*Purpose: To analyze the impact of steatotic livers with conventional cold storage (CS) and hypothermic oxygenated perfusion (HOPE) approaches, and further to investigate the rescue of steatotic livers applied by HOPE and elucidate the potential mechanism.

*Methods: Sprague – Dawley rats, male, weighing 220±10 g. 44 rats were randomly divided into 4 groups: group A, control. Non-steatotic livers were procured from SCD rats and preserved at University of Wisconsin solution (0-4℃) for 45 min ex-vivo. Group B: CS. Steatotic livers were excised and cold stored in UW (0-4℃) for 4 hours. Group C: HOPE. Steatotic livers perfused with 3-h HOPE followed by 1-h CS. Group D: DF. On the basis of Group C, defatting cocktail were added to the perfusate. All the livers were then taken for orthotopic liver transplantation (OLT) after being preserved. The expression of KLF-2, eNOS, ICAM-1, HMGB1, TLR4, PRDX6, Caspase-3, PP2A, PPY, GRP78, CHOP and ATF-6 in liver were examined by Western blot. Apoptosis was assessed using Tunel. ATP and glycogen were detected by ELISA kits. The post-transplantation survival was assessed in all groups.

*Results: Compared with group B, the expression of microcirculation related proteins KLF2 and eNOS were increased in group C and group D (p<0.05), accompanying with the deceased expression of ICAM-1. Expression of HMGB1, PRDX6 and TLR4 had statistical significance between CS group and HOPE group. Expression of endoplasmic reticulum stress proteins (ATF-6, PPY, GRP78, and CHOP) were down-regulated in group C compared to group B (p<0.05), the proteins expression was further being down regulated in group D. The PP2A expression showed opposite trend. According to Tunel, the hepatocytes apoptosis was obveiously being inhibited in group C and group D compared with group B. The contents of ATP in group C was obviously increased, the differences revealed statistical significance when comparing to group B (p<0.05). Meanwhile, the overall post-transplantation survival rates in group C and group D significantly improved comparing to group B.

*Conclusions: The mechanisms of protection of steatotic donor livers by hypothermic oxygenated machine perfusion refer to protect from microcirculation disturbance and endoplasmic reticulum stress and then promote ATP and glycogen synthesis to improve mitochondrial function. Meanwhile, under the ischemia reperfusion injury conditions, prevents nuclear injury and endothelial damage through suppressing the release of inflammatory mediator. The HOPE approach dramatically boosts long-term survival rate, which appears to be a very promising and powerful theoretical foundation to improve steatotic liver in clinical transplant and explores an effective therapeutic target for assessment of donor quality.

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