This study aimed to explore potential relationships between gut microbiota composition and hepatic metabolic disturbances in mice with methotrexate (MTX)-induced hepatotoxicity.Hepatotoxicity was induced in healthy Kunming mice using MTX, followed by measurements of plasma ALT and AST levels and histopathological examination of liver tissue. An integrated approach combining gas chromatography–mass spectrometry (GC-MS) and 16S rRNA gene sequencing was employed to assess MTX-induced alterations in gut microbiota and liver metabolic profiles. Spearman correlation analysis was used to identify associations between gut microbial changes and hepatic metabolomic profiles.MTX administration led to elevated ALT and AST levels and evident liver damage. MTX disrupted multiple metabolic pathways, including amino acid biosynthesis (valine, leucine, isoleucine, arginine) and metabolism (alanine, aspartate, glutamate, histidine, beta-alanine, glycine, serine, threonine), aminoacyl-tRNA biosynthesis, pantothenate and CoA biosynthesis, as well as energy, glutathione, porphyrin, and chlorophyll metabolism. Gut microbiota analysis revealed increases in Staphylococcus, Enterococcus, Collinsella, Streptococcus, and Aerococcus, alongside decreases in Lactobacillus, Ruminococcus, norank_f_Muribaculaceae, unclassified_f_Lachnospiraceae, norank_f_Lachnospiraceae, A2, Eubacterium_xylanophilum_group, Phascolarctobacterium, Bifidobacterium, and Faecalibaculum. Correlation analysis indicated that shifts in the abundance of Phascolarctobacterium, Faecalibaculum, norank_f_Muribaculaceae, Streptococcus, Enterococcus, Staphylococcus, and Collinsella were associated with liver injury.The findings suggest that MTX-induced hepatotoxicity is closely linked to alterations in gut microbiota composition and hepatic metabolite profiles, highlighting potential strategies for mitigating MTX-related liver damage.
