Multi-omics analysis identifies gut microbiota-glutamine axis contributing to the pathogenesis of reflux esophagitis
Background: Reflux esophagitis (RE), a common gastroesophageal reflux disease characterized by esophageal mucosal inflammation, is closely associated with gut microbiota dysbiosis and metabolic abnormalities. The glutamine-glutamate metabolic pathway regulates inflammation and mucosal barrier function, but its role in RE and association with gut microbiota remain unclear. This study aimed to characterize gut microbiota and serum metabolites in RE patients via integrated multi-omics (focusing on the gut microbiota-glutamine axis), and verify the activation status of this pathway in RE inflammatory models and the anti-inflammatory effect of its targeted inhibition.Methods: RE patients and healthy controls (HCs) were enrolled. Fecal metagenomic sequencing and serum untargeted metabolomics (LC-MS/MS) were performed to identify differential gut microbiota and serum metabolites between the two groups, followed by Pearson correlation analysis to explore their associations. In vitro experiments were conducted on human esophageal epithelial cells (HEECs) divided into four groups: normal, inflammatory, glutamine-supplemented, and inflammatory + glutamine + glutaminase inhibitor (BPTES) groups. qPCR was used to detect the mRNA expression of glutamine-glutamate pathway molecules (GLS, c-Myc, SLC1A5), mucosal barrier markers (ZO-1, Occludin), and pro-inflammatory cytokines (IL-8, IL-6, IL-1beta, TNF-alpha). Intracellular concentrations of glutamine, glutamate, and alpha-ketoglutarate were measured, and the anti-inflammatory effect of BPTES was verified.Results: RE patients showed significant differences in gut microbiota diversity and composition compared with HCs, with Bacteroidota, Pseudomonadota, Escherichia coli, and Klebsiella pneumoniae as dominant taxa. Serum metabolomics revealed elevated glutamine and glutamate in RE patients, which were identified as key differential metabolites related to RE pathogenesis. Pearson analysis revealed that alterations in serum metabolite profiles of RE patients were significantly correlated with changes in gut bacterial abundance. Notably, glutamate-glutamate (Glu-Glu) metabolism exhibited negative correlations with multiple bacterial genera (Acrocarpospora, Limnobacter, Pseudobacter, Shewanella, and Tropicimonas). In vitro, inflammatory HEECs exhibited increased intracellular glutamine, glutamate, and alpha-ketoglutarate, upregulated glutamine-glutamate pathway molecules and pro-inflammatory cytokines, and downregulated mucosal barrier markers. Exogenous glutamine alone failed to alleviate inflammation, while combined with BPTES significantly reversed pathway activation and mitigated inflammation in inflammatory HEECs.Conclusion: RE patients exhibit significant gut microbiota dysbiosis (dominated by Bacteroidota, Pseudomonadota, Escherichia coli, and Klebsiella pneumoniae) and abnormal glutamine metabolism (elevated serum glutamine and glutamate). Pearson analysis reveals that the glutamine-glutamate pathway correlates negatively with multiple bacterial genera (Acrocarpospora, Limnobacter, Pseudobacter, Shewanella, and Tropicimonas). The glutamine-glutamate pathway is activated in inflammatory esophageal epithelial cells, and targeted GLS inhibition by BPTES reverses pathway activation and mitigates inflammation. These findings highlight the gut microbiota-glutamine axis as potential diagnostic biomarkers and therapeutic targets for RE, providing new insights into pathogenesis and a basis for novel clinical interventions.