background: The gut-liver axis plays a crucial role in various liver diseases. Therefore, targeting this crosstalk may provide a new treatment strategy for liver diseases. However, the exact mechanism underlying this crosstalk and its impact on drug-induced liver injury (DILI) requires clarification.

Aim: This study aimed to investigate the potential mechanism and therapeutic effect of MgIG on MTX-induced liver injury, which is associated with the gut-liver axis and gut microbiota.

Methods: An MTX-induced liver injury model was generated after 20-mg/kg/3d MTX application for 30 days. Meanwhile, the treatment group was treated with 40-mg/kg MgIG daily. Histological examination, aminotransferase, and aspartate aminotransferase enzyme levels were estimated to evaluate liver function. Immune cells infiltration and inflammatory cytokines were detected to indicate inflammation levels. Colon histological score, intestinal barrier leakage, and expression of tight junctions were employed to assess the intestinal injury. Bacterial translocation was observed using fluorescent in situ hybridisation, colony-forming unit counting, and lipopolysaccharide detection. Alterations in gut microbial composition were analysed using 16s rDNA sequencing and relative quantitative polymerase chain reaction. Short-chain-fatty-acids and lactic acid concentrations were then utilized to validate changes in metabolites of specific bacteria. Lactobacillus sp. supplement and fecal microbiota transplantation were used to evaluate gut microbiota contribution.

Results: MTX-induced intestinal and liver injuries were significantly alleviated using MgIG treatment. Bacterial translocation resulting from the intestinal barrier disruption was considered a crucial cause of MTX-induced liver injury and the therapeutic target of MgIG. Moreover, MgIG was speculated to have changed the gut microbial composition by up-regulating probiotic Lactobacillus and down-regulating Muribaculaceae, thereby remodelling the intestinal barrier and inhibiting bacterial translocation.

Conclusion: The MTX-induced intestinal barrier was protected owing to MgIG administration, which reshaped the gut microbial composition and inhibited bacterial translocation into the liver, thus attenuating MTX-related DILI.

Keywords: bacterial translocation; gut microbiota; gut-liver axis; magnesium isoglycyrrhizinate; methotrexate.

Graphical Abstract

Schematic illustration of the therapeutic effects of magnesium isoglycyrrhizinate (MgIG) on methotrexate (MTX)-induced intestinal and liver injuries: MTX induces intestinal barrier damage by altering the microbiota composition, which causes bacterial translocation and liver inflammation. However, MgIG treatment elevated Lactobacillus levels and restored the intestinal barrier, which acts as a protective barrier in MTX-induced liver injuries.

Figure 1

MgIG alleviated MTX-induced liver injury. (A) Experiment design. (B) Bodyweights during mouse model development (n = 8, Two-way ANOVA). (C) Liver index (liver weight/body weight) of each group (n = 8). (D, E) ALT and AST levels in serum(n = 8). (F) Representative H&E staining images of liver tissue from each group. Arrows and asterisks in the sections indicate histopathological differences between groups: inflammatory infiltration (arrow) and necrosis (asterisk). Scale bar, 100μm. (G) Histological score of liver tissue in each group (n = 6, Kruskal-Wallis test). ***p < 0.001, compared with Control group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with MTX group.

Figure 2

MgIG reduced MTX-induced inflammation levels. (A–D) FACS staining of immune cells (neutrophils, monocytes and macrophages) in the liver represented as percentages of CD45+ cells (n = 4-6, One-way ANOVA). (E) Relative mRNA expression of cytokines in liver tissues from each group (n = 3-6). (F) Heatmap of specific cytokine and chemokine levels in serum, including IFN-γ, IL-4, IL-6, GM-CSF, IL-1β, IL-17, IL-10, TNF-α, MIP-2, MCP-1 (n = 4). (G) Cytokines and chemokines from panel F and comparisons of MTX and other groups. Purple entries indicate cytokines/chemokines that were less abundant in various groups compared to the MTX group. *p < 0.05, **p < 0.01, ***p < 0.001, compared with Control group. ^#p < 0.05, ^###p < 0.001, compared with MTX group. ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Figure 3

MgIG alleviated MTX-induced intestinal injury. (A) Representative H&E staining of colon tissue sections from each group, Scale bar, 200μm. (B) Histological score in each group (n = 6, Kruskal-Wallis test). (C) Representative In vivo imaging of FITC-Dextran. (D) Intestinal leakage measured by FITC-Dextran concentration in serum (n = 6, One-way ANOVA). (E) Relative mRNA expression of tight junctions in liver tissue from each group (n = 3-5). (F) Immunoblot analysis of ZO-1, E-cadherin and Claudin-1 in colon tissues (n = 3). (G) Immunofluorescence analysis on ZO-1, E-cadherin and Claudin-1 in colon sections from different groups. Representative images are shown. Scale bar, 100μm. *p < 0.05, **p < 0.01, ***p < 0.001, compared with Control group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with MTX group.

Figure 4

MTX-induced intestinal barrier disruption was detected in advance of hepatic lesion (A) Experiment design. (B) Histological score of colon tissue (n = 6, Kruskal-Wallis test). (C) Representative in vivo imaging of FITC-Dextran leakage. (D) Quantification of intestinal leakage measured by FITC-Dextran concentration in serum (n = 5, One-way ANOVA). (E) Immunoblot analysis of ZO-1, E-cadherin and Claudin-1 in colon sections from different groups (n = 3, One-way ANOVA). (F) Quantification of Figure 3E. (G) Histological score of liver tissue (n = 6, Kruskal-Wallis test). (H, I) ALT and AST levels in serum (n = 6, One-way ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001, compared with D0 or Control.

Figure 5

Bacterial translocation is the prerequisite of MTX-induced hepatic lesion (A) Representative images of Fluorescent in situ hybridization of the colon at different timepoints. Arrows indicate detected bacteria. (B) Lipopolysaccharide concentration in plasma (n = 5, One-way ANOVA). (C) Fluorescent in situ hybridization of the liver (D) Counting of colony-forming units in liver tissue of each group (n = 5, One-way ANOVA). ***p < 0.001, compared with D0 or Control group. ^###p < 0.001, compared with MTX group.

Figure 6

16s rDNA sequencing revealed altered microbiota composition after MTX or MgIG treatment (A) PCoA analysis with the ANOSIM significant test for each group (n = 5). (B) Relative abundance of microbial taxa was determined at the genus level. Top 10 abundances are shown. (C) Significance test of multiple groups using Kruskal-Wallis H test. (D, E) Multilevel species-level bar chart from LEfSe analysis, showing biomarker taxa at the genus level (LDA score) of >4 and a significance of P < 0.05 determined by the Wilcoxon signed-rank test. (F) 16s rDNA relative expression measured by quantitative PCR. (G) Fecal lactic acid concentration of each group (n = 6). ***p < 0.001, compared with Control group. ^##p < 0.01, ^###p < 0.001, compared with MTX group.

Figure 7

Lactobacillus. sp. can protect the liver and intestine against MTX-induced toxicity. (A) Experiment design. (B, C) Representative H&E staining of liver tissues and histological score, Scale bar, 100μm. (D, E) Representative H&E staining of colon tissue sections and histological score in each group, Scale bar, 200μm (n=6, Kruskal-Wallis test). (F) In vivo imaging of FITC-Dextran leakage. **p < 0.01, compared with Control group. ^#p < 0.05, ^##p < 0.01, compared with MTX group.

Figure 8

Lactobacillus. sp. reduced MTX-induced inflammation levels in liver and serum. (A–D) FACS staining of immune cells in the liver represented as percentages of CD45+ cells. (E) Heatmap of specific cytokine and chemokine levels in serum, including IFN-γ, IL-4, IL-6, GM-CSF, IL-1β, IL-17, IL-10, TNF-α, MIP-2, MCP-1 (n=4-5). (F) Cytokines and chemokines from panel L and comparisons of MTX and other groups. Blue entries indicate cytokines/chemokines that were less abundant in various groups compared to the MTX group. *p < 0.05, **p < 0.01, ***p < 0.001, compared with Control group. ^#p < 0.05, ^###p < 0.001, compared with MTX group.

Figure 9

FMT helped restoring intestinal barrier function and ameliorating hepatic inflammation. (A) Experiment design. (B) Immunofluorescence analysis on ZO-1, E-cadherin and Claudin-1 in colon sections from different groups. Representative images are shown. Scale bar, 100μm. (C) Relative mRNA expression of tight junctions and cell adhesion protein in colon. (D, E) ALT and AST levels in serum (ALT, n = 6. AST, n = 4, One-way ANOVA). (F) Relative mRNA expression of cytokines in liver (n = 6, One-way ANOVA). (G, H) Quantification of immune cells in the liver represented as percentages of CD45+ cells (n = 6-8). *p < 0.05, **p < 0.01, ***p < 0.001, compared with Control group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared with M→C group. FMT, fecal microbiota transplantation.