Male Sprague–Dawley rats (aged 8–10 weeks, weighing 220–250 g) were purchased from the Medical Experimental Animal Center of Guangdong Province, China. The study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1985)  and was approved by the local Ethical Committee of the Third Affiliated Hospital, Sun Yat-Sen University.
Preparation of experimental animal models
The rats were kept under observation in a constant environment (room temperature 25–27°C) for 1 week prior to the start of the experiments. The OALT model was established in Sprague–Dawley rats as previously reported . Briefly, under anesthesia, the falciform ligament of the liver of the rat was severed. The first hepatic portal was dissected, and the portal vein (PV) was liberated. The hepatic artery and biliary tract were liberated together based on their anatomic relationship. Vascular clamps were applied at the convergence of the inferior mesenteric, splenic veins, hepatic artery, supra hepatic vena cava (SVC), and inferior vena cava (IVC). The PV was punctured with a needle in preparation for reperfusion using pre-cooled Ringer lactate solution. Finally, the needle was extracted, and the PV, SVC, IVC, and hepatic artery were unclamped. All rats were sacrificed 8 h after liver reperfusion for collection of blood and lung samples.
Grouping and drug treatment
Forty-eight rats subjected to OALT were randomly divided into six groups (n = 8 in each group) that received 10 µg/kg Dex (group D1), 50 µg/kg Dex (group D2), 50 µg/kg Dex + nonspecific α2-AR antagonist atipamezole (group B1), 50 µg/kg Dex + specific α2B/C-AR antagonist ARC-239 (group B2), 50 µg/kg Dex + specific α2A-AR antagonist BRL-44408 (group B3), or the same amount of normal saline (model group or group M) . The rats were intraperitoneally injected with Dex at different doses 30 min before OALT, and 500 µg/kg atipamezole, 1.5 mg/kg BRL-44408, or 50 µg/kg ARC-239 40 min before OALT, according to the group designations. The dose selection of antagonists was based on the antagonists’ affinity and dose–effect relationship with Dex. The sham rats (n = 8; the sham group or group S) underwent anesthesia induction, laparotomy, and separation of the portal vein without liver ischemia and reperfusion.
Histological analysis of lung tissues
Lung tissues were fixed, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (HE). The sections were scored as previously reported, and the histological scoring parameters included edema of the alveoli, edema of the alveolar mesenchyme, intra-alveolar cell infiltration, alveolar hemorrhage, and atelectasis .
Lung wet-to-dry (W/D) weight ratio
The wet (W) and dry (D) weights (after drying in ventilated oven at 80°C for 24 h) of the right middle lung lobes were measured. The lung W/D weight ratio was calculated by dividing the wet weight by the dry weight.
Western blotting analysis of TLR4 expression
Protein samples were extracted from the lung tissues. Samples (20 μg protein) were loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% fat-free in Tris-buffered saline-Tween (TBS-T) blocking solution (containing 0.2% Tween-20, 20 mmol/L Tris–HCl, and 150 mmol/L NaCl, pH 7.14) at 37°C for 1 h, followed by incubation with primary antibody (1:1,000; Abcam, USA) at 37°C for 2 h. Membranes were washed with TBS solution containing 1% milk four times and then incubated at room temperature for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (dilution 1:2,000, Santa Cruz Biotechnology, CA, USA). The bands on the membranes were visualized by an enhanced chemiluminescence (ECL) system. The density of each band was quantified by an image analyzer (Lab Works Software, CA, USA) and corrected by reference to the expression value for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Immunofluorescent assay and laser scanning confocal microscopy (LSCM)
Paraffin-embedded sections of lung tissue were dewaxed and rehydrated. Antigen retrieval was performed by heating in citrate buffer (pH = 6.0). Then, the sections were treated with 3% hydrogen peroxide (H2O2) for 10 min and incubated with a primary rabbit phospho-NF-κB p65 antibody (Cell Signaling Technology, MA, USA) at a dilution of 1:100, overnight at 4°C. The sections were washed with phosphate-buffered saline (PBS) and then further incubated with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody at a dilution of 1:100 (Life Technologies, USA) for 2 h at room temperature in the dark. The sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Images were captured by LSCM (Zeiss LSM 510 META, Jena, Germany).
Enzyme-linked immunosorbent assay (ELISA)
The lung tissues were homogenized and centrifuged. The TNF-α and IL-1β levels in the lung tissues were measured using commercially available ELISA kits (Keygen Biotech, Nanjing, China), according to the instructions provided by the manufacturer.
Detection of myeloperoxidase (MPO) activity
MPO activity, an indicator of polymorphonuclea (PMN) infiltration, was determined as previously described . MPO activity was defined as the quantity of enzyme that degraded 1 mmol H2O2 at 37°C, and it was expressed as U/g wet tissue.
All statistical analyses were performed using SPSS 13.0 (SPSS, Inc., IL, USA). Tests for normality and homogeneity of variances were performed. For normally distributed data, quantitative data are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to test for differences among the seven groups. A statistical difference was considered when the P value was less than 0.05.