A total of 6 adult male Sprague-Dawley rats (age, 8–10 weeks; weight, 250–300 g) were obtained from the Experimental Animal Culture Center of Hubei Centers for Disease Control (Wuhan, China). All animals were housed under standard laboratory conditions (temperature, 25±2°C; relative humidity, 55±5%; a 12-h light/dark cycle) with free access to food and water. All animal procedures were approved by the Animal Ethics Committee of Wuhan University and handled in accordance with the Experimental Animal Regulations of the People's Republic of China and the Guide for the Care and Use of Laboratory Animals of the USA (20).

A photograph of the recirculating perfusion system is presented in Fig. 1. It consisted of a thermostat water bath (Jiaxing Zhongxin Medical Instruments Co., Ltd., Jiaxing, China) containing water or ice water mixture, a liver container for perfusion installed in the water bath, a temperature sensor (Jiangsu Singhe Electronic Technology Co., Ltd., Suqian, China) inserted into the container to measure the fluid temperature, a peristaltic pump (Shanghai Jingke Industrial Co., Ltd., Shanghai, China), a hollow-fiber membrane oxygenator (Dongguan Kewei Medical Instrument Co., Ltd., Dongguan, China), a self-made bubble trap used to communicate between the ‘├’ shunt tube and the oxygenator to deliver the bubble, a flow meter (Nanjing Careermen Instrument Co., Ltd., Nanjing, China) connected to one side of the ‘├’ shunt tube to measure the hepatic inflow, and the other side of the ‘├’ shunt tube was used as a probe for liver inflow sample collection. To measure the perfusion portal pressure, a portal vein cannula was connected to the baroreceptor of a BL-420F biological functional system (Chengdu Techman Software Co., Ltd., Chengdu, China).

The HMP system (Fig. 1A and B) consisted of the above-mentioned recirculating perfusion system without an oxygen supply. The water bath was powered off and filled with ice water mixture to maintain the temperature of perfusate at 0–4°C.

The IPRL system (Fig. 1A and C) consisted of all of the above-mentioned parts of the recirculating perfusion system. In the present study, the top of the water in the water bath, where a container for the rat liver was installed, had a lower temperature compared with the bottom. According to the results of a preliminary experiment, the water bath was set at 40°C to maintain the temperature of perfusate in the liver container at 36–37°C. Krebs-Henseleit buffer (Macgene Biotechnology, Ltd., Beijing, China) with 4% dextran was added into the liver container for reperfusion (21). The perfusate was oxygenated (95% O₂ and 5% CO₂; pO₂ >500 mm Hg) and recirculated for 2 h at 36–37°C under a portal venous perfusion pressure of 10.3 mm Hg, as described previously (22).

Rats were anesthetized with chloral hydrate (10%; 3 ml/kg; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) by intraperitoneal injection. A midline incision was used to expose the abdomen, the liver was freed from all ligamentous attachments and the common bile duct was cannulated with an epidural guiding tube (Jiangsu Changfeng Medical Industry Co., Ltd., Yangzhou, China) to collect the total bile outflow during reperfusion.

Cardiac death was induced by hypoxia following incision of the diaphragm without portal clamping or prior heparinization (23). The warm ischemia time in situ began from the point of cardiac arrest. After 30 min, an intravenous injection of 2 ml saline containing 100 IU heparin (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) via the right iliac vein was performed (24). Subsequently, the hepatic artery was ligated, the portal vein was cannulated by a self-made polyethylene (PE) catheter (outer diameter, 2.1 mm; inner diameter, 1.8 mm) and flushed in situ with 20 ml 4°C histidine-tryptophan-ketoglutarate (HTK) solution (Dr. Franz Köhler Chemie GmbH, Bensheim, Germany). The liver was then removed and a PE catheter (internal diameter, 3 mm) was cannulated into the suprahepatic inferior vena cava for the collection of hepatic effluent (25). Finally, the liver was randomly assigned either to the CS or HMP group.

In the CS group (n=3), livers underwent static CS in 150 ml HTK solution at 0–4°C for 3 h. In the HMP group (n=3), livers were connected to the HMP system (Fig. 1B). HMP was performed via the portal vein with 0–4°C HTK solution (150 ml) for 3 h at a rate of 0.5 ml/g/min without oxygenation, as detailed previously (25).

IPRL is an in vitro system that permits the investigation of physiological and pathophysiological conditions in rat livers, including IRI in DCD livers after transplantation (26). After 3 h of CS or HMP preservation, the viability of all livers was evaluated using IPRL, as detailed previously (23). To simulate the slow rewarming period in liver transplantation, livers were left untouched on a petri dish at room temperature for ~15 min prior to reperfusion (23). During the normothermic reperfusion (NR), which was performed for 2 h, the portal pressure and portal flow were measured for the calculation of the intrahepatic resistance (IHR) using the following formula: IHR (mmHg/ml/min)=portal pressure (mmHg)/portal flow (ml/min).

Hepatic effluent was collected through the PE catheter cannulated in the suprahepatic vena cava every hour and analyzed for the enzyme activities of alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH), and oxygen (O₂) consumption rate. The enzyme activities were assessed using ALT (cat. no. C009-2), AST (cat. no. C010-2) and LDH assay kits (cat. no. A020-2; all Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol.

Hepatic O₂ consumption was used to assess the metabolic activity of the livers. Perfusate samples were respectively collected from the portal inflow and the PE catheter placed in the suprahepatic vena cava. Subsequently, the O₂ content in perfusate samples was measured immediately by a pH-blood gas analyzer (i-STAT; Abbott Point of Care, Inc., Princeton, NJ, USA). O₂ consumption by livers was calculated according to standard formulas, as detailed previously (22).

Bile was collected through the epidural guiding tube placed in the common bile duct every hour. As the density of bile was equal to the water, the bile flow was gravimetrically estimated and expressed as µl/h/g liver.

At the end of the 2 h in vitro isolated perfusion period (the end of the IPRL assay), livers were weighed and a portion was fixed in 10% formaldehyde for 24 h at room temperature for histological analysis. The rest of the liver was immersed in liquid nitrogen and stored at −80°C until subsequent experiments.

Liver samples (20 mg) stored at −80°C were homogenized using 4.2% perchloric acid and 1 mM diethylenetriamine pentaacetic acid. Subsequently, samples were centrifuged at 14,000 × g and 4°C for 5–10 min, and the supernatants were collected and brought to pH 6 by 69% K₂CO₃. Finally, ATP content was measured using an ATP assay kit (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol. Protein concentration was determined using the bicinchoninic acid (BCA) method; ATP levels were expressed as µmol/g protein.

Frozen liver samples were prepared and centrifuged as described for determination of ATP levels, subsequently, the supernatants were collected and used immediately for MDA content and SOD activity assays, according to the standard protocol of the MDA assay (cat. no. A003-1; Nanjing Jiancheng Bioengineering Institute) and SOD assay kits (cat. no. A001-1-1; Nanjing Jiancheng Bioengineering Institute).

The formaldehyde (10%)-fixed liver samples were paraffin-embedded and serially cut to 5-µm sections, which was followed by staining with hematoxylin and eosin (HE; hematoxylin staining for 5–15 min and eosin staining for 1–3 min; all performed at room temperature) for histological analysis at ×200 magnification using a light microscope.

Rat liver tissues were homogenized and lysed with lysis buffer (50 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40 and 5 mM protease inhibitor cocktail; pH 7.4; Beyotime Institute of Biotechnology, Jiangsu, China). Protein concentration was determined by a BCA assay (Beyotime Institute of Biotechnology). The proteins (30 µg/lane) were combined with β-mercaptoethanol and bromophenol blue for electrophoresis, and separated via 10% PAGE. Proteins were then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA), which were blocked with 5% skim milk for 2 h at room temperature. Membranes were then incubated overnight at 4°C with the following primary antibodies: Rabbit anti-SIRT-1 (cat. no. bs-0921R; 1:400; Beijing Biosynthesis Biotechnology Co., Ltd, Beijing, China), rabbit anti-NF-κB p65 (cat. no. bs-20355R; 1:400; Beijing Biosynthesis Biotechnology Co., Ltd.), rabbit anti-acetyl (K310) p65 (cat. no. ab19870; 1:300; Abcam, Cambridge, UK) and rabbit anti-β-actin (cat. no. GB13001-3; 1:1,500; Wuhan Goodbio Technology Co. Ltd., Wuhan, China). This was followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. GB23303; 1:5,000; Wuhan Goodbio Technology Co. Ltd.) for 1 h at room temperature. The reactive bands were visualized using an Electrochemiluminescence kit (cat. no. P0018; Beyotime Institute of Biotechnology). Quantification of protein bands was performed using ImageJ v1.42q software (National Institutes of Health, Bethesda, MA, USA).

RNA was extracted from the frozen liver tissue of rats using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol, and quantified using a NanoDrop instrument (Thermo Fisher Scientific, Inc.). RNA was reverse-transcribed into cDNA using the Thermo Scientific RevertAidä First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) with a total volume of 20 µl, containing total RNA (1 µg), Oligo(dT)₁₈ primer (1 µl), 5X Reaction Buffer (4 µl), RiboLock RNase Inhibitor (20 U/µl; 1 µl), 10 mM dNTP Mix (2 µl), RevertAid M-MuLV RT (200 U/µl; 1 µl) and water (nuclease-free). The reverse transcription reactions were performed with the following conditions: 42°C for 1 h and 75°C for 5 min. qPCR was performed using the SYBR^® Select Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) in a StepOnePlus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. All of the reactions were performed 3 times in triplicate. The relative mRNA expression was analyzed by the comparative threshold cycle (2^−∆∆Cq) method (27) and the results were normalized to those of β-actin. The primer sequences used were as follows: TNF-α, 5′-AGTCCGGGCAGGTCTACTTT-3′ (forward) and 5′-TTCAGCGTCTCGTGTGTTTC-3′ (reverse); IL-6, 5′-GGTCTTCTGGAGTTCCGTTTC-3′ (forward) and 5′-TCCATTAGGAGAGCATTGGAA-3′ (reverse). β-actin, 5′-TGCTATGTTGCCCTAGACTTCG-3′ (forward) and 5′-GTTGGCATAGAGGTCTTTACGG-3′ (reverse).

The levels of NAD⁺ and NADH in liver tissue homogenate were measured using the Coenzyme I NAD (H) content test kit (Nanjing Jiancheng Bioengineering Institute). Total NAD⁺ and NADH were quantified according to the manufacturer's protocol.

Statistical analysis was performed using SPSS v16.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean. After the assumption of normality and equal variance between groups was confirmed, the differences between CS and HMP groups were analyzed using independent samples t-tests. P<0.05 was considered to indicate a statistically significant difference.

The portal pressure and portal flow were used to calculate the IHR of DCD livers preserved by static CS or HMP at various time points during NR. A significant difference was observed between the CS and HMP groups at each analyzed time point during NR (Fig. 2A). IHR in the livers in the CS group was significantly higher compared with the HMP group at 0 (9.36±1.08 vs. 5.20±0.78 mmHg/ml/min; P=0.036), 60 (4.43±0.57 vs. 0.63±0.19 mmHg/ml/min; P=0.003) and 120 min (2.67±0.67 vs. 0.56±0.13 mmHg/ml/min; P=0.037).

To characterize the hepatocyte viability, the release of ALT, AST and LDH enzymes was determined during reperfusion. Increasing levels of ALT, AST and LDH enzymes were observed during the course of NR in CS and HMP groups. However, a significant reduction of ALT enzyme leakage was observed in the HMP group compared with the CS group at 0 (24.67±5.61 vs. 66.33±8.67 U/l; P=0.016), 60 (56.00±13.58 vs. 176.67±19.68 U/l; P=0.007) and 120 min (208.67±20.28 vs. 435.00±63.89 U/l; P=0.028), as presented in Fig. 2B. Similar patterns were observed for AST and LDH levels in the HMP and CS groups. AST levels were significantly reduced in the HMP group at 0 (29.33±7.54 vs. 98.00±13.58 U/l; P=0.011), 60 (75.33±17.42 vs. 167.33±16.95 U/l; P=0.019) and 120 min (218.33±25.99 vs. 448.67±47.91 U/l; P=0.013; Fig. 2C). Similarly, LDH levels were significantly decreased in the HMP group compared with the CS group at 0 (469.77±92.05 vs. 1,301.97±193.53 U/l; P=0.018), 60 (1,096.50±115.13 vs. 2,058.33±199.25 U/l; P=0.014) and 120 min (2,588.50±237.96 vs. 3,784.07±172.23 U/l; P=0.015; Fig. 2D).

Efficient O₂ consumption is crucial for ATP production, which maintains the energy status of cells. The influence of CS and HMP on the O₂ consumption of DCD rat livers was analyzed during reperfusion (Fig. 3). DCD livers in the HMP group exhibited significantly higher rates of O₂ consumption when compared with the CS group at 0 (1.50±0.21 vs. 0.59±0.10 µmol O₂/min/g liver; P=0.017), 60 (1.71±0.16 vs. 0.73±0.06 µmol O₂/min/g liver; P=0.005) and 120 min (1.74±0.14 vs. 0.73±0.07 µmol O₂/min/g liver; P=0.003). Anaerobic metabolism leads to rapid depletion of ATP, which in turn leads to the disruption of homeostasis. Therefore, the present study also determined ATP levels in HMP or CS preserved DCD livers. In the HMP group, ATP levels in liver tissues obtained after 2 h reperfusion were significantly higher compared with the CS group (21.80±2.01 vs. 4.16±0.93 µmol/g protein; P=0.001; Fig. 4A). However, no significant difference in the production of bile flow was observed between livers preserved by HMP or CS (Fig. 4B). Notably, the bile flow production was marginally higher in the HMP group compared with the CS group, however, this did not reach statistical significance after 120 min reperfusion (9.29±2.48 vs. 7.90±2.36 µl/h/g liver; P=0.704).

MDA, an end-product of lipid oxidation, serves as an important indicator for the assessment of oxidative damage. There was a significant reduction in MDA levels in livers preserved by HMP compared with those preserved by CS (2.57±0.45 vs. 10.52±1.86 nmol/mg protein; P=0.014; Fig. 5A), which indicates lower levels of oxidative stress following HMP. In addition, an increase in the activity of SOD was observed in livers of the HMP group compared with the CS group (431.41±35.99 vs. 163.34±48.97 U/mg protein; P=0.012; Fig. 5B), indicating a higher antioxidant activity in the cells preserved by HMP.

HE staining demonstrated a more intact hepatic cytoarchitecture in tissues preserved by HMP compared with those preserved by CS. In the HMP group, hepatic sinusoids were dilated due to the perfusion pressure, however, few sinusoidal endothelial cells exhibited signs of shrinkage and detachment. In addition, areas of hydropic changes and necrosis were limited in livers preserved by HMP compared with those preserved by CS (Fig. 6).

The present study subsequently determined the protein expression of NF-κB p65 and observed a significant decrease in the expression in livers preserved by HMP compared with the CS group (0.41±0.06 vs. 1.05±0.09; P=0.004; Fig. 7). The activation of NF-κB p65 is reversed by deacetylation. Therefore, to investigate whether HMP attenuated hepatic NF-κB p65 activity via SIRT-1-mediated deacetylation of NF-κB p65, the protein expression of SIRT-1 and acetylated-NF-κB p65 (an active form of post-translationally modified NF-κB p65) was also determined by western blotting. SIRT-1 expression levels were increased in the HMP group compared with the CS group (1.16±0.16 vs. 0.53±0.12; P=0.035; Fig. 7). In addition, the protein expression of acetylated-NF-κB p65 was significantly reduced by HMP preservation when compared with CS preservation (0.24±0.04 vs. 1.06±0.23; P=0.023; Fig. 7). IL-6 and TNF-α are NF-κB-mediated genes. Therefore, to assess the level of the NF-κB p65-induced inflammatory response, the mRNA expression of the inflammatory cytokines IL-6 and TNF-α was determined. The mRNA expression of IL-6 was significantly reduced in the HMP group compared with the CS group (0.26±0.03 vs. 1.01±0.08; P=3.06×10⁻⁸; Fig. 8A). Similar results were obtained for TNF-α expression in the HMP and the CS groups (0.54±0.04 vs. 1.03±0.06; P=2.59×10⁻⁶; Fig. 8B). These results indicated that the inflammatory response induced by NF-κB p65 was attenuated by HMP preservation.

In addition to the protein expression of SIRT-1 and acetylated-NF-κB p65, SIRT-1 activity indicators (hepatic NAD⁺ levels and NAD⁺/NADH ratio) were also measured. Consistent with results for SIRT-1 protein expression (Fig. 7), SIRT-1 activity was increased in the DCD livers preserved by HMP compared with the CS group, as indicated by higher levels of NAD⁺ (188.14±16.89 vs. 90.61±12.03 nmol NAD⁺/g protein; P=0.009; Fig. 9A) and NAD⁺/NADH ratio (2.13±0.43 vs. 0.42±0.11; P=0.018; Fig. 9B). These results indicate that HMP preservation may attenuate the hepatic NF-κB p65-induced inflammatory response in DCD rats via SIRT-1-mediated deacetylation of NF-κB p65.