Pathogen-free adult male Sprague–Dawley rats were obtained from the Laboratory Animal Center of China Medical University. All experimental animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health (NIH) Guide publication no. 85–23, revised 1985, http://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf and approved by the Ethics Review Committee for Animal Experimentation of the China Medical University.
Isolation, culture and characterization of ADSCs
ADSCs were isolated from the inguinal fat pad by enzyme digestion and cultured as described previously with minor modifications . Briefly, the adipose tissues were minced into small <1 mm3 fragments and incubated with 0.1% collagenase type I (Sigma-Aldrich, USA) in water bath under continuous shaking (37°C, 90 min) to digest the tissue. Collagenase was then neutralized by adding an equivalent DMEM/F12 (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco) to the tissue sample. The digested tissues were separated from the floating adipocytes by two centrifugations at 600 g for 5 min at room temperature. The mature adipose cells on the upper layer were removed and the pellet, as the stromal vascular fraction, was resuspended and filtered through a 40-μm filter. The fraction was then centrifuged for 5 min (50 g) to remove supernatant containing the cell debris. The cell pellets were plated in 25-cm2 culture flasks (Corning, USA) filled with 5 ml DMEM/F12 containing 15% FBS and 100 μg/ml penicillin/streptomycin. Cell cultures were maintained in a humidified tissue culture incubator (37°C, 5% CO2) and the medium was subsequently changed every 3 days for further cultivation. When ADSCs reached 90% confluence, the cells were passaged by 0.25% trypsin and 0.05% EDTA (Gibco) for analysis or transplantation. This study used ADSCs at their third passage.
To induce osteogenic differentiation, cells were cultured for 3 weeks in osteogenic medium (DMEM supplemented with 10% FBS, 10 mM β-glycerophosphate, 0.1 mM dexamethasone, and 50 mM ascorbic acid), as described previously . Early mineralization was detected using Alizarin Red S. For adipogenic differentiation, cells were cultured in adipogenic differentiation medium (DMEM containing 10% FBS, 0.5 mM isobutylmethylxanthine, 200 μM indomethacin, 10 μM bovine insulin, and 1 μM dexamethasone), and Oil Red-O staining was performed after 21 days .
For phenotypic characterization, approximately 1 × 105 cells were incubated for 30 min with monoclonal antibodies to CD29, CD105, CD90, CD45 and CD14 labeled with fluorescein isothiocyanate or phycoerythrin. Cells were analyzed using fluorescence-activated cell sorter (FACSCalibur, BD Biosciences, USA) and Cell Quest software.
Induction of VILI and administration of ADSCs
Two weeks after surgery, Sprague–Dawley (body weights 260 ± 16 g) rats were anesthetized by intraperitoneal injection of pentobarbital 75 ml/kg (Sigma-Aldrich, USA). The rectal temperature of the rats was maintained within the range of 36.5–37.5°C throughout the procedure. Rats were placed supine and ventilated through a tracheotomy tube (16-gauge, Becton Dickinson, USA) with a volume-controlled ventilator (Model 683; Harvard Apparatus, South Natick, MA) at the following settings. A total of 72 animals were randomly assigned to four groups (n = 18 each). Group I: sham group; animals received no VILI or treatment and were kept under spontaneous breathing for the entire duration of the experiment. Group II: HVT; this experimental protocol has been reported previously . Briefly, rats were ventilated for 60 min with HVT 40 mL/kg to peak airway pressures of 35 cm H2O and a respiratory rate of 40 breaths/min 100% oxygen. When high-stretch ventilation was discontinued, the animals were allowed to recover, and subsequently returned to their cages. Group III: HVT + low tidal volume (LVT); rats received HVT as in Group II, plus an additional low tidal volume of 6 mL/kg (LVT 6 h) to peak airway pressures of approximately 8 cm H2O and 100% oxygen. Group IV: HVT + ADSCs; rats received HVT as in Group II, plus autologous ADSCs (5 × 106) were slowly infused approximately 5 min via a jugular venous cannula after 1 h of HVT 40 mL/kg. After ADSCs were infused, the animals were allowed to recover as in Group II. Anesthesia was maintained with repeated pentobarbital (500 μg/kg/h intravenous) and muscle relaxation was achieved with cisatracurium besylate 0.5 mg/kg. All animals were sacrificed 7 after the experiments lasted for 7 hours. Bronchoalveolar lavage fluid (BALF) was collected and lungs were obtained for analysis.
Obtaining and processing bronchoalveolar lavage fluid (BALF)
Each animal was sacrificed by eyeball removal and blood letting. After exsanguination, the right main bronchus of rats in each group (n = 6) was tied with string at the right hilum. BALF was obtained from the left lung (n = 6) by using a 20-gauge angiocatheter ligated into the trachea and then flushing through it 3 mL of 0.9% NaCl three times. BALF recovery was always greater than 90% from each animal and was centrifuged at 1,200 g for 10 min at 4°C to remove cell debris. Total cell numbers per milliliter in the BALF were counted, and differential cell counts were performed. The supernatant was stored at -80°C for subsequent analysis. The concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-10, transforming growth factor (TGF)-β1 and keratinocyte growth factor (KGF) in BALF fluid were determined using enzyme-linked immunosorbent assay (ELISA, R&D Systems, USA) kits. Total protein content of BAL supernatant was determined using a BCA protein assay kit (Pierce, USA).
Lung wet/dry weight ratio
The right upper lobe of the same rats (n = 6) was ligated, excised, and weighed in a tared container immediately after exsanguination. The right upper lobe was then dried in an oven at 80°C until a constant weight was obtained and the wet-to-dry lung weight ratio was calculated.
The right lower lung lobes of the same rats (n = 6) were resected and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 24 h embedded in paraffin and stained with hematoxylin and eosin (H&E) staining kit (Beyotime Institute of Biotechnology, China) for microscopic observation. Two random tissue sections from four different lungs in each group were examined by a blinded investigator. Each subject was assessed according to a five-point scale: 0 = no injury; 1 = slight injury (25%); 2 = moderate injury (50%); 3 = severe injury (75%); and 4 = very severe injury (almost 100%).
RNA extraction and real-time polymerase chain reaction (PCR) for α,β, and γ-ENaC
Total RNA from the residual right frozen tissues of the same rats (n = 6) was isolated using Trizol reagent. Quantitative real-time polymerase chain reaction (qPCR) was performed using SYBR Premix Ex Taq (Takara, Japan) in a total volume of 20 μl on a 7300 Real-Time PCR System (Takara, Japan): 95°C for 30 s, then 40 cycles of 95°C for 5 s, 60°C for 31 s. The sequences of the primer pairs were: α-ENaC forward, 5'-CATGCAAGGACTGGGGAAGG-3', reverse, 3'-TGGTCATGATCCTGCTGCTTAG-5'; β-ENaC forward, 5-AGAAGAAGGCCATGTGGTTCC-3', reverse, 3'-GCTCAGGTAGGTCTG- GATGAAG-5'; γ–ENaC forward, 5'-AGAAGAAGGCCATGTGGTTCC-3', reverse, 3'-GCTCAG- GTAGGTCTGGATGAAG-5'.
The comparative Ct method was used as described by Livak  for quantitation of gene expression. The Ct values of samples (group II–IV) and sham (group I) were normalized to the glyceraldehyde phosphate 3 dehydrogenase (GAPDH). The relative levels of gene expression were calculated as ∆Ct1 = Ct of ENaC – Ct of GAPDH (group II–IV) and ∆Ct2 = Ct of ENaC – Ct of GAPDH (group I), and the fold change of gene expression was calculated by the 2-∆∆Ct method where ∆∆Ct = ∆Ct1–∆Ct2. Experiments were repeated in triplicate.
Western blotting analysis
The right lung tissues proteins of other rats in each group (n = 6) were obtained with 1 ml of lysis buffer and 1 ml of extraction buffer using a protein extraction kit (Pierce) according to the manufacturer’ s instructions and stored at -80°C for analysis. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% nonfat dried milk in Tris-buffered (Sigma-Aldrich) saline containing 0.05% Tween 20 (Sigma-Aldrich), the membranes were incubated with primary antibodies α, β, γ-ENaC (1: 300 Santa Cruz Biotechnology, USA), and β-actin (1:500) overnight at 4°C, and then reacted with horseradish peroxidase-conjugated secondary antibody (1:5000) (Santa Cruz Biotechnology) at room temperature for 1 h. Three 10-min TBS-Tween washes were performed at 24°C after each incubation. Using a western blot enhanced chemiluminescence method , protein bands were visualized with a UVP Gel imaging system (Upland, CA, USA) and analyzed with Labworks software.
The residual left lung basolateral membrane proteins (BLMs) of the same rats (n = 6) were obtained from basolateral cell membranes isolated from the 1–2 mm of peripheral homogenizing lung tissue . Na,K-ATPase activity was quantified by comparing the amount of inorganic phosphate (Pi) liberated from ATP over 1 h by 20 μg of BLMs in the absence or presence of the Na,K-ATPase inhibitor ouabain under conditions that maximize Na,K-ATPase activity (Vmax), as described previously . Conditions used maximize Na,K-ATPase activity (Vmax) to produce an index of functional, membrane-bound receptor number.
AFC from the alveolar airspace was performed by the residual rats in each group as described previously . Clearance was expressed by the change in concentration of Evan’s blue-tagged albumin in an iso-osmolar alveolar instillate placed into the alveolar airspace over a 30-minute period of measurement (n = 6). AFC was calculated as follows: AFC = (1-C0/C30), where C0, is the protein concentration of the instillate before instillation, and C30 is the protein concentration of the sample obtained at the end of 30-min of mechanical ventilation. Clearance was expressed as a percentage of total instilled volume cleared in 30 min.
All data were described as mean ± SEM. Statistical analysis was performed using SPSS16.0 software. Results were compared by one-way ANOVA followed by Student-Newman-Keuls test, or by one-way ANOVA on ranks followed by Bonferroni-Dunn test. Statistical significance was confirmed at a two-tailed p value of <0.05.