Cerebral ischemia-reperfusion (I/R) injury is one of the leading causes of death in cases of severe hypotension resulting from cardiac arrest, drowning, and excessive blood loss. Electro-acupuncture (EP) treatment, a practice used in stroke rehabilitation in China for thousands of years, was examined in this study for its effects on cerebral ischemia-reperfusion injury. Our objective was to identify urinary protein biomarkers associated with global cerebral I/R injury and to investigate the dynamic changes in the urinary proteome following EP treatment in a rat model of cerebral ischemia-reperfusion (CI/R) injury. A rat model of global cerebral I/R injury was established using the well-established Pulsinelli’s four-vessel occlusion (4-VO) method. Through data-independent acquisition (DIA) proteomics, we identified a total of 164 urinary proteins that significantly changed in the 4-VO rat urine samples compared to the control samples (with a 1.5-fold change, p < 0.05). After validation by parallel reaction monitoring (PRM), 15 differential proteins with human orthologs were confirmed as potential urinary markers of cerebral I/R injury. Among these biomarkers, eight proteins had previously been reported to be closely associated with cerebral I/R injury. In addition, 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 nine proteins could serve as early screening biomarkers for cerebral I/R injury, potentially allowing for preventive interventions to avoid progression to cerebral damage. KNG1, CATZ, PTGDS, PRVA, and HEPC showed an overall trend of upregulation or downregulation at 12 and 48 h after I/R injury, indicating their potential role as biomarkers for assessing the progression of cerebral I/R injury. In healthy control rats, 47 urinary proteins (23 up-regulated, 24 down-regulated) were differentially expressed following EP treatment (1.5-fold change, p < 0.05). In contrast, in the I/R rat model, 80 urinary proteins (27 up-regulated, 53 down-regulated) were significantly altered compared to controls. Among these differentially expressed proteins, 23 proteins (17 up-regulated, 6 down-regulated) exhibited significant changes following EP treatment (1.5-fold change, p < 0.05). The primary biological processes associated with these proteins included aging, immune response, acute-phase response, liver regeneration, protein catabolism, and oxidative stress response. Several metabolic pathways were enriched, as revealed by KEGG analysis. These findings offer important insights into the monitoring of cerebral I/R injury and contribute to a deeper understanding of its molecular mechanisms. Our results suggest that EP may mitigate ischemia-reperfusion-induced cerebral damage through anti-inflammatory pathways and by modulating metabolic processes.

Changes of Urinary Proteome in Global Cerebral Ischemia-Reperfusion Injury

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发表日期：

2025年

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Weiwei Qin,
Mingshan Wang

摘要

Cerebral ischemia-reperfusion (I/R) injury is one of the leading causes of death in cases of severe hypotension resulting from cardiac arrest, drowning, and excessive blood loss. Electro-acupuncture (EP) treatment, a practice used in stroke rehabilitation in China for thousands of years, was examined in this study for its effects on cerebral ischemia-reperfusion injury. Our objective was to identify urinary protein biomarkers associated with global cerebral I/R injury and to investigate the dynamic changes in the urinary proteome following EP treatment in a rat model of cerebral ischemia-reperfusion (CI/R) injury. A rat model of global cerebral I/R injury was established using the well-established Pulsinelli’s four-vessel occlusion (4-VO) method. Through data-independent acquisition (DIA) proteomics, we identified a total of 164 urinary proteins that significantly changed in the 4-VO rat urine samples compared to the control samples (with a 1.5-fold change, p < 0.05). After validation by parallel reaction monitoring (PRM), 15 differential proteins with human orthologs were confirmed as potential urinary markers of cerebral I/R injury. Among these biomarkers, eight proteins had previously been reported to be closely associated with cerebral I/R injury. In addition, 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 nine proteins could serve as early screening biomarkers for cerebral I/R injury, potentially allowing for preventive interventions to avoid progression to cerebral damage. KNG1, CATZ, PTGDS, PRVA, and HEPC showed an overall trend of upregulation or downregulation at 12 and 48 h after I/R injury, indicating their potential role as biomarkers for assessing the progression of cerebral I/R injury. In healthy control rats, 47 urinary proteins (23 up-regulated, 24 down-regulated) were differentially expressed following EP treatment (1.5-fold change, p < 0.05). In contrast, in the I/R rat model, 80 urinary proteins (27 up-regulated, 53 down-regulated) were significantly altered compared to controls. Among these differentially expressed proteins, 23 proteins (17 up-regulated, 6 down-regulated) exhibited significant changes following EP treatment (1.5-fold change, p < 0.05). The primary biological processes associated with these proteins included aging, immune response, acute-phase response, liver regeneration, protein catabolism, and oxidative stress response. Several metabolic pathways were enriched, as revealed by KEGG analysis. These findings offer important insights into the monitoring of cerebral I/R injury and contribute to a deeper understanding of its molecular mechanisms. Our results suggest that EP may mitigate ischemia-reperfusion-induced cerebral damage through anti-inflammatory pathways and by modulating metabolic processes.