🎗Time resolved proteomic signatures of ischemia–reperfusion injury in a porcine donation after circulatory death simulated liver transplant model using ex vivo machine perfusion

Ex vivo machine perfusion (EVMP) has emerged as a promising alternative to static cold storage (SCS) for preservation of donation after circulatory death (DCD) liver grafts, yet its time‑dependent molecular effects and their implications for graft assessment remain poorly defined. In a porcine DCD liver model, we directly compared SCS and normothermic EVMP using an identical 6‑hour preservation period followed by 3 hours of ex vivo reperfusion and integrated serial functional, histologic, and tandem mass tag–based quantitative proteomic analyses from baseline through preservation and reperfusion. EVMP attenuated ischemia–reperfusion injury relative to SCS, as reflected by lower transaminase and lactate levels, more favorable coagulation and lactate dehydrogenase profiles, higher bile production, and reduced hepatocyte apoptosis during late reperfusion, indicating superior early graft functional recovery. Global proteomic profiling of 2,383 high‑confidence proteins demonstrated that EVMP constrained temporal deviation from the baseline liver proteome, whereas SCS induced larger, less reversible shifts.

Focusing on the end‑of‑preservation time point, we identified eight differentially regulated proteins that formed three trajectory‑defined modules: a sustained EVMP‑associated stress‑adaptive program involving activating transcription factor 3, sequestosome 1, and heat shock protein family H member 1; a transient EVMP priming module including stress‑associated endoplasmic reticulum protein 1 and Bcl‑2‑like protein 2; and a transient SCS‑enriched sinusoidal endothelial/macrophage‑related module comprising liver and lymph node sinusoidal endothelial cell C‑type lectin, macrophage mannose receptor 1, and stabilin 1.

These findings show that EVMP promotes a more controlled and reversible pattern of liver injury and adaptation during DCD graft preservation and support the development of small, preservation‑phase protein panels as molecular adjuncts to conventional viability criteria for marginal livers.