Animals and tissue preparation
Sprague-Dawley rats (SD, 90–100 days old, 250–300 g) were provided by the Animal Center of the Jiangsu University (Zhenjiang, China). The BPD animal model was constructed as previously described[16]. Newborn SD rats were randomly divided into two groups, and were exposed to hyperoxia (80–85% O2; hyperoxia group) and room air (21% O2; normoxia group) at the beginning of the day of their birth. Three to five newborn rats per group were dissected and their lungs were removed at the postnatal day 7 and day 14 (P7 and P14). The left lungs were fixed with 4% paraformaldehyde and the right lungs were stored at -80 ˚C.
Histological analysis
Tissues were fixed with 4% paraformaldehyde for 24 h at 4˚C and washed with PBS. Subsequently, samples were dehydrated using an alcohol gradient (75% alcohol, 1.5 h; 95% alcohol, 1.5 h; 100% alcohol, 1.5 h; 100% alcohol, 1 h; two xylene washes, 0.5 h each) and embedded in paraffin. Sections were sliced at 3 µm, followed by conventional dewaxing in water. Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) and boiled for 20 min. The tissue sections were stained with hematoxylin and eosin (Solarbio Science & Technology Co., Ltd., China) 23 min each for histological analysis. All steps were performed at a room temperature. The sections were acquired by confocal light microscopy (Olympus Corporation, Japan) at magnification of x400. Radial alveolar counts (RACs), representing alveolar septation and alveologenesis, were determined by standard morphometric techniques[17]. From the center of the respiratory bronchiole, a perpendicular was drawn to the edge of the acinus, defined by a connective tissue septum or the pleura, and the number of septa intersected by this line were counted.
TUNEL analysis for lung tissue
TUNEL assay was applied to detect the apoptotic cells in lung tissue samples. Lung tissues were fixed with 4% paraformaldehyde, embedded in paraffin wax, and sectioned into 4 µm thick slices. The tissue section was then dewaxed and rehydrated. A Dead End Fluorometric TUNEL System (Vazyme Biotech Co., Ltd., China) was used to perform the TUNEL assay on the isolated lung tissue, following the manufacturer’s instructions. Finally, 5 random fields were selected in each section and the TUNEL-positive cells were calculated by an inverted fluorescent microscope at magnification of x400. The apoptosis index was determined as the percentage of the total cells positive for TUNEL.
AECII isolation and culture
At the age of 19–20 days, the lung of prenatal rats was in the small tubular phase. In this period, AECII produce a large number of lamellar bodies and secrete large amounts of alveolar surfactant. The structure and functional characteristics of human fetal lung at 34–35 weeks are similar with that of prenatal day 19–20. So we selected 19 fetal rat for this study. Isolation and culture of fetal AECIIs was performed as previously described[18, 19]. In brief, lungs of 19-day gestation fetal rats (Term 22 days) were removed, dissected free from connective and nonparenchymal pulmonary tissues. Cells were dispersed using a solution of trypsin, Dnase and collagenase. AECIIs were extracted from a cell suspension utilizing a property of fibroblasts and other lung cells to adhere to plastic. Freshly isolated AECIIs were plated at 5×105 cells/mL in 50 ml culture flasks in 2 ml of MEM containing 10% fetal bovine serum. The cells were incubated for18-20 h at 37°C in 5%CO2 atmosphere. AECIIs were characterized by their morphologic appearance and the presence of lamellar bodies. All cultures contained 94 ± 2% (mean ± SE) AECIIs as determined under phase contrast microscope.
Cell grouping and MG132 treatment
AECIIs were divided into two groups according to the conditions of their maintenance, “normoxia group” and “hyperoxia group”. The normoxia group was cultured in 5% CO2 incubator at 37oC.The hyperoxia group was exposed to the stream of 95% O2 and 5% CO2 at a speed of 3 l/min for 10 minutes, then sealed and cultured in a parallel with normoxia group in 5% CO2 incubator for 24, 48 and 72 hours at 37° C. To explore ubiquitin proteasome pathway during hyperoxia exposure, AECII cells were divided into Control group (hyperoxia group), DMSO group (hyperoxia + DMSO) and MG132 group (hyperoxia + MG132). MG132 was purchased from MCE (MedChemExpress, USA) and dissolved in dimethyl sulfoxide (DMSO). The method of hyperoxia exposure is the same as before. The oxygen concentration in the cells of hyperoxia group was detected by CYS-1 digital oxygen monitor when the cells were harvested. The samples with oxygen concentration less than 90% were discarded and the remaining cells were harvested for the next experiment.
Cell viability assay
AECII cells were treated by MG132 with varying concentration (0, 5, 10, 15 and 50µmol/L) and cell viability was detected by MTT assay. The MTT assay involves the reduction of the soluble yellow dye (MTT) to an insoluble purple formazan salt. Cells were cultured in a sterile 96 wells plate in 100 µl media and incubated overnight for attachment. AECII cells were treated by MG132 for 24 h, and at the end of reaction 50 µl of MTT dye (5 mg/ml) was added to each well and incubated further for 4 h at 37°C in a CO2 incubator. The formazan products formed in cells were dissolved in DMSO (100 µl) and absorbance was measured at 540 nm using multimode plate reader (Perkin Elmer, USA).
AECIIs apoptosis assay
The cells were treated with 0.25% trypsin non-supplemented with EDTA (Invitrogen, USA), washed and resuspended in PBS. Apoptotic cells were identified by double supravital staining with recombinant FITC-conjugated Annexin V and PI, using the Annexin V/PIFITC Apoptosis Detection kit (Becton, Dickinson and Company, USA) according to the manufacturer’s recommendations. After 15 min incubation in the dark, the samples were subjected to flow cytometry analysis using BD FACS CantoⅡ flow cytometer (Becton, Dickinson and Company, USA), the data obtained were analyzed by Flow Jo software.
Protein extraction and Western blotting
All protein extraction handling was performed on ice, washed with cold PBS, lysed in proper volume of cell lysis buffer containing protease inhibitor PMSF (1:100, Sigma, Germany), and centrifuged at 12,000 g at 4 ˚C for 15 min; next, 5x loading buffer was added to the supernatant. Lysates were boiled for 8 min, then separated by 12% SDS-PAGE at a constant voltage of 70 V, and transferred to polyvinylidene difluoride (PVDF) membranes for 90 min under a constant current of 350 mA. The PVDF membranes were blocked with 5% non-fat milk in TBST containing 0.1% Tween at 37 ˚C for 1 h, and incubated at 4 ˚C overnight with the following primary antibodies: anti-Ubiquitin (#3933, 1:1000, CST, USA), anti-GRP-78(ab229317, 1:1000, Abcam, UK), anti-PERK (#3192, 1:1000, CST, USA), anti-ATF4 (#11815, 1:1000, CST, USA), anti-ATF6 (#65880, 1:1000, CST, USA), anti-CHOP (#2895, 1:1000, CST, USA) and anti-β-actin (#3700, 1:1000, CST, USA). The membranes were washed three times with TBST and then incubated with biotinylated secondary antibodies (1:5000, Fcmacs Biotech, China) for 1 h at 37 ˚C. Eventually, the immunoreactive bands were visualized by FluorChem FC3 chemiluminescence (Protein Simple, USA). Protein contents were densitometrically calculated using the LANE 1D software (Sage, China), and the relative protein expression levels were calculated as target protein/β-actin.
Measurement of 20S Proteasome Activity
Activity assays were carried out in a 200 µL reaction volume. Different concentrations of test compounds were added to a black flat/clear bottom 96-well plate containing 1nM of constitutive 20S proteasome in 50 mM Tris-HCl at pH 7.5 and allowed to sit for 10 min at RT. Fluorogenic substrates were then added and the enzymatic activity measured at 37°C on a Spectra Max M5e spectrometer by measuring increase in fluorescence unit per minute for 1h at 380/460 nm. The fluorescence units for the vehicle control were set to 100%, and the ratio of MG132-treated sample set to that of vehicle control was used to calculate the fold change in enzymatic activity.
Statistical analysis
Values are presented as mean ± SD. All data were analyzed by the SPSS 19.0 statistical software. Comparisons between two groups were performed by the Independent Samples t-test, while comparisons among multiple groups were performed using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparison post hoc test. Differences were considered statistically significant when P < 0.05.