Discovery of metabolite biomarkers for cerebral ischemia-reperfusion injury based on metabolomics: neurotoxic mechanism and functional validation of a flavopiridol -related metabolite

Abstract

To investigate the role and potential mechanism of metabolite alterations during cerebral ischemia-reperfusion injury (CIRI) and to explore novel candidate molecular markers for the diagnosis of this disease. A rat model of middle cerebral artery occlusion was established using the modified Longa EZ intraluminal suture method. The infarct status was comprehensively evaluated by neurological deficit score (mNSS), TTC staining, HE staining, and Nissl staining. Serum metabolite changes after CIRI were detected using non-targeted metabolomics, and the potential toxic effects of significantly altered metabolites were predicted using the ProTox tool. Subsequently, bioinformatics analysis was employed to identify core targets and key pathways associated with the metabolite of interest, followed by molecular docking and molecular dynamics simulations to predict their interactions. Metabolomic analysis identified 41 significantly upregulated and 232 downregulated metabolites. Among the significantly altered metabolites, a flavopiridol‐related metabolite (FRM) exhibited potential neurotoxic characteristics. A total of 193 CIRI-related core targets were associated with this metabolite, from which 10 core targets and 15 key pathways were further screened. ROC analysis suggested that this metabolite possessed good discriminatory ability (AUC = 0.94). Molecular docking results showed binding energies of less than −7 kcal/mol for all core targets. A 100 ns molecular dynamics simulation indicated that the complex formed by the FRM and SRC remained structurally stable, as evidenced by balanced RMSD, Rg, SASA, and P-L-RMSD values. The calculated binding free energy was −32.48 kcal/mol, with key residues LEU-393, MET-341, and GLU-339 contributing significantly to the interaction, suggesting potential biological activity. This study reveals a complex metabolic reprogramming phenomenon during CIRI and preliminarily identifies a FRM as a potential candidate molecular marker and possible interventional target, providing a new theoretical basis and directions for future research on the diagnosis and treatment of CIRI.