Blood Cell Secretome Associated with Remote Ischemic Conditioning: Proteomic Profiling of Processes Linked to Neuroprotection in Experimental Stroke

Ischemic stroke rapidly disrupts neuronal and synaptic integrity through excitotoxicity, oxidative stress, and inflammation, yet no effective neuroprotective therapy exists. Remote ischemic conditioning (RIC) is linked to systemic protective responses, but the molecular mechanisms by which circulating blood cells (BCs) contribute to RIC-mediated neuroprotection remain poorly understood. To address this gap, we examined whether RIC is associated with changes in BC properties and whether these changes correspond to alterations in the composition and function of their secretome. RIC-treated donor BCs exhibited biochemical features consistent with reduced hemo-lysis, maintained stable glutathione levels during incubation, and showed increased catalase activity, suggesting improved redox resilience. Secretomes from RIC-treated BCs showed elevated levels of the exosome-associated tetraspanin markers CD9 and CD81. Quantitative proteomics identified 680 proteins, of which 40 were differentially abundant (DAPs) between tolerant (TS) and non-tolerant secretomes (NTS). Most DAPs were downregulated in TS. GO enrichment analysis did not identify any significantly enriched terms after FDR correction. Because no GO term passed multiple testing cor-rection, GO results were not interpreted further. Functionally, TS treatment reduced infarct volume from 62.5% to 41.6%, improved neurological scores, and significantly decreased neuronal pyknosis, degeneration, and loss in the penumbra. TS also improved systemic redox balance, lowering oxidized glutathione, reducing the oxidative stress index from 3.5% to 1.9%, and diminishing the oxidative burden, as reflected in lymphocyte DNA damage, to near-control levels. Together, these findings indicate that TS derived from RIC treated donors exhibits neuroprotective potential in an experimental stroke model and is associated with dis-tinct proteomic signatures. The results are exploratory and do not establish a definitive mechanism or therapeutic readiness.