Male Sprague-Dawley (SD) rats, weighing 250–300 g, were provided by the Experimental Animal Center of the Third Military Medical University (Chongqing, China). Rats were housed with free access to food and water under a natural day/night cycle. Rats were acclimated for seven days prior to any experimental procedures. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Third Military Medical University.

Different concentrations of hydrogen gas (1, 2 and 3%) were established by combining hydrogen with air, and subsequently compressed into oxygen bottles. During the process of hydrogen administration, rats were placed into a glass container which was connected to a pipeline. The hydrogen was administered to the conscious rats through the pipeline at a rate of 1.5 ml/h. The duration of hydrogen administration was 1, 3 or 6 h.

Immediately following the administration of hydrogen, the rats underwent an I/R injury procedure under general anesthesia with Sevofrane as described by Lord et al (9). A midline incision was created and the portal triad to the left lobe and the left middle lobe of the liver was occluded with a vascular microclamp to induce partial liver ischemia. Occlusion was verified visually by the color change of the left side of the liver to a paler shade. The abdominal muscles and peritoneum were closed with 5.0-nylon sutures. Following 90 min of ischemia, a second laparotomy was performed to remove the clamp. The abdomen was closed in the same manner and followed by 180 min of reperfusion. Then, immediately after I/R injury, syngeneic orthotopic liver transplants (OLTs) were performed using livers that were harvested from SD rats and stored for 18 h in University of Wisconsin (UW) solution, prior to being transplanted into syngeneic SD recipients with revascularization without hepatic artery reconstruction. There were three groups of rats that received liver transplants (each n=6), namely the hydrogen-treated donor and recipient group in which the donor and recipient rats both received hydrogen, the hydrogen-treated donor group in which only the donor rat received hydrogen, and the hydrogen-treated recipient group in which only the recipient rat received hydrogen. Right hepatic lobe tissue samples were taken at 1-h intervals following surgery and venous blood samples were collected after 3 h. Liver tissues were placed in 4% formaldehyde and stored in liquid nitrogen.

Fixed livers were dehydrated and embedded in paraffin. Tissues were sectioned (4-µm thickness) and stained with H&E.

Following clotting, each blood sample was centrifuged at 1,100 × g for 5 min. The clear top layer was centrifuged again under the same conditions to prepare the serum. The activities of serum ALT and AST were examined using a Wako Transaminase CII-Test kit (Wako Pure Chemical Industries Ltd., Osaka, Japan).

The liver tissues were lysed with TRIzol® (Invitrogen Life Technologies, Carlsbad, CA, USA) and vigorously mixed with chloroform for 15 sec, then stored at room temperature for 3 min. Subsequently, they were centrifuged at 12,000 × g for 15 min at 4°C and the RNA was precipitated in the aqueous phase with isopropanol. The upper aqueous phase was transferred to a new microcentrifuge tube. The RNA was precipitated by adding 0.75% ethanol and centrifuged at 12,000 × g for ≤5 min at 4°C. The supernatant was removed and the RNA was dried at room temperature for 5–10 min. The mRNA expression levels of zinc finger protein A20 (A20), nuclear factor κB (NF-κB), heme oxygenase-1 (HO-1) and B-cell lymphoma 2 (Bcl-2) were determined using qPCR with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as a control. The expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, early growth response protein 1 (Egr-1) and IL-1β mRNA were also determined. The qPCR reactions were performed using an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with the following conditions: 95°C, 10 min for one cycle; and then 95°C, 15 sec, 60°C, 1 min for 40 cycles. The expression levels of mRNA were quantified with 2^−∆∆CT. The primer sequences are listed in Table I.

ELISA (R&D Systems, Minneapolis, MN, USA) was used to determine the expression levels of IL-6 and TNF-α in serum according to the manufacturers' instructions. The absorbance was measured at 450 nm using a microplate reader (Model 680; Bio-Rad, Hercules, CA, USA).

Liver samples were fixed in 2.5% glutaraldehyde for 2 h and then rinsed in phosphate buffer. This was followed by a postfixation step for 2 h with 1% osmium tetroxide in phosphate buffer at 4°C. Afterwards, samples were dehydrated and embedded in resin. The samples were then trimmed and sectioned into slices (50∼60 nm). Ultrastructural features were observed on a transmission electron microscope (TEM; Philips CM120 TEM; Philips, Amsterdam, The Netherlands) at 60 kV.

After treatment with different concentrations of hydrogen (1, 2 and 3%) for different durations (1, 3 and 6 h), total cell lysates were prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For western blot analysis, the primary antibodies used included anti-A20, anti-NF-κB, anti-HO-1, anti-Bcl-2 (Cell Signaling Technology, Inc., Beverly, MA, USA) and anti-GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). An anti-rabbit or anti-mouse secondary antibody conjugated with horseradish peroxidase was also used (Pierce Biotechnology, Inc., Rockford, IL, USA). Immunoreactive bands were detected with an enhanced chemiluminescence (ECL) kit for western blot detection using the ChemiGenius Bio Imaging System (Syngene, Frederick, MD, USA).

Data are presented as mean ± standard error of the mean (SEM) and were statistically analyzed using SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA). Comparisons between groups were made using analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference.

The serum activities of ALT and AST were measured by ELISA. For rats that were administered different concentrations of hydrogen gas (1, 2 and 3%), the results revealed that gas inhalation at all concentrations significantly reduced the ALT and AST activities compared with those in the control group (Fig. 1). In particular, a gas inhalation concentration of 2% achieved the optimal effect. Furthermore, for the rats treated with hydrogen at different time points, the results demonstrated that 1 h administration of gas prior to surgery led to a significant reduction in ALT and AST activities (Fig. 1).

The mRNA expression of IL-6, TNF-α, Egr-1 and IL-1β was measured using qPCR. Results revealed that hydrogen administration at 2% concentration significantly downregulated the mRNA levels of IL-6, TNF-α, Egr-1 and IL-1β (Fig. 2). In addition, the expression levels of these cytokines were clearly decreased after hydrogen treatment for a period of 1 h (Fig. 2). The expression levels of IL-6 and TNF-α in serum were further examined using an ELISA. As demonstrated in Fig. 3, hydrogen gas inhalation at a concentration of 2% and a duration of 1 h led to marked reductions in the levels of IL-6 and TNF-α expression.

The effect of hydrogen gas treatment on histopathological changes in the livers of rats with I/R is demonstrated in Fig. 4. Morphological examination revealed that the liver tissues of rats with I/R injury were severely damaged in the control group with severe edema, alveolar hemorrhage and extensive inflammatory cell infiltration. In the experimental groups, hydrogen treatment significantly alleviated liver edema, alveolar hemorrhage and inflammatory cell infiltration, suggesting that the liver injury induced by I/R was reduced by hydrogen treatment. In addition, following exposure to hydrogen, the morphological changes in subcellular structures in the liver tissues were further examined using a TEM. Gross morphological changes in the liver were clearly observed in the control group while hydrogen administration markedly reduced the I/R injury in the experimental groups (Fig. 5).

The current study then investigated whether the NF-κB signaling pathway was involved in initiating the protective effect of hydrogen on liver I/R injury. The rats were randomly divided into three groups: hydrogen-treated donor and recipient, hydrogen-treated donor, and hydrogen-treated recipient. The mRNA expression levels of NF-κB, HO-1, Bcl-2 and A20 were measured by qPCR. The results revealed that the mRNA expression levels of NF-κB, HO-1, Bcl-2 and A20 in the hydrogen-treated donor group were significantly increased compared with those in the other groups (Fig. 6). The expression levels of the corresponding proteins were then determined by western blot and demonstrated similar results (Fig. 6).