Integrated Microbiome and Metabolome Analysis Reveals Correlations Between Gut Microbiota Components and Metabolic Profiles in Mice with Methotrexate-Induced Hepatoxicity

Changshui Wang,1,* Shuzhen Zhao,2,* Yuan Xu,3 Wenxue Sun,4 Yuanyuan Feng,2 Deshuai Liang,5 Yun Guan3 1Department of Neurosurgery, Affiliated Hospital of Jining Medical University, Jining Medical University, Jining, 272000, People’s Republic of China; 2Children’s Rehabilitation Center, Jining Maternal and Child Health Family Planning Service Center, Jining, 272000, People’s Republic of China; 3Department of Hematology, Jining NO. 1 People’s Hospital, Jining, 272000, People’s Republic of China; 4Institute of Clinical Pharmacy and Pharmacology, Jining NO. 1 People’s Hospital, Jining Medical University, Jining, 272000, People’s Republic of China; 5Department of pharmacy, Jining NO. 1 People’s Hospital, Jining, 272000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yun Guan; Deshuai Liang, Jining NO. 1 People’s Hospital, 6 Jiankang Road, Jining, Shandong, 272000, People’s Republic of China, Tel/Fax +86-0537 2087092, Email gyuanun1989@163.com; 13468322037@163.comPurpose: We designed this study to investigate the potential correlations between gut microbiota compositions and hepatic metabolomic disorders in mice with methotrexate (MTX)-induced hepatoxicity.Methods: We used MTX to induce hepatoxicity in healthy Kunming mice, and we determined plasma ALT and AST levels and assessed the liver tissue histopathology. We applied an integrated gas chromatography-mass spectrometry (GC-MS) and 16S ribosomal RNA (rRNA) gene sequencing approach to evaluate the effects of MTX on the gut microbiota and hepatic metabolic profiles of mice. We uncovered correlations between the gut microbiota and hepatic metabolomic profiles by calculating the Spearman correlation coefficient.Results: MTX caused ALT and AST level elevations and hepatoxicity in our mouse model. MTX disrupted amino acid metabolic pathways (including biosyntheses of valine, leucine, and isoleucine; and arginine; and, metabolism of alanine, aspartate, and glutamate; histidine; beta-alanine; and glycine, serine, and threonine); biosyntheses of aminoacyl-tRNA; and pantothenate, and CoA; and, metabolic pathways of energy, glutathione, and porphyrin; and chlorophyll. In addition, MTX increased the abundances of Staphylococcus, Enterococcus, Collinsella, Streptococcus, and Aerococcus, but decreased the amounts of Lactobacillus, Ruminococcus, norank_f_Muribaculaceae, unclassified_f_Lachnospiraceae, norank_f_Lachnospiraceae, A2, Eubacterium_xylanophilum_group, Phascolarctobacterium, Bifidobacterium, and Faecalibaculum. Our correlation analyses showed that different flora abundance changes including those of Phascolarctobacterium, Faecalibaculum, norank_f_Muribaculaceae, Streptococcus, Enterococcus, Staphylococcus, and Collinsella were associated with liver injury.Conclusion: We present evidence supporting the notion that MTX causes hepatoxicity by altering the gut microbiota and hepatic metabolite profiles, our findings provide new venues for the management of MTX-induced hepatoxicity.Keywords: gas chromatography-mass spectrometry, 16S ribosomal RNA, methotrexate, hepatoxicity