Hypothermic oxygenated machine perfusion alleviates liver injury in donation after circulatory death through activating autophagy in mice

Hypothermic oxygenated machine perfusion (HOPE) is a safe and reliable method that could alleviate liver injury in donation after circulatory death (DCD). This study focuses on the role of Autophagy in HOPE's protective effect on DCD liver injury. A 30-minute warm ischemic liver model was established in mice. After 4 hours of cold storage (CS), 1 hour of hypothermic machine perfusion (HMP) with 100% O₂ or 100% N₂ was employed. During 2 hours of reperfusion, liver tissue and perfusate were collected to evaluate liver function, oxidative stress level, Apoptosis, and necrosis. Western blotting was used to explore the level of Autophagy. When the liver experienced warm ischemic injury, LC3B-II expression was significantly enhanced. Compared with the CS, HOPE induced lower release of AST and ALT, as well as lower oxidative stress levels, Apoptosis, and necrosis cell numbers, and led to higher tissue ATP content. Meanwhile, expression of autophagy-related proteins, such as ULK1, Atg5, and LC3B-II, increased. When oxygen was completely replaced by nitrogen, the washout effect of HMP did not activate Autophagy and did not relieve DCD liver injury. When the Autophagy Inhibitor 3-methyladenine was used in HOPE, the protective effect of HOPE was attenuated. In conclusion, DCD liver injury activated Autophagy compared with healthy liver, while HOPE alleviated DCD liver injury by increasing Autophagy levels further in this mouse model. However, HMP with 100% of N₂ had no beneficial effect on DCD liver injury or on Autophagy levels compared with CS. The research on Autophagy may provide a new strategy for alleviating DCD liver injury in clinical practice.