Animals
Male Sprague Dawley rat (6 weeks old, total n=34) were purchased from Charles River Laboratories (Beijing, China), and housed at the specific pathogen-free (SPF) facility at the Animal Center of Harbin Medical University (Harbin, China) at room temperature (22 ± 1 ˚C) with a 12/12 hours light/dark cycle and access to food and water ad libitum. Rats were randomly assigned into the sham group, LIRI group and lipoxin A4 (LA4) group. Rats in the sham group only received the anesthesia and thoracotomy, while rats in the LIRI and LA4 groups received the orthotopic rat left lung transplantation and injection of saline and lipoxin A4, respectively, as our previous studies15, 16. This study was approved by the Secondary Affiliated Hospital of Harbin Medical University. All treatments were carried out in accordance with the Institutional Animal Care and Use Committee of Second Affiliated Hospital of Harbin Medical University and followed national guidelines for the treatment of animals.
Donor graft preparation
A total of 18 donor rats were anesthetized with intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg body weight). After the disappearance of clip tail reflection, the rats were intubated and ventilated with the following parameters: tidal volume 10 ml/kg and 50 breaths per minute (50% O2 +50% N2) with 2 cm H2O positive end-expiratory pressure and inspiratory expiratory ratio of 1:1. Under analgesia with 1% lidocaine, rats were subjected to the thoracotomy. After heparization with 300U/kg heparin via femoral vein, the heart-lungs was collected, and followed by the flushing with 4°C cold saline at 20 cm H2O pressure. Under the microscopy view, the left pulmonary artery, vein, and left bronchus were clipped on the CUFF tube (24 G central vein catheter). The graft was preserved at 4 °C for 60 min with end tidal volume.
Lung transplantation to recipients and intervention
A total of 16 recipient rats (8 rats in the LIRI group and 8 rats in the LA4 group) were anesthetized as the donors and intubated with 12G catheter. The femoral artery and vein were cannulated for blood collection and drug administration. After injection of 0.5 mg/kg atracurium, the recipients were ventilated with the same parameters as described for the donor rats. After left thoracotomy within 3 to 4 rib, the left lung was dislodged, and the pulmonary artery, vein and bronchus were dissociated. After blocking the blood and ventilation with clamps, the artery, vein and bronchus were anastomosed to CUFF tube of the lung graft. During this process, the tidal volume was decreased to 6 ml/kg and returned to 10 ml/kg after reperfusion. After closing the thoracotomy, the recipients were extubated, and followed with spontaneous breath. Rats in the sham and LIRI groups received intravenous injection of saline (1 ml each), while rats in the LA4 group received intravenous injection of lipoxin A4 (Cayman Chemical, Ann Arbor, MI, USA) at the dose of 100 μg/kg (diluted into 1 ml saline) in 30 min after reperfusion, as previously described 12, 14 .
Lung samples collection
After 24 hours of reperfusion, all the rats were anesthetized and cannulated. The arterial blood gas analysis was performed, and the peripheral blood was collected. After sacrificing the rats with overdose of anesthetic, the left lungs were collected and divided into 3 parts. Upper part of the graft (the LIRI and LA4 groups) or control lung (the sham group) was stored at liquid nitrogen for further analysis of protein expression; the middle part was prepared for the histological and apoptotic evaluation; the lower part was homogenized with 0.9% saline (1: 9 weight) for testing the cytokines levels in the 10% homogenate. The peripheral blood and homogenate were centrifuged at 4℃, 1000 g/min for 10 min, and the supernatant was collected for further analysis.
The alveolar capillary permeability
The partial pressure of O2 (PaO2) was analyzed by the Bayer Rapidlab 348 Blood Gas Analyzer (Bayer Diagnostics, Germany). The partial pressure of O2 to fraction inspiratory O2 (PaO2/FiO2) ratio was calculated. The lung tissues were weighed (wet weight) before drying for 72 h at 50°C (dry weight). The wet/dry weight ratio was calculated by dividing the wet weight by the dry weight. The protein concentrations in the homogenate of lung samples were measured by the Bradford method.
Histological estimation
Part of lung samples was preserved in the paraffin, and the lung tissue was prepared for the HE staining to evaluate the histological injury, which was scored by 2 independent investigators. The score of lung injury was based on the Table 1 which included 5 variables such as lung hemorrhage, peri-bronchial infiltration of inflammatory cells, pulmonary interstitial edema, pneumocyte hyperplasia and intra-alveolar infiltration of inflammatory cells. Each criterion was scored on a semiquantitative scale of 0–4, where 0=normal, 1=minimal change, 2=mild change, 3=moderate change and 4=severe change. An overall histological score was calculated by summing the scores for criterion 1 through 5.
Table 1
lung injury evaluation variables
Parameters
|
Score
|
Hemorrhage
|
0 or 1
|
Peri-bronchial infiltration
|
0 or 1
|
Interstitial edema
|
0 to 2
|
Pneumocyte hyperplasia
|
0 to 3
|
Intra-alveolar infiltration
|
0 to 3
|
Oxidative stress response
The activity of myeloperoxidase (MPO), superoxide dismutase (SOD), xanthine oxidase (XO), and the concentration of malondialdehyde (MDA) in the homogenate samples were measured with the specific kits (Nanjing Jiancheng, Nanjing, China) per the manufacturer’s instructions.
Inflammation assays
The systemic and local inflammation were assessed by testing the cytokines levels in homogenate and serum. The levels of TNF-α, IL-1β and IL-10 in homogenate, as well as the levels of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein 1 (MCP-1) in serum, were determined by the Enzyme-Linked Immunosorbent Assays with commercial kits (Wuhan Boster Bio-Engineering Limited Company, Wuhan, China) following the manufacturer’s protocols. The activity of NF-κB in lung tissues was measured using the Transcription Factor Assay Kit (Abcam, Toronto, Canada) according to the manufacturer’s instructions. The expression of NF-κB in lung tissue was determined with Western Blot.
Western blot
Protein samples were prepared from the lung tissues using 1× cell lysis buffer (Cell Signaling Technology, USA). Equivalent amounts of proteins (10-20 µg) were used to assess protein expressions as described previously11, 12. Briefly, protein was denatured by boiling, separated by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE, 12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 8% non-fat milk/PBS-T (PBS with 0.5% Tween-20) for 2 h and respectively incubated with the following rabbit-derived primary antibodies: NF-κB, Bax, Bcl-xL, cleaved-Caspase-3 and β-actin (all 1:1000 dilution; Sigma Aldrich, St. Louis, MO, USA). After washing, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000 dilution; Cell Signaling Technology, USA). Proteins of interest were visualized using the enhanced chemiluminescence kit (EMD Millipore, USA), and the band intensities were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Apoptosis assays
The lung tissue section for TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) apoptosis assay was prepared according to the instruction of a commercial TUNEL kit (Roche Diagnostics GmbH, Science, Mannheim, Germany). Briefly, the lung tissue sections were treated with proteinase K and incubated in terminal deoxyribonucleotidyl transferase enzyme. And then, the sections were stained with diaminobenzidine–hydrogen peroxidase and Mayer’s hematoxylin. The nuclei with brown staining indicated apoptosis. Ten random fields of each section were selected, and two pathologists counted the apoptotic cells independently. The apoptosis index was calculated as the ratio of positive cells to total cells. In addition, the expressions of apoptosis associated proteins including Bax, Bcl-xL and cleaved-caspase-3 in lung tissues were evaluated by Western blot assays.
Endothelial viability and tube formation assays
Rat pulmonary microvascular endothelium cells were purchased from PriCells (Wuhan, China). The endothelium cells were cultured in normal endothelial cell growth medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 1% endothelial cell growth factors under standard cell culture conditions (21% O2, 5% CO2, and 74% N2). To mimic hypoxemia and reoxygenation, cells seeded in 96-well plates with a density of 1×104 cells per well were cultured in glucose-deprived medium in a hypoxic chamber (5% CO2, and 95% N2; Biospherix hypoxia chamber, NY, USA) for 1 hour, and then returned to the normal condition for 24 hours. Subsequently, lipoxin A4 (100 ng/ml) was added in the culture medium of the LA4 group as previously described17, and the cells in the LIRI group were treated with the vehicle PBS. Twenty-four hours after reoxygenation, all the cells were collected to test the viability and tube formation capacity.
The endothelial viability was evaluated using the cell counting kit-8 (CCK-8) commercial kit. Briefly, endothelium cells with a density of 1×104 cells/well were plated in the 96-well plates. After incubation with the CCK-8 solution for additional 4 hours, the absorbance of the culture medium was detected at 450nm with a microplate reader (Quant Bio Tek Instruments, Winooski, Vermont, USA). The tube formation activity of endothelium was determined using the commercial assay kit (Abcam, Toronto, Canada) following the manufacture’s instructions. Briefly, 50μl matrigel was added into the 96-well plate, and then the plate was incubated at 37 °C for 30 min. The endothelium cells (104 cells/well) were seeded and cultured for 12 hours. Then, the endothelium was washed with PBS and the tube network was imaged using the IX51 research microscope. Meanwhile, the tube formation was quantitatively measured using Image J software (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis
All the data were presented as mean ± standard deviation (SD). All the variables were analyzed by one-way analysis of variance. Data were analyzed using the IBM SPSS Statistics 19.0 (SPSS, Chicago, IL, USA). A two-tailed p-value of <0.05 was considered statistically significant.