Animal modelling and grouping
Healthy New Zealand white rabbits weighing 2.18 ± 0.20 kg were purchased from the Animal Center of Second Military Medical University (SCXK 2012-0007, Shanghai, China). Rabbits were group-housed at 20–22 °C on a 12 h light–dark cycle with free access to a standard diet and water. Animals remained in the housing facilities for at least one week to acclimatize prior to initiation of the experimental protocols. Freshwater was collected from Dianshan Lake, Shanghai (the water quality essentially met the third-level national surface water quality standard, pH 6.5–8.5, chloride content lower than 0.01 mg/l).
Freshwater drowning animal modelling: The rabbits were anaesthetized by left ear vein injection of sodium pentobarbital (30 mg/kg, Sigma-Aldrich, St. Louis, MO, USA). A 14-F endotracheal tube (Smiths Medical International Ltd. Kent, UK) was inserted into the trachea, and the groin was cleanly shaven. A 23-gauge PE-50 polyethylene catheter (Becton, Dickinson and Company, NJ, USA) was inserted into the right femoral artery to measure arterial blood pressure and obtain blood samples. During the operation, an electric blanket was used to maintain the animal's anal temperature at 38 ± 0.1 °C. Following 15 min of stabilization, freshwater (18 ml/kg) was instilled into both lungs over 5 min through the endotracheal tube. After instillation, the rabbits were maintained in a supine position with the head elevated 30°. Arterial blood gas was measured after 15 min of freshwater instillation was complete. When the PaO2/FiO2 (P/F), which was calculated by the ratio of arterial partial pressure of oxygen (PaO2) to the fraction of inhaled oxygen (FiO2) (21%), had decreased to less than 300 and remained at this level for over 1 h, the freshwater instillation-induced ADLI model was successfully established.
Rabbits (n = 30) were randomly divided into five groups: i) control group (C, n = 6), no instillation; ii) freshwater group (F, n = 6), freshwater-induced ADLI; iii) small-dose (2.5 × 104 U/kg) Ulinastatin treatment group (SU, n = 6), Ulinastatin (2.5 × 104 U/kg) administered intravenously immediately following successful establishment of freshwater-induced ADLI; iv) medium-dose (5 × 104 U/kg) Ulinastatin treatment group (MU, n = 6); v) large-dose (10 × 104 U/kg) Ulinastatin treatment group (LU, n = 6). The dose of Ulinastatin used was determined on the basis of a previous study [15].
Blood Gas Analysis
In each group, arterial blood gas analysis was performed before modelling (baseline) and at 0 h (T0), 0.5 h (T0.5), 1 h (T1), 3 h (T3), and 6 h (T6) after freshwater instillation. Then, PaO2 was measured with a blood gas analyser (GEM Premier 3000, Instrumentation Laboratory Company, USA).
Wet-to-dry Weight (w/d) Ratio
The W/D ratio was used to represent the severity of pulmonary oedema. At the end of the experiment, the left lower lungs were weighed and then dried to a constant weight at 80 °C for 72 h. The W/D ratio was finally calculated by dividing the wet weight by the dry weight.
Assessment Of The Lung Permeability Index (lpi)
At the end of the experiment, the rabbits were anaesthetized, and the left upper lungs of rabbits in all groups were lavaged twice with 10 ml of normal saline. The recovery rate of the lavage fluid was approximately 90%. The bronchoalveolar lavage fluid (BALF) was centrifuged at 3000 rpm for 10 min at 4 °C, after which the supernatant was collected. The total protein (TP) content in the lavage fluid and serum was detected by using an enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Gen. Med. Corporation, Shanghai, China). The LPI was then calculate as follows [16]:
LPI = TP lavage fluid/TP serum
Histological And Pathological Injury Score
The Smith score was used to evaluate lung injury, including alveolar, bronchial, and vascular injury, characterized by inflammatory infiltration, alveolar wall rupture and fusion [17]. The total lung injury score (LIS) was the sum of the scores for the above items assigned as followed: score of 0, no injury; score of 1, injured area of < 25%; score of 2, injured area of 25%-50%; score of 3, injured area of 50%-75%; score of 4, injury observed in the full vision field. At the end of the experiments, the right lower lung tissues were extracted, fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. After deparaffinization and dehydration, the lungs were cut into 5 µm sections and stained with haematoxylin and eosin (HE). The sections were scored by observing 10 visual fields for each section from each animal at high magnification (200×), and the average of the total scores was recorded.
ELISA
The concentrations of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the BALF were detected by using ELISA kits (R&D Systems; Minneapolis, MN, USA). After freshwater instillation, the rabbits were anaesthetized, and the left upper lungs of rabbits in all groups were lavaged twice with 10 ml of normal saline. The recovery rate of the lavage fluid was approximately 90%. The lavage fluid was centrifuged at 3000 rpm for 10 min at 4 °C, after which the supernatant was collected and processed for ELISA according to the manufacturer’s instructions.
Western Blotting
Western blotting
The lung tissues were lysed in RIPA lysis buffer (containing 1/100 PMSF, Solarbio, China). The lysates were centrifuged at 12,000 rpm for 30 min at 4 °C, after which the supernatants were collected. The protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific Inc., USA). TP extracts were separated on 8% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels before being electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, USA). After they had been blocked for 1 h with 5% milk in Tris-buffered saline with Tween-20 (TBST), the membranes were incubated with various primary antibodies overnight at 4 °C. The following day, the membranes were washed three times with TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Cell Signalling Technology) for 1 h at room temperature. Reactive bands were visualized using an enhanced chemiluminescence kit (Millipore Corporation, USA), and image acquisition was performed using an Amersham Imager 600 (GE, USA). To ensure the loading of equal samples in each lane, membranes were stripped and re-probed with anti-GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) antibody. Signal intensity was semi-quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA). The following primary antibodies were used according to the manufacturers’ instructions: anti-HIF-1α (1:1000; Millipore, USA), anti-VEGF (1:1000; LSBio, USA), and anti-GAPDH (1:1000; Cell Signalling Technology, USA).
Quantitative Real-time Polymerase Chain Reaction (q-pcr)
Total RNA was extracted from 100 mg of right upper lung tissue with a TRIzol RNA extraction kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA was converted to cDNA using a cDNA synthesis kit (Bio-Rad). Reverse transcriptase-generated cDNAs encoding VEGF, HIF-1a, and GAPDH (used as a control for RNA integrity and internal standard) were amplified by Q-PCR. The sequences of the primers used for amplification were as follows: VEGF, (forward) 5'-CGGTTCCAGAAGGGAGAGGA-3' and (reverse) 5'-CTGGGACCACTTGGCATGG-3'; HIF-1a, (forward) 5'-AGAGTCAAGCCCAGAGTCAC-3' and (reverse) 5'-TGGGACTGTTAGGCTCAGGT-3'; and GADPH, (forward) 5'-GGGCTCTCTGCTCCTCCCTGT-3' and (reverse) 5'-ACGGCCAAATCCGTTCACACC-3'. Forty amplification cycles consisting of 15 s of denaturation at 95℃, 30 s of annealing at 60℃, and 30 s of extension at 72℃ were performed. The relative mRNA expression of each target gene was determined by the 2 -ΔΔCT method.
Statistical analysis
All data are expressed as the mean ± SD or mean ± SEM. The statistical significance of differences was determined by one-way analysis of variance using GraphPad Prism (v.5.0.) (GraphPad Software, USA). A P value of less than 0.05 indicated significance.