Dynamic proteomic landscape of local ischemic conditioning reveals a sequential dominance of signal transduction, protein synthesis, and mitochondrial metabolism.

Ischemic conditioning confers systemic protection through complex humoral, neural, and immune mechanisms; however, the proteomic signatures in the local venous effluent during conditioning cycles remain poorly defined. This study aimed to establish a comprehensive proteomic atlas of the local response to direct ischemic conditioning in a healthy, young animal model. Three adult male beagles underwent five cycles of 5-minute left hindlimb ischemia-reperfusion induced by a tourniquet, with the contralateral right limb serving as a control. Blood samples were collected from the bilateral femoral veins at the end of the 1st, 3rd, and 5th occlusion cycles. Plasma proteomic profiling was performed using magnetic nanoparticle-based enrichment followed by liquid chromatography-tandem mass spectrometry. Differentially expressed proteins were subjected to Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, and protein-protein interaction network analyses. A total of 155, 66, and 26 differentially expressed proteins were identified after the 1st, 3rd, and 5th cycles, respectively, with the number of differentially expressed proteins decreasing as the cycles progressed. Seven proteins, including RTK, SRP9, MAP1S, eEF, UPF2, HNRNPR, and GAPDH, showed overlapping differential expression across two cycles. Protein-protein interaction network analysis highlighted Grb2, RPS27A, and RPS3, as well as NDUFS3 and COX4I1, as key hubs. Functional enrichment analysis revealed a temporal shift from signal transduction during the early cycles to protein synthesis during the middle cycles and, finally, to mitochondrial energy metabolism during the later cycles. This is the first study to profile local proteomic responses during sequential cycles of ischemic conditioning. The identified biomarkers suggest a coordinated, sequential pattern of dominant biological processes involving signal transduction, protein synthesis, and mitochondrial metabolism. These findings provide a foundation for understanding the mechanistic basis of ischemic conditioning and warrant further validation in disease and aging contexts.