A total of 48 male inbred Sprague Dawley rats (weight, 250–300 g) aged 7–8 weeks were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Rats were raised in the Animal Experiment Center of The Zhongnan Hospital of Wuhan University (Wuhan, China). All rats were maintained under standard animal care conditions at 24±3°C and 60% humidity, with a 12-h dark/light cycle and free access to food and water. All animal experiments and protocols were approved by the Committee on the Experimental Animal Regulations of the Zhongnan Hospital of Wuhan University (approval no. A237; Wuhan, China) and conformed to the Guide for the Care and Use of Laboratory Animals (15).

To investigate the effects of Alda-1 on HIRI, rats were treated intraperitoneally 30 min before warm liver ischemia with either 10 mg/kg Alda-1 (cat. no. HY-18936; MedChemExpress) (16) or 100 mg/kg Daidzin (an ALDH2 inhibitor; cat. no. HY-N0018; MedChemExpress). Both Alda-1 and Daidzin were dissolved in 0.1% DMSO (Sigma-Aldrich; Merck KGaA). A total of 48 rats were divided into four groups (n=12/group): i) A sham group, in which rats were given laparotomies without HIRI; ii) an IR group, where rats were treated with vehicle (0.1% DMSO) 30 min before laparotomy with HIRI; iii) an IR-Alda-1 Group, where rats were treated with 10 mg/kg Alda-1 30 min before laparotomy with HIRI; and iv) an IR-Daidzin group, where rats were treated with 100 mg/kg Daidzin 30 min before laparotomy with HIRI (Fig. 1A).

Briefly, rats were fasted for 12 h before the experiment but allowed free access to water. Rats were then anesthetized with intraperitoneal (IP) injection of 50 mg/kg sodium pentobarbital. Normal body temperature was maintained using a heating pad at 37±0.5°C and was monitored with a rectal probe. Under anesthesia, an abdominal midline laparotomy was performed on all rats. Partial (70%) liver ischemia was induced in the HIRI groups by clamping the median and left lobes for 60 min with a microvascular clip. Hepatic ischemia was confirmed by color changes in the tissue (Fig. 1B). After the 60 min period of warm ischemia, the clamp was removed, the abdomen was then closed, and the rats were allowed to recover in a warm environment. No rats died within 24 h of the laparotomy and no antibiotics were administrated over the course of the experiment.

After 6 or 24 h of reperfusion, rats were sacrificed using 100 mg/kg pentobarbital sodium (IP). After cessation of breathing and heartbeat, 5 ml blood samples were collected through inferior vena cava punctures using K2-EDTA test tubes, then centrifuged at 3,000 × g for 15 min at 4°C to extract serum. Liver tissue samples were harvested from the median and left lobes of the ischemic liver. Serum and liver tissue samples were frozen in liquid nitrogen, then stored at −80°C. A section of each liver tissue sample was immersed in 4% paraformaldehyde at 25°C for 48 h for histopathology.

The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) released into the serum were detected using an ALT ELISA kit (cat. no. E-BC-K235-M; Elabscience Biotechnology, Co., Ltd.) and an AST ELISA kit (cat. no. E-BC-K236-M; Elabscience Biotechnology, Co., Ltd.) following the protocols of the clinical laboratory of the Zhongnan Hospital of Wuhan University. The serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and 4-HNE in the serum were measured using TNF-α ELISA kit (cat. no. E-EL-R0019c), IL-1β ELISA kit (cat. no. E-EL-R0012c), IL-6 ELISA kit (cat. no. E-EL-R0015c) and 4-HNE ELISA kit (cat. no. E-EL-0128c; all from Elabscience Biotechnology, Co., Ltd.) according to the manufacturers' protocols.

The samples after paraffin immersion were embedded in paraffin wax and cut into 5-µm sections for histological analysis via hematoxylin-eosin staining (HE; hematoxylin staining for 5–15 min and eosin staining for 1–3 min; all performed at room temperature). The degree of liver injury was assessed according to Suzuki's grading scale (17): i) Grade 0, no congestion, no vacuolization and no necrosis; ii) grade 1, minimal congestion, minimal vacuolization and single-cell necrosis; iii) grade 2, mild congestion, mild vacuolization and <30% necrosis; iv) grade 3, moderate congestion, moderate vacuolization and ≤60% necrosis; and v) grade 4, severe congestion, severe vacuolization and >60% necrosis. Two blinded pathologists independently evaluated the sections under a light microscope (magnification, ×100).

ALDH2 activity was measured using an ALDH2 activity assay kit (cat. no. A075-1-1; Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol. Briefly, the activity of the ALDH2 enzyme was determined by monitoring the conversion of NAD⁺ to NADH at a spectrophotometric absorbance of 340 nm. ALDH2 activity is expressed as nmol NADH/min per mg protein.

The levels of 4-HNE (cat. no. H268), MDA (cat. no. A003-1-2) and ROS (cat. no. E004-1-1) in liver tissue samples were measured using specific rat colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute), according to the manufacturers' protocol.

Western blotting was performed as previously described (18). Briefly, total protein from liver tissues was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology), supplemented with protease inhibitor and phosphatase inhibitor (Roche Applied Science). Protein concentration of the supernatant was determined by the bicinchoninic acid kit (cat. no. G2026; Wuhan Servicebio Technology Co., Ltd.). Subsequently, 50 µg protein/lane was loaded and separated by SDS-PAGE on 8–12% gels, then transferred onto PVDF membranes. The PVDF membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated overnight at 4°C with one of the following primary antibodies: Rabbit anti-ALDH2 (1:1,000; cat. no. 15310-1-AP; ProteinTech Group, Inc.), rabbit anti-4-HNE (1:500; cat. no. ab46545; Abcam), rabbit anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 10829-1-AP; ProteinTech Group, Inc.), rabbit anti-toll-like receptor 4 (TLR4; 1:1,000; cat. no. 19811-1-AP; ProteinTech Group, Inc.), rabbit anti-beclin1 (1:500; cat. no. 11306-1-AP; ProteinTech Group, Inc.), rabbit anti-autophagy-related 7 (ATG7; 1:1,000; cat. no. 67341-1-IG; ProteinTech Group, Inc.), rabbit anti-p62 (1:1,000; cat. no. 18420-1-AP; ProteinTech Group, Inc.), rabbit anti-Rab7 (1:1,000; cat. no. 55469-1-AP; ProteinTech Group, Inc.), rabbit anti-microtubule associated protein 1 light chain 3 α (LC3; 1:500; cat. no. 14600-1-AP; ProteinTech Group, Inc.), mouse anti-AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), rabbit anti-phosphorylated (p-)AKT at Thr308 (1:1,000; cat. no. 13038T; Cell Signaling Technology, Inc.), rabbit anti-mTOR (1:300; cat. no. 20657-1-AP; ProteinTech Group, Inc.), rabbit anti-p-mTOR at Ser2448 (1:1,000; cat. no. 2971S; Cell Signaling Technology, Inc.), rabbit anti-S6 (1:1,000; cat. no. 14823-1-AP; ProteinTech Group, Inc.), rabbit anti-p-S6 at Ser240/244 (1:1,000; cat. no. 5364T; Cell Signaling Technology, Inc.), mouse anti-AMP-activated protein kinase (AMPK; 1:1,000; cat. no. 2793S; Cell Signaling Technology, Inc.), rabbit anti-p-AMPK at Thr172 (1:1,000; cat. no. 2535T; Cell Signaling Technology, Inc.) or rabbit anti-β-actin (1:1,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.). The following day, the PVDF membranes were washed three times and incubated with goat anti-mouse (1:2,000; cat. no. SA00001-1, ProteinTech Group, Inc.) or goat anti-rabbit (1:2,000; cat. no. SA00001-2, ProteinTech Group, Inc.) horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Finally, bands were detected using a chemiluminescent ECL reagent (cat. no. G2020; Wuhan Servicebio Technology Co., Ltd.). Protein expression bands were quantified using densitometry analysis with ImageJ v6.0 software (National Institutes of Health) and normalized to β-actin.

Hepatic HMGB1 and TLR4 expression levels were also evaluated by immunohistochemical staining. Briefly, paraffin-embedded 5-µm liver tissue sections were treated with 3% hydrogen peroxide for 15 min at 37°C to block endogenous peroxidase activity. Nonspecific binding was blocked by incubating the sections with 5% BSA (Wuhan Goodbio Technology Co. Ltd.) for 20 min at room temperature. Liver tissue sections were then incubated with a polyclonal rabbit anti-HMGB1 (1:100; cat. no. 10829-1-AP; ProteinTech Group, Inc.) antibody or anti-TLR4 (1:200; cat. no. 19811-1-AP; ProteinTech Group, Inc.) antibody at 4°C overnight, followed by incubation with polymer-HRP-conjugated anti-rabbit IgG (1:1,000; cat. no. SA00001-2, ProteinTech Group, Inc.) for 30 min at room temperature. Lastly, slides were stained for 3 min at room temperature using a diaminobenzidine (DAB) kit with HRP as the substrate. Finally, the sections were dehydrated in a graded ethanol solution and cleared with xylene. The HMGB1 and TLR4 protein expression levels were viewed under a light microscope (magnification, ×200).

For immunofluorescence, Liver tissue 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.) overnight at 4°C. 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 (cat. no. C1002; Shanghai Biyuntian Biological Co., Ltd.) at room temperature for 1 h. Results were visualized using a fluorescence microscope (magnification, ×400) and images were analyzed by ImageJ software (version 1.51; National Institutes of Health).

Total RNA was extracted from 100 mg frozen liver tissue samples using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), 20% chloroform and 50% isopropyl alcohol. Total RNA was subsequently reverse transcribed into cDNA using a reverse transcription kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RT was performed at 42°C for 1 h followed by an incubation at 75°C for 5 min. 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. RT-PCR was subsequently performed using SYBR Green (Shanghai Yeasen Biotechnology Co. Ltd.) to determine the expression levels of target genes. The relative expression of genes was calculated using the 2^−∆∆Cq method. Gene expression was normalized against that of the β-actin gene (19). All primers used for the RT-qPCR are listed in Table I.

The liver tissue was fixed in 4% paraformaldehyde solution at room temperature for 24 h, paraffin-embedded, dehydrated in a graded ethanol series and coronally cut into 5 µm sections. Sections were deparaffinized using xylene and rehydrated in a descending ethanol series. Tunel assay (Roche Diagnostics) was used to detect quantitatively the apoptotic hepatocytes according to the manufacturer's protocol. Liver tissue sections were incubated with fluorescein-dUTP at 37°C for 1 h. 6 visual fields were randomly selected under a fluorescence microscope. The normal hepatocytes were stained blue and the apoptosis-positive cells green. Total hepatocytes and TUNEL-positive cells were counted under a fluorescence microscope (magnification, ×200). The apoptotic rate was calculated as: (The number of TUNEL-positive cells/the total number of hepatocytes) ×100%. Analysis was carried out using Image Pro Plus 6.0 software (Media Cybernetics, Inc.).

The 1–2 mm³ fresh liver tissue fixed in 2.5% glutaraldehyde solution was rinsed in PBS and fixed with 1% osmium tetroxide for 2 h at room temperature. Following dehydration with a graded acetone series and embedding in Pon812 epoxy resin for 12 h at room temperature, the liver tissue was cut into 1 µm sections. The sections were stained with 3% uranyl acetate at room temperature for 30 min and washed with double distilled water. Then, the sections were stained with 3% lead citrate at room temperature for 10 min and washed with double distilled water. The mitochondrial injuries was observed by Tecnai G² 20 Twin TE microscope (FEI; Thermo Fisher Scientific, Inc.) at room temperature: 5 fields were selected for each specimen for statistical analysis.

Statistical analysis was performed using SPSS 16.0 statistical software (SPSS, Inc.). Data are presented as the mean ± SD. For experimental data following a normal distribution, statistical differences were determined using an ANOVA, followed by a Tukey's post-hoc test. Otherwise, nonparametric data were analyzed using the Kruskal-Wallis test, followed by a Dunn's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

After 70% warm hepatic ischemia for 60 min, followed by 6 or 24 h of reperfusion, the serum levels of ALT and AST were significantly increased in the IR group compared with the sham group (Fig. 2A and B). However, the pretreatment with Alda-1 prior to HIRI decreased the serum levels of ALT and AST after 6 and 24 h of reperfusion compared with the IR and IR-Daidzin groups. These results were supported by the histological lesions observed in the liver tissues with H&E staining (Fig. 2C and D). Extensive necrosis (indicated by the white arrows), congestion and neutrophil infiltration were observed in the IR and IR-Daidzin groups after 6 and 24 h of reperfusion. These lesions were significantly reduced in the IR-Alda-1 group compared with the IR group, based on Suzuki's HIRI scoring system (17). These results suggested that ALDH2 may serve a role in HIRI and that ALDH2 activation may confer protection from HIRI.

Changes in the ALDH2 expression levels were then evaluated in HIRI rats. ALDH2 protein expression levels were not significantly different among the four groups at either 6 or 24 h (Fig. 3A and B). However, HIRI significantly decreased ALDH2 activity in the IR group compared with rats in the sham group at both 6 and 24 h. This effect was partially rescued by ALDH2 activation in the IR-Alda-1 group (Fig. 3C).

The accumulation of ROS, MDA and 4-HNE aldehydes has been discovered to promote toxic effects associated with HIRI (4,8,9). A significant increase in 4-HNE protein expression levels, serum levels and tissue accumulation was observed following IR and 6 or 24 h reperfusion compared with the sham group (Fig. 4A-D), while these increases were partially reversed in the IR-Alda-1 group. To investigate the relationship between the ALDH2-mediated liver protection and oxidative stress, the levels of ROS and MDA were measured in the liver tissue; Alda-1 pretreatment reduced the production of ROS and MDA in hepatic tissues following 6 and 24 h of reperfusion compared with the IR and IR-Daidzin groups (Fig. 4E and F).

Mitochondrial changes in the hepatocytes were observed after 24 h of reperfusion using TEM. Substantial mitochondrial damage occurred in the IR group hepatocytes compared with the sham group (Fig. 5A). However, decreased mitochondrial edema was observed in the IR-Alda-1 group (indicated in the figure by arrows). TUNEL staining also indicated that Alda-1 pretreatment attenuated hepatocyte apoptosis 24 h after reperfusion compared with the IR and IR-Daidzin groups (Fig. 5B and C).

HMGB1 and TLR4 have been reported to be involved in inflammatory responses associated with HIRI (4). After 6 and 24 h of reperfusion, the protein expression levels of HMGB1 and TLR4 were significantly upregulated in the IR group compared with the sham group (Fig. 6A-C). Meanwhile, Alda-1 pretreatment significantly inhibited the protein expression levels of HMGB1 and TLR4 compared with the IR group.

RT-qPCR was then used to analyze the mRNA expression levels of HMGB1, TLR4 and other downstream inflammatory factors. Alda-1 pretreatment significantly reduced the mRNA expression levels of HMGB1, TLR4, interferon-γ (IFN-γ), IL-1β, IL-6 and TNF-α at 6 and 24 h after reperfusion, compared with the mRNA expression levels of these genes in the IR and IR-Daidzin groups (Fig. 6D-I). Similar trends were also observed in the serum levels of IL-1β, IL-6 and TNF-α after 6 and 24 h of reperfusion (Fig. 6J-K). The results of the immunohistochemical staining of HMGB1 and TLR4 expression levels, and of IL-6 immunofluorescence, were consistent with the western blotting, ELISA and RT-qPCR data (Fig. 7). Thus, these findings indicated that Alda-1 pretreatment may protect the liver against HIRI by inhibiting the HMGB1/TLR4 inflammatory pathway.

To evaluate the effects of Alda-1 on HIRI-induced autophagy, the protein expression levels of Beclin1 (important for autophagy nucleation) (20), ATG7 (a mediator of autophagosome formation) (12), p62 (a specific protein adaptor degraded by autophagy) (20), Rab7 (a small GTPase protein that stimulates lysosomal biogenesis and autophagic vacuole maturation) (12) and LC3 (important for autophagolysosome formation) (12) were evaluated. The expression level of Beclin1 protein showed no significant differences between the groups (Fig. 8B). Compared with the sham group, the IR group had significantly downregulated expression levels of ATG7 and Rab7, a significantly reduced LC3II/I ratio and significantly upregulated protein expression levels of p62 after 6 and 24 h of reperfusion (Fig. 8A, C-F). These effects were significantly reversed in the IR-Alda-1 group (Fig. 8A, C-F). Thus, these results strongly suggested that Alda-1 pretreatment may significantly alleviate HIRI by inducing autophagy.

To assess the potential regulatory factors underlying autophagy induction, the activity levels of the AKT/mTOR and AMPK signaling pathways were analyzed. Compared with the sham group, the IR group had significantly downregulated phosphorylation levels of AKT at Thr308 and of AMPK at Thr172, while upregulated expression levels of p-mTOR at Ser2448 and p-S6 at Ser240/244 were observed (Fig. 9A-E). These effects were significantly reversed in the IR-Alda-1 group. Thus, these findings suggested that ALDH2-induced autophagy in HIRI may be dependent on the AKT/mTOR and AMPK signaling pathways.