The present experiment was conducted with the ethical approval of the Animal Experimental Ethics Committee of Wuhan University (Wuhan, China) and all experimental procedures were performed in accordance with the Guidance for the Care and Use of Laboratory Animals (20). A total of 24 male Sprague-Dawley (SD) rats (weighing 250–300 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained at 22–24°C, with a 12-h light/dark cycle and ad libitum access to food and water.

Rats were randomly divided into 4 groups (n=6 each), and all rats were fasted for 12 h prior to the initiation of the experimental procedures (but without drinking restrictions). For the control group, the rats were pretreated with intraperitoneal injections of 0.1% dimethyl sulfoxide (DMSO) as the vehicle for 30 min prior to the surgical operations, and then anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally) and the livers without warm ischemia exposure were obtained by midline laparotomy, followed by 24 h storage at 4°C in University of Wisconsin (UW) solution (DowDuPont, Inc.). Subsequently, the livers were connected to an isolated perfused rat liver system (IPRL) and perfused at 37±0.5°C for 1 h. For the TAK242 group, TAK242 was dissolved in DMSO and diluted with physiological saline. Doses of TAK-242 were determined based on preliminary tests (using 0.1, 0.5 and 1.0 mg/kg) and on previous studies (18,21). The preliminary tests revealed that 0.5 and 1.0 mg/kg TAK-242 induced protective effects in DCD livers, and that 1.0 mg/kg TAK-242 was more effective. The rats were pretreated with an intraperitoneal injection of TAK242 (1.0 mg/kg) at 37°C for 30 min prior to all surgical operations and all other operations were performed consistent with the control group. For the DCD group, the livers were subjected to in situ warm ischemia at 37°C for 30 min, but all other operations were performed in the same manner as in the control group. For the DCD+TAK242 group, the rats were pretreated with TAK242 (1.0 mg/kg) at 37°C for 30 min, as previously described (22), but all other procedures were similar between the DCD+TAK242 group and the DCD group.

Following midline laparotomy, cardiac arrest was induced by an incision of the diaphragm (23), and simultaneously, curved vascular clamping was used to prevent blood flow in the thoracic aorta. Following 30 min of warm ischemia at 37°C, the livers were perfused with 40 ml cold heparinized Ringer's solution (Shanghai Guandao Biological Engineering Co., Ltd.) via the aorta abdominalis. Subsequently, the portal vein and infrahepatic vena cava were cannulated with polyethylene pipes, followed by the ligation of the superior hepatic caval and the right adrenal veins. The rats in the control group underwent the same procedure but with an absence of warm ischemia.

Following cold storage, the livers were connected to the IPRL system and subjected to normothermic perfusion for 60 min at 37±0.5°C, as previously described (24). The IPRL system was based on previously described methods (24). Freshly prepared Krebs-Henseleit bicarbonate buffer (Sigma-Aldrich; Merck KGaA) with 95% O₂ and 5% CO₂ was pumped into the liver via the portal vein with a peristaltic pump (Shanghai Jingke Industrial Co., Ltd.). The flow velocity of the perfusate was set at a flow rate of 15 ml/min. To ensure that portal pressure did not exceed 8 mmHg, pressure sensors (Chengdu Techman Software Co., Ltd.) were used to monitor portal pressure. The perfusate was serially collected from the intrahepatic vena at 5, 30 and 60 min. Following perfusion, the perfusate and tissue samples were stored at −80°C for later analyses.

The perfusates were stored in a refrigerator at −80°C for 24 h and tested after all liver perfusions have been completed. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the perfusate were determined using an automatic biochemical analyzer (Hitachi, Ltd., Tokyo, Japan) in the clinical laboratory of Zhongnan Hospital of Wuhan University.

Liver samples were sliced into small pieces following 1 h of perfusion and were fixed in 4% buffered paraformaldehyde at room temperature for 24 h, embedded in paraffin at 60°C for 1 h and stained with haemotoxylin and eosin (H&E) at room temperature for 10 min. The severity of liver IRI was blindly graded according to Suzuki's criteria (25). Briefly, the degree of sinusoidal congestion, vacuolization/ballooning and hepatocyte necrosis are graded on a scale from 0 to 4 (25). Histopathological changes were evaluated by two pathologists who were blinded to this experiment. Paraffin-embedded tissue sections were examined for apoptosis using a TUNEL staining assay (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's protocol and tissue sections were incubated with fluorescein-dUTP at 37°C for 1 h. Total hepatocytes and TUNEL-positive cells were counted in 6 randomly selected fields of view, and the apoptotic index (the percentage of apoptotic cells in all hepatocytes in each view) was calculated with the use of Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

The protein expression levels of high-mobility group box protein b1 (HMGB1) and TLR4 in liver tissues were analyzed by immunohistochemistry using polyclonal rabbit primary antibodies and secondary polymeric horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) antibodies. Livers were fixed in 4% buffered paraformaldehyde at room temperature for 24 h and embedded in paraffin at 60°C for 1 h. The paraffin-embedded liver tissues were cut into 3–4 µm-thick sections. Sections were deparaffinized and rehydrated with a graded xylene and alcohol series. Antigen retrieval was performed by high temperature and pressure; 1 mM EDTA (Beijing Solarbio Science & Technology Co., Ltd.) was heated to ~100°C, the sections were soaked in EDTA and heated for ~3–4 min at a pressure of 110 kPa. Sections were incubated in 3% hydrogen peroxide at room temperature for 10 min. Subsequently, sections were blocked in 10% BSA (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 30 min. Section were incubated with rabbit anti-HMGB1 (cat. no. 10829-1-AP; 1:100; ProteinTech Group, Inc.) or rabbit anti-TLR4 (cat. no. 19811-1-AP; 1:200; ProteinTech Group, Inc.) at 4°C overnight, and incubated with HRP-conjugated anti-rabbit IgG antibodies at room temperature for 30 min (cat. no. SA00001-2; 1:200; ProteinTech Group, Inc.). Sections were incubated with diaminobenzidine (Shanghai Yeasen Biotechnology Co., Ltd.) at room temperature for 5 min. Subsequently, sections were stained using 1.5 mg/ml hematoxylin at room temperature for 2 min. Sections were visualized using light microscopy (magnification, ×200). HRP activity was measured using 3,3′-diaminobenzidine substrate. The expression of interleukin-6 (IL-6) in liver tissues was analyzed by immunofluorescence and eight fields of view were randomly selected to quantify IL-6 expression. Livers were fixed, embedded and sectioned using the same method mentioned above. Sections were blocked in 5% BSA (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 h and were incubated with rabbit anti-IL-6 (cat. no. 21865-1-AP; 1:100; ProteinTech Group, Inc.) at 4°C overnight. The sections were then incubated with Alexa Fluor 555-conjugated goat anti-rabbit (cat. no. P0179; 1:100; Shanghai Biyuntian Biological Co., Ltd.) and DAPI at room temperature for 1 h (cat. no. C1002; Shanghai Biyuntian Biological Co., Ltd.). Results were visualized using a fluorescence microscope (magnification, ×400) and images were analyzed by Image J software (version 1.51; National Institutes of Health).

Tissues were cut into 1–2 mm sections and were fixed in 2.5% glutaraldehyde at 4°C for 24 h. Subsequently, tissues were immersed in 1% osmium tetroxide at 4°C for 2 h and were dehydrated using a graded ethanol series. Later, tissue were embedded in Epon resin (Thermo Fisher Scientific, Inc.) at 60°C for 36 h. Tissues were cut into sections (thickness, ~70 nm) using a microtome (Leica Microsystems Inc.) and stained with uranylacetate and lead citrate. The extent of liver IRI was evaluated by observing damaged subcellular organelles under a TEM (Tecnai G2 20 TWIN; FEI; Thermo Fisher Scientific, Inc., Waltham, MA, USA), performed by the Wuhan Institute of Virology, Chinese Academy of Sciences (Wuhan, China). The severity of mitochondrial damage was blindly graded according to Flameng criteria which evaluates mitochondrial function based on mitochondrial morphology and grades this on a scale from 0 to 4 (26).

Total RNA from liver tissues was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.). RT was performed at 42°C for 1 h followed by an incubation at 75°C for 5 min. cDNA was separated by 1% agarose gel electrophoresis. 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. The relative quantity of the assessed genes to the housekeeping gene were analyzed using the SYBR green PCR Master Mix (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol, and performed on a SYBR green quantitative RealTime-PCR system (Yeasen Biotechnology Co., Ltd., Shanghai, China). The relative expression of genes was calculated using the 2^−∆∆Cq method and β-actin was used as internal standard (27). Primers used are listed in Table I.

Frozen liver tissues were disrupted in phenylmethylsulfonyl fluoride (Roche Applied Science, Penzberg, Germany)-containing RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) using a tissue lyser, followed by centrifugation (24,000 × g for 15 min at 4°C). Following heat denaturation, Protein concentration was determined using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). An equal amount of protein (50 µg) was separated on 10% SDS-PAGE gels, followed by an electrotransfer onto PVDF membranes. Subsequent to blocking in 5% bovine serum albumin at room temperature for 2 h, the immunoblots were incubated with primary antibodies at 4°C overnight, followed by incubation with the secondary HRP-conjugated anti-rabbit IgG antibody (cat. no. SA00001-2; 1:8,000; ProteinTech Group, Inc.) for 2 h at room temperature. All primary and secondary antibodies were from ProteinTech Group, Inc. (Chicago, IL, USA) and include rabbit anti-HMGB1 (cat. no. 10829-1-AP; 1:1,000; ProteinTech Group, Inc.), rabbit anti-TLR4 (cat. no. 19811-1-AP; 1:1,000; ProteinTech Group, Inc.), rabbit anti-IL-1β (cat. no. 16806-1-AP; 1:500; ProteinTech Group, Inc.), rabbit anti-IL-6 (cat. no. 21865-1-AP; 1:500; ProteinTech Group, Inc.), rabbit anti-mitochondrially encoded cytochrome c oxidase II (COX2; cat. no. 12375-1-AP; 1:500; ProteinTech Group, Inc.) and rabbit anti-β-actin (cat. no. 20536-1-AP; 1:1,000; ProteinTech Group, Inc.). The bands were visualized using enhanced chemiluminescent reagent (Wuhan Boster Biological Technology, Ltd.) and quantified by using the Image-J software (version 1.51; National Institutes of Health).

To evaluate oxidative stress and lipid peroxidation, the levels of MDA and ROS were measured using commercial colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol.

Data were analyzed using SPSS 15.0 statistical software (SPSS, Inc., Chicago, IL, USA) and the results are expressed as the mean ± standard deviation. Differences between groups were analyzed using a one-way analysis of variance followed by a Kruskal-Wallis test. P<0.05 was considered to indicate a statistically significant difference.

First, the present study determined the leakage of ALT and AST into the liver perfusate, which are indicative of DCD liver injury and altered metabolic functions. As presented in Fig. 1A and B, the levels of two transaminase in the perfusate were significantly increased at each time point assessed in the DCD group when compared with those in the control group (P<0.05). There was no statistically significant difference in the level of transaminases between the control group and the TAK242 group (P>0.05). However, TAK242 pretreatment for 30 min significantly decreased the levels of ALT and AST in the perfusate compared with those in the DCD group (P<0.05), suggesting that TAK242 pretreatment improves the metabolic functions of the DCD livers. H&E staining revealed that there were significantly more infiltrated inflammatory cells and necrotic hepatocytes in the DCD group compared with in the control group, whereas they were significantly attenuated in the DCD+TAK242 group compared with the DCD group (Fig. 1D). Furthermore, the Suzuki scores, used for the assessment of liver damage and hepatocyte death (25), identified no statistically significant differences between the control group and the TAK242 group (P>0.05) but were significantly lower in the DCD+TAK242 group when compared with the DCD group (P<0.05; Fig. 1C). These results indicate that pretreatment with TAK242 alleviates the IRI of the DCD livers.

Next, the present study determined the redox status and hepatic apoptosis in the DCD livers pretreated with or without TAK242. When compared with the control group, the levels of ROS and MDA were not significantly different in the TAK242 group (P>0.05) but significantly increased in the DCD group (P<0.05), whereas these changes were significantly reversed following TAK242 pretreatment (P<0.05; Fig. 2A and B), indicating an antioxidant effect of TAK242. Furthermore, as presented in Fig. 2C and D, the numbers of TUNEL-positive apoptotic cells in the DCD group were significantly increased compared with the control group, whereas a significant decrease in the numbers of apoptotic cells were observed in the DCD+TAK242 group compared with the DCD group (P<0.05). These results indicate that pretreatment with TAK242 attenuates oxidative stress and hepatocytic apoptosis in DCD livers.

Next, the present study determined the subcellular structures of the DCD livers pretreated with or without TAK242 by TEM. As presented in Fig. 3A, when compared with the control group, the subcellular structure in group A was essentially normal without obvious changes, whereas the DCD group exhibited more serious damage to the subcellular structures, including the appearance of mitochondria swelling, nuclei irregularity, cellular edema and transparent electronic density area, whereas these structural changes, including mitochondrial damage and cellular edema, in the DCD livers were substantially relieved with the pretreatment with TAK242. As presented in Fig. 3B, the Flameng scores exhibited no statistically significant difference between the control group and the TAK242 group (P>0.05) but were significantly lower in the DCD+TAK242 group when compared with the DCD group (P<0.05). These results indicate that the TLR4 inhibitor protects DCD-exposed hepatocytes, partially via minimizing subcellular organelle damage.

To understand the mechanism behind the protective effects of TAK242 on the DCD livers, the present study further determined the expression of HMGB1, TLR4 and their downstream target genes at the mRNA levels using RT-qPCR analysis. As presented in Fig. 4, it was revealed that TAK242 pretreatment, as expected, significantly inhibited the gene expression of TLR4, IL-1β, IL-6, COX2 and tumor necrosis factor-α (TNF-α) in comparison with the DCD-alone group (P<0.05). However, TAK242 did not significantly influence the gene expression of the cytokine HMGB1. These results support the concept that TAK242 inhibits the TLR4 signaling pathway and associated inflammation in DCD livers.

Next, the present study determined the effect of TAK242 on the expression of HMGB1, TLR4 and their downstream inflammatory mediators in DCD livers at the protein level using western blot analysis. As presented in Fig. 5, compared with the control group, TLR4 and their downstream inflammatory mediators did not change significantly in TAK242 group. But TAK242 pretreatment significantly downregulated the protein expression levels of TLR4 and its downstream inflammatory mediators IL-1β, IL-6 and COX2, compared with the DCD group (P<0.05). Again, there was no statistically significant difference in HMGB1 expression levels between the DCD group and the DCD+TAK242 group (P>0.05). These results further confirmed that the TLR4 inhibitor, TAK242, reduces the production and expression of proinflammatory cytokines and enzymes in the DCD livers subjected to 24-h cold storage and 1 h warm perfusion.

Finally, the present study demonstrated the downregulation of TLR4 in the DCD livers by immunohistochemical analysis. When compared with the control group, there were no significant changes in HMGB1 or TLR4 in the TAK242 group (Fig. 6A). There was no statistically significant difference in the percentage of HMGB1 and TLR4-positive cells between the TAK242 and the control group (P>0.05; Fig. 6B and C). The DCD groups exhibited a significantly increased expression of HMGB1 and TLR4 compared with the control group (P<0.05; Fig. 6). TAK242 pretreatment in the DCD livers significantly decreased the percentage of TLR4 positive cells compared with the DCD group (P<0.05; Fig. 6C), However, TAK242 pretreatment in the DCD livers did not significantly decrease the percentage of HMGB1 positive cells compared with the DCD group (P>0.05; Fig. 6B). Immunofluorescence revealed that TAK242 pretreatment in DCD livers reduced the release of IL-6 when compared with the DCD group (Fig. 6D). These results further indicate that TAK242 reduces TLR4 expression in DCD livers.