Study design
All the procedure of this study was approved by the Animal Research and Care Committee of Harbin medical University. Thirty-two SD rats which were 13 weeks old and had body weights of 300 ± 20 g were randomized into 4 groups: the sham group (S), mechanical ventilation group (V), mechanical ventilation/ Ac2-26 group (VA) and mechanical ventilation/ Ac2-26/L-NIO group (VAL) (n = 8). All rats received anesthesia with 3% pentobarbital sodium (30 mg/kg intraperitoneal injection). After anesthesia and local infiltration of lidocaine, the caudal artery and vein were cannulated to analyze the arterial blood gas analysis, collect blood samples and inject saline.
The rats in the S group only received anesthesia and intubation. The rats in the V, VA and VAL groups first received an intravenous injection of endotoxin (Sigma-Aldrich, St, Louis, MO, USA) to simulate ARDS[18]. After 30 min of injection with endotoxin, the rats were analyzed for arterial blood gas to detect the PaO2/FiO2 ratio. ARDS establishment was judged to be successful when the PaO2/FiO2 ratio was less than 300[1]. Then, these rats received a large tidal volume of MV for 4 h to induce VILI. During MV, the tidal volume was set to 30 ml/kg according to previous study[18, 19], the respiratory rate was set to 50/min, and the inspiratory/expiratory ratio was set to 1:1 without positive end-expiratory pressure (PEEP). Based on results of previous studies, the dosage of Ac2-26 [13] and inhibitor of eNOS [20, 21] were confirmed at 1 mg/kg and 10 mg/kg. At initiation of mechanical ventilation, the saline, Ac2-26 (1 mg/kg) (Sigma, America) and Ac2-26 (1 mg/kg) combined with N5-(1-iminoethyl)-l-ornithine (L-NIO) (Santa Cruz) (10 mg/kg) [20, 21] were injected into the rats in the V, VA and VAL groups immediately. During the MV procedure, the anesthesia was maintained with 3% pentobarbital sodium (10 mg/kg) and 0.6 mg/kg rocuronium per hour.
Randomization
Thirty-two number from 1 to 32 were labeled in 32 cards, which were stored in envelope. The number 1 to 8, 9 to 16, 17 to 24, and 25 to 32 were allocated into S, V, VA and VA/L group respectively. The rat was randomly selected a card after anesthetized and allocated into S, V, VA or VA/L group according to the number on card.
Sample collection
The peripheral blood and arterial blood analysis were conducted at baseline, 30 min after injection of endotoxin, and 4 h after ventilation. All rats were sacrificed with an overdose of anesthetics after 4 h of ventilation, and the lung tissues and blood were collected. The right lung tissue was collected to analyze lung injury, apoptosis and protein analysis. The left lung was injected with saline to collect the bronchoalveolar lavage fluid (BALF). The serum and BALF were centrifuged at 1,000 g at 4°C for 15 min, and the supernatants were stored at -80°C for further analysis. After the procedure,rats were injected with large doses of anesthetic drugs.
The alveolar-capillary permeability
We performed the arterial blood gas analysis with a Rapidlab 348 system (Bayer Diagnostics, Germany) to calculate the PaO2/FiO2 ratio. Moreover, we also detected the protein concentration and lung tissue wet/dry weight ratio. Part of the right upper lung tissue of all rats was collected to weigh and then dry this lung tissue at 60°C for 48 h. The weight ratio of wet lung tissue to dry lung tissue was calculated. We also tested the protein levels of BALF using the BCA method. These data were calculated to evaluate the effect of AnexA1 on pulmonary alveolar-capillary permeability.
Local and systemic inflammation analysis
To evaluate the effect of AnexA1 on systemic and local inflammation in VILI, we collected peripheral blood samples and BALF. The inflammatory factors, including TNF-α, IL-1β, IL-6 and IL-10, in peripheral blood and BALF were detected with ELISA kits (Wuhan Boster Bio-Engineering Limited Company, Wuhan, Hubei, China). The ICAM-1 and IL-8 levels in serum were also estimated. Furthermore, after centrifugation, BALF deposits were stained with Giemsa by an independent pathologist to count the number of macrophages and neutrophils.
Oxidative stress response
We collected part of the right lung tissues and homogenized them with saline. The homogenate was centrifuged, and the homogenized supernatants were collected to analyze the malondialdehyde (MDA) concentration and the myeloperoxidase (MPO) and NADPH activities by using specific kits (Nanjing Jiancheng).
Histopathologic lung injury evaluation
We analyzed lung tissue histological injury with hematoxylin and eosin (HE) staining. First, part of the right lung middle lobe was collected and fixed with paraformaldehyde. After dehydration and dealcoholization, the lung tissue was embedded in paraffin. The lung tissue was cut into 4-μm sections and preserved on a slide. Slides were stained with HE. The severity of lung injury was estimated by two independent pathologists who did not participate in this study. The lung histological injury was scored according to the following system (0, minimum damage; 1, mild damage; 2, moderate damage; 3, severe damage; and 4, maximum damage).
Apoptosis evaluation
We assessed lung tissue apoptosis by TUNEL staining with commercial kits (Roche Diagnostics GmbH, Science, Mannheim, Germany). The slide with lung tissue section was immersed into proteinase K solution, and then, the slide was rinsed with phosphate-buffered saline (PBS). After the slide was rinsed with PBS, it was immersed in TUNEL reaction solution. Then, the slide was rinsed 3 times with PBS and was washed with H2O2 to inhibit the endogenous peroxidase activity. Finally, the slide was immersed in extravidin peroxidase and diaminobenzidine solution. Apoptosis was estimated by an independent pathologist by the brownish staining of the nuclei under a microscope. The apoptosis of endothelium and epithelium was identified and figured out by the independent pathologist to distinguish the apoptosis endothelium and epithelium from inflammatory cells.
Western blotting
To observe the expression of various proteins in lung tissues, we collected part of the lung tissue and extracted the protein from the lung tissue. The concentrations of proteins were tested with the Bradford assay. We added equivalent protein levels to each the polyacrylamide gel well. After electrophoresis, all the proteins were transferred onto polyvinylidene fluoride membranes (PVDF). The PVDF membrane containing the target protein was cut and blocked with 5% dry milk for 24 h. The PVDF membrane was washed with PBS 3 times and incubated with endothelin-1, phosphorylated-endothelial nitric oxide synthase (p-eNOS), AKT-1 and phosphorylated myosin light chain (pMLC) (pSer18, Sigma Aldrich). The PVDF membrane was incubated with primary antibodies for 12 h at 4 ℃ and washed with PBS 3 times, and then, the membrane was incubated with horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology) for 1 h. Finally, the bands on the PVDF were visualized via enhanced chemiluminescence.
Cell culture
To investigate the possible mechanism of Ac2-26 on VILI in ARDS, we cultured the human alveolar epithelial cell A549 (American Type Culture Collection, Manassas, CA, USA).
To avoid the influence of apoptotic protein extracted from inflammatory cells on the results of Ac 2-26 on the epithelium, the human epithelium (A549) was cultured, and received the endotoxin to simulate the ARDS. The A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, CA, USA) and cultured in Dulbecco’s modified Eagles medium (DMEM) combined with Glutamax (Gibco, Grand Island, NY, USA), 10% fetal bovine serum (FBS, Gibco), penicillin (100 units/ml) and of streptomycin (0.1 mg/ml) (Beyotime, Shanghai, China), under 95% air, 5% CO2 atmosphere at 37 °C condition. Then the cells were washed 3 time with serum-deprived DMEM medium when the cells were cultured to 80% confluency at a density of 104 cells/cm2. All the cells were divided into 4 groups: sham group(S), endotoxin group (ARDS), endotoxin/Ac2-26(ARDS/A) group and endotoxin/Ac2-26/L-NIO group (ARDS/A/L). The cells in sham did not receive any treatment. The cells in other groups received 15 μg/ml of endotoxin for 4 hours [22]. The cells in ARDS group, ARDS/A group and ARDS/A/L group respectively received the treatment of vehicle, Ac2-26 (0.3 μM), or Ac2-26 (0.3 μM)[13] and L-NIO 10 μM [23] in medium.
Four hours after stimulation of endotoxin, the cells were washed and changed the fresh medium. After that, the cell proliferation and viability were detected with the cell counting kit-8 (CCK-8). Briefly, 100μl 10% CCK-8 solution was added into the 96-well plate for 1 hour. After that, the absorbance of the solution was detected at 450nm with a microplate reader (Quant Bio Tek Instruments, Winooski, Vermont, USA). Moreover, the protein was extracted from epithelium and effect of Ac2-26 on apoptotic regulated protein Bax, Bcl-2 and cleaved caspase-3 were detected and evaluated.
Apoptosis assay
The Annexin V-FITC/PI apoptosis detection kit (BestBio, Shanghai, China) was purchased from the commercial company and used to investigate the apoptotic cells with flow cytometry (FACScan, Becton Dickinson, USA). The epitheliums of 4 groups were suspended in Annexin-binding buffer and stained with Annexin V-FITC and PI for 30 min in dark room at room temperature. The adherent and floating cells were measured by flow cytometer (Beckman Coulter, USA) to distinguish the apoptotic cells (Annexin-V positive and PI-negative) from necrotic cells (Annexin-V and PI-positive).
Statistical analysis
The primary outcome was the PaO2/FiO2 ratio after 4 h of ventilation. Our previous study indicated that the PaO2/FiO2 ratio after 4 h of ventilation was 166±15 mmHg[18]. A power analysis was performed to detect an increase of 50 mmHg in the PaO2/FiO2 ratio with an alpha error of 0.05 and a power of 90%. Based on the power analysis, 7 rats were required in each group. To compensate for the potential missed participants, we enrolled 8 rats in each group.
The normally distributed data are presented as the mean ± standard deviation (SD), and the skewed data are presented as medians (IQR). The continuous data with normally distributed data were analyzed with two-way repeated ANOVA. The skewed data were analyzed with a nonparametric Friedman test. When differences were noted, Bonferroni post hoc analysis was performed to identify the source of the difference. All statistical analyses were performed using SPSS 19.0 for Windows (SPSS, Inc., USA). A P value less than 0.05 was considered statistically significant.