Urinary Proteome Analysis of Global Cerebral Ischemia–Reperfusion Injury Rat Model via Data-Independent Acquisition and Parallel Reaction Monitoring Proteomics

Abstract

Cerebral ischemia–reperfusion (I/R) injury is the leading cause of death in severe hypotension caused by cardiac arrest, drowning, and excessive blood loss. Urine can sensitively reflect pathophysiological changes in the brain even at an early stage. In this study, a rat model of global cerebral I/R injury was established via Pulsinelli’s four-vessel occlusion (4-VO) method. Overall, 164 urinary proteins significantly changed in the 4-VO rat urine samples compared to the control samples by data-independent acquisition (DIA) proteomics technique (1.5-fold change, p < 0.05). Gene Ontology annotation showed that the acute-phase response, the ERK1 and ERK2 cascade, endopeptidase activity, blood coagulation, and angiogenesis were overrepresented. After parallel reaction monitoring (PRM) validation, 15 differential proteins having human orthologs were verified as the potential urinary markers associated with cerebral I/R injury. Of these potential biomarkers, 8 proteins were reported to be closely associated with cerebral I/R injury. Nine differential proteins changed even when there were no clinical manifestations or histopathological cerebral damage, including FGG, COMP, TFF2, HG2A, KNG1, CATZ, PTGDS, PRVA, and HEPC. These 9 proteins are potential biomarkers for early screening of cerebral I/R injury to prevent the development of cerebral injury. KNG1, CATZ, PTGDS, PRVA, and HEPC showed an overall trend of upregulation or downregulation at 12 and 48 h after I/R injury, reflecting the progression of cerebral I/R injury. These 5 proteins may serve as potential biomarkers for prognostic evaluation of cerebral I/R injury. These findings provide important clues to inform the monitoring of cerebral I/R injury and further the current understanding of its molecular biological mechanisms.