Multi-omics and machine learning identify neutrophil annexin A3 as a therapeutic target of ginsenoside Rb1 for comorbidity of ischaemic stroke and ulcerative colitis.

BACKGROUND AND PURPOSE: Epidemiological evidence suggests a bidirectional relationship between ischaemic stroke (IS) and ulcerative colitis (UC), yet the underlying molecular mechanisms remain poorly understood. EXPERIMENTAL APPROACH: Four IS and three UC datasets from the gene expression omnibus (GEO) database were analysed to identify key comorbidity genes. Two-sample Mendelian randomisation (MR) was conducted to infer causal relationships. Clinical validations utilised peripheral blood neutrophils from UC-IS patients, assessed via western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The direct binding of ginsenoside Rb1 to annexin A3 (ANXA3) was verified through molecular docking, molecular dynamics simulations and cellular thermal shift assay (CETSA). KEY RESULTS: MR analysis indicated a causal association between ANXA3 expression and increased risk of IS and UC. Immune infiltration analysis demonstrated a strong positive correlation between ANXA3 expression and neutrophil infiltration in both IS and UC. ScRNA-seq confirmed that ANXA3 was expressed in neutrophils within diseased tissues. Circulating neutrophils from UC-IS comorbidity patients showed significantly elevated ANXA3 mRNA and protein levels. Molecular dynamics simulations revealed a stable ginsenoside Rb1-ANXA3 complex with favourable binding free energy. CETSA confirmed the direct binding of ginsenoside Rb1 to ANXA3 in primary human neutrophils. UC-IS serum induced neutrophil hyperactivation, including excessive migration, NETosis, myeloperoxidase (MPO) release and reactive oxygen species (ROS) production, which was significantly attenuated by ginsenoside Rb1 in vitro. CONCLUSIONS AND IMPLICATIONS: ANXA3 is a predominant neutrophil-derived biomarker linking UC-IS comorbidity. Ginsenoside Rb1 mitigates comorbidity-induced neutrophil hyperactivation by directly binding ANXA3, representing a promising therapeutic strategy.