Integrated multi-omics analysis reveals distinct microbiota-metabolite signatures and a novel HCN2-2-hydroxybutyric acid interaction in inflammatory bowel disease.

Introduction: Gut microbiota-derived short-chain fatty acids (SCFAs) exert critical regulatory functions in inflammatory bowel disease (IBD). However, integrated profiling of fecal SCFA signatures alongside gut microbiota composition in ulcerative colitis (UC) and Crohn's disease (CD) remains insufficiently characterized. Furthermore, the molecular mechanisms through which microbiota metabolites engage host protein targets warrant systematic investigation. Methods: This study enrolled 30 patients with UC, 20 with CD, and 30 healthy controls, with paired fecal collection. Gut microbiota composition was analyzed by deep metagenomic sequencing, and SCFA concentrations were quantified by gas chromatography-mass spectrometry. Multi-omics integration, correlation network analysis, and Bayesian kernel machine regression were employed to resolve microbiota-metabolite associations. An integrated computational pipeline incorporating molecular dynamics simulations was constructed to evaluate the thermodynamic stability and binding modalities of metabolite-protein interactions. Results: Both UC and CD patients exhibited significantly reduced gut microbial α-diversity and characteristic community structure alterations. Fecal metabolomic profiling revealed synchronous elevation of 2-Hydroxybutyric acid (2-HB) and isocaproate in both patient groups, whereas butyrate reduction was restricted to UC. Multi-omics correlation analysis identified significant associations between 2-HB and unclassified Veillonella species as well as specific functional modules. Molecular dynamics simulations with an aggregate sampling time of 100 ns revealed a structural basis for the formation of a stable complex between 2-HB and the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2). This interaction was primarily mediated by electrostatic interactions involving Arg659, Arg618, and Arg617 residues alongside hydrophobic contacts, suggestive of potential allosteric modulation. Conclusions: This study identifies 2-HB and isocaproate as shared fecal metabolic markers across IBD and provides a structural rationale for the interaction between 2-HB and HCN2. The druggability profile of HCN2 supports its prioritization for mechanistic investigation, with the caveat that functional validation is prerequisite to any inference of therapeutic relevance.