Screening of potential targets of (2S)-2'-methoxykurarinone against sepsis and in vivo verification in zebrafish model.

OBJECTIVE: To screen the potential core targets of (2S)-2'-Methoxykurarinone, a dimethyldihydroflavonoid derived from Sophora flavescens, against sepsis. Furthermore, we explore the in vivo pharmacological efficacy and potential mechanism of action through bioinformatics analysis, clinical sample verification, and an LPS-induced zebrafish sepsis model, aiming to provide experimental evidence for the development of this compound as a candidate lead compound against sepsis. METHODS: Differentially expressed genes (DEGs) associated with sepsis were screened based on the GSE310929 sepsis transcriptome dataset, and weighted gene co-expression network analysis (WGCNA) was performed to identify key sepsis-related modules. Multiple databases were used to predict compound targets. The intersection of DEGs, key module genes, and compound targets was obtained to screen core anti-sepsis genes. A protein-protein interaction (PPI) network was constructed, followed by Gene Ontology (GO) functional enrichment analysis. A total of 22 patients with sepsis and 10 healthy controls were enrolled for peripheral blood transcriptome sequencing. After batch correction by integrating multiple GEO datasets, 12 machine learning algorithms, SHAP interpretability analysis, and an artificial neural network (ANN) model were applied to screen and verify core diagnostic and prognostic genes. Single-cell transcriptome analysis was used to clarify the cellular localization of core gene expression in immune cells. Molecular docking was conducted to evaluate the binding affinity between the compound and core target proteins. An LPS-induced zebrafish sepsis model was further established, and the in vivo protective effects of the compound were evaluated in terms of embryonic survival rate, macrophage infiltration, intracellular ROS levels, and behavioral phenotypes. RESULTS: A total of 1617 sepsis-related DEGs were identified, and the greenyellow module was determined as the core co-expression module of sepsis. A total of 2124 target genes were predicted for the compound, and 36 potential core anti-sepsis targets were obtained from the intersection of the three gene sets, which were mainly enriched in immune-related processes including T cell activation, lymphocyte differentiation, and kinase activity regulation. After screening and validation via machine learning and ANN models, five core genes (PRKCQ, DYRK2, CD2, BACH2, AAK1) were identified. All five genes were significantly downregulated in the peripheral blood of sepsis patients, enabling efficient discrimination of sepsis, and their high expression was closely correlated with favorable patient prognosis. These core genes were predominantly localized to peripheral blood T cells and NK cells. Molecular docking confirmed that (2S)-2'-Methoxykurarinone exhibited favorable binding affinity with all five core target proteins. In vivo experiments demonstrated that (2S)-2'-Methoxykurarinone markedly improved the embryonic survival rate of septic zebrafish, inhibited excessive macrophage infiltration and abnormal ROS accumulation, and effectively ameliorated behavioral abnormalities in larval zebrafish. CONCLUSION: (2S)-2'-Methoxykurarinone exerts significant protective effects against sepsis in vivo by targeted regulation of the core genes PRKCQ, DYRK2, CD2, BACH2, and AAK1, mediating immune modulation and oxidative stress inhibition. It possesses promising potential for development as a natural candidate drug for sepsis treatment.