METABOLOMICS COMBINED WITH SINGLE-CELL SEQUENCING REVEAL THE METABOLIC REPROGRAMMING IN INTESTINAL FIBROSIS

Objective: This study aims to systematically elucidate the metabolic landscape of intestinal fibrosis in Crohn's disease, revealing the key reprogrammed metabolic pathways in the fibrotic microenvironment.
Methods: We collected normal and fibrosis tissues from 122 Crohn's disease patients undergoing surgery for fibrosis-induced intestinal obstruction, and established DSS/TNBS-induced murine intestinal fibrosis model. To measure metabolites level in normal and fibrotic tissues, untargeted metabolomics was performed. C13-labeled glutamine was used for metabolic tracing to delineate metabolic fluxes in vitro and in vivo. We performed integration analysis by merging metabolomics and metabolic tracing data with established single-cell transcriptomic profiles. Isolation and further validation of human intestinal stromal cells were carried out, and gene expression was assessed using qRT-PCR and immunofluorescence.
Results: Significant metabolic heterogeneity was observed between normal and fibrotic tissues, with differential metabolites enriched in glucose and amino acid metabolism. Notably, intermediates of glycolysis were significantly increased, while tricarboxylic acid (TCA) cycle intermediates were decreased in fibrotic regions (p<0.05). Amino acids were broadly downregulated in fibrosis, whereas glutamate and glutamine were highly enriched (p<0.05). Mechanistic insights into these changes were provided by isotope tracing, revealing enhanced glycolysis and weakened TCA flux attributed to transition from TCA intermediate α-KG to glutamate. Moreover, catabolism of glutamine to glutamate was enhanced. Glutamate from above pathways converted to proline, which is the ingredient of extracellular matrix (ECM).
Single-cell transcriptomic analysis indicated a high enrichment of genes related to glutamine metabolism in stromal cells, with significant upregulation of enzymes such as GLS (Glutaminase) and PYCR1 (Pyrroline-5-carboxylate Reductase 1) involved in glutamine and proline synthesis. Immunofluorescence and qRT-PCR confirmed the upregulation of GLS and PYCR1. Further experiments using primary stromal cells and murine models demonstrated that inhibiting GLS activity reduced ECM synthesis, alleviating intestinal fibrosis.
Conclusion: In intestinal fibrosis, glycolysis and glutamine catabolism enhanced. Stromal cells utilized the reprogrammed metabolic pathway to synthesize glutamate, thereby promoting pathological ECM anabolism. Targeting the key enzyme GLS represent a potential therapeutic strategy for intestinal fibrosis.