Hypothermic machine perfusion ameliorates inflammation during ischemia-reperfusion injury via sirtuin-1-mediated deacetylation of nuclear factor-kappa B p65 in rat livers donated after circulatory death

摘要

Hypothermic machine perfusion (HMP) effectively reduces ischemia-reperfusion injury (IRI) in livers donated after circulatory death (DCD) when compared with cold storage (CS). However, the underlying mechanisms remain unclear. The current study aimed to investigate the cellular mechanisms by which HMP ameliorates the inflammatory response during IRI. Adult male Sprague-Dawley rat livers were exposed to 30 min of warm ischemia following cardiac arrest and preserved by CS or HMP for 3 h (n=3 per group). The severity of IRI was assessed in vitro on normothermic reperfusion for 2 h, and intrahepatic resistance (IHR) and bile production were subsequently recorded. The perfusate was analyzed for transaminase leakage and oxygen consumption. Livers were subsequently subjected to histological examination, and measurement of adenosine triphosphate (ATP) levels, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, nicotinamide adenine dinucleotide (NAD)(+) levels and the ratio of NAD(+)/NADH. In addition, the protein expression of sirtuin-1 (SIRT-1), acetylated-nuclear factor-kappa B (NF-kappa B) p65 and NF-kappa B p65 was detected by western blotting, and the mRNA expression of the inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha was determined by reverse transcription-quantitative polymerase chain reaction. Compared with CS, HMP resulted in significantly lower IHR, transaminase leakage and MDA levels, and higher oxygen consumption, ATP levels and SOD activity. In addition, improved preservation of hepatic histology was observed in HMP compared with CS. The mRNA expression of NF-kappa B p65, IL-6 and TNF-alpha was significantly decreased in the HMP group compared with CS samples. Under HMP preservation, SIRT-1 activity and protein expression were increased, while the protein expression of acetylated-NF-kappa B p65 was decreased, compared with CS. These results indicate that HMP may reduce the inflammatory response during IRI via SIRT-1-mediated deacetylation of NF-kappa B p65. These findings may provide a theoretical basis for the clinical application of HMP as an effective strategy to preserve DCD livers.