*Purpose: It has been reported that activation of apoptosis signaling-regulating kinase 1 (ASK1)/p-p38 axis during tissue cold storage results in ferroptotic cell death. Ferroptosis is a novel type of iron-dependent programmed cell death mechanism, leading to the multiple danger-associated molecular patterns (DAMPs) release. Several studies have recently reported that ferroptotic cell death aggravates ischemia-reperfusion injury (IRI) in various organs, such as heart, kidney, and intestine. However, the contribution of ferroptosis in liver IRI after transplantation remains to be elucidated. In the current study, we demonstrate the role of ferroptosis inhibitor during cold storage using mouse orthotopic liver transplantation (OLT) model.

*Methods: Wild type C57BL/6 mouse livers were subjected to the extended cold storage (4°C/18 hours in UW solution with or without adjunctive ferroptosis inhibitor: ferrostatin-1). Liver grafts were perfused with Ringer solution and the liver perfusate was collected after cold storage as liver flush; the hepatic expression of ASK1, p-p38, and high morbility group box 1 (HMGB1) in liver flush were analyzed by Western blots. After cold preservation with or without ferrostatin-1, the livers were orthotopically transplanted into syngeneic mice, and liver/serum samples were collected at 6 hours after reperfusion.

*Results: Western blot-assisted analysis revealed that HMGB1 in liver flush, hepatic ASK1 and p-p38 expression markedly increased after cold storage (4°C/18 hours) as compared with naive untreated liver. To optimize the appropriate dose that reduces cold stress, HMGB1 expression in liver flush after cold preservation with 1, 2.5, 10uM of ferrostatin-1 and DMSO control in UW solution were analyzed. Among these concentrations, 2.5uM of ferrostatin-1 showed dramatically reduced HMGB1 expression level in liver flush. With 2.5uM of ferrostatin-1, IRI was mitigated in the transplanted liver grafts compared with liver grafts stored without ferroptosis inhibitor, as evidenced by serum ALT and AST levels (ALT: 8995±2917 vs 3903±772 IU/L; AST: 4151±593 vs 1771±913 IU/L, p<0.05), reduced infiltrating CD11b positive macrophage/ Ly6G positive neutrophil, and frequency of TUNLE positive cells.

*Conclusions: Cold preservation triggered the increase of ASK1/p-p38 expression in WT mouse livers, suggesting activation of ferroptotic hepatocellular death during cold storage. Inhibition of ferroptosis during cold stress might alleviate IRI after liver transplantation. This study provides the basis for novel therapeutic strategy to mitigate IRI by using ferroptosis inhibitor for cold preserved donor livers.

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