Candidate metabolites in sepsis-associated encephalopathy: Network analysis and in vitro validation of glycitein

Sepsis-associated encephalopathy (SAE) is a severe neurological complication of sepsis. Cerebrovascular endothelial injury serves as a key contributor to its pathogenesis, and metabolite-mediated inflammatory regulation may be involved in this process. However, the specific candidate metabolites and their underlying pathways remain poorly characterized. This study aimed to identify candidate metabolites related to SAE through network pharmacology and to provide preliminary in vitro validation.

A "microbiota-substrate-metabolite-target-signal pathway" (MMTS) network was constructed by integrating metabolite databases, target prediction resources, and sepsis-related encephalopathy gene databases. Candidate metabolites were screened through protein-protein interaction analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, drug-likeness assessment, toxicology screening, and molecular docking. Glycitein was selected as a representative candidate for preliminary validation in a lipopolysaccharide (LPS)-induced human brain microvascular endothelial cell (hCMEC/D3) injury model. Cell activity, migration, tube formation, inflammatory factor release, intracellular reactive oxygen species levels, and NF-κB-related molecule expression were assessed.

A total of 206 gut microbiota-related metabolites and 1518 potential targets were identified. Network analysis predicted 20 key targets related to SAE. PPI network analysis indicated TNF, IL6, CXCL8, NFKB1, and TLR4 as central nodes. KEGG analysis was particularly enriched in the Toll-like receptor, NOD-like receptor, and NF-κB signaling. 14 candidate metabolites were retained after integrated screening. In LPS-treated hCMEC/D3 cells, glycitein partially restored cell viability, migration, and tube formation, reduced IL-6 and TNF-α levels, alleviated intracellular ROS accumulation, and was associated with downregulation of NFKB1, CXCL8, and IL-6 expression, together with reduced phosphorylation of NF-κB p65 and IκBα.

This study prioritized candidate metabolites associated with SAE through database-supported network analysis. Preliminary in vitro validation suggested that glycitein was associated with attenuation of inflammatory endothelial injury-related phenotypes, providing a basis for further mechanistic studies.