2.1. Experimental animals and groups
All animal experiments were conducted in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animals were supplied with food and water and were subjected to the same day and night light cycles. The wild-type male C57BL/6J mice (20–28 g; Orient Bio, Sungnam, Korea) and NOX4 knockout (KO) male mice (7–9 weeks 20–28 g, provided by Prof. Ji Hwan Ryu, Severance Biomedical Science Institute, Yonsei University College of Medicine) were divided into the following five groups:
(1) Control group: wild-type mice + no ventilation
(2) High tidal ventilation (HTV) group: wild-type mice + HTV
(3) Normal ventilation control (NVC) NOX4 KO: NOX4 KO mice + no ventilation
(4) NOX4 KO with HTV group: NOX4 KO mice + HTV
(5) NOX4 inhibitor during HTV group: wild-type mice + HTV + anti-GKT 137831 inhibitor (Fig S1).
Mice groups using ventilator were anesthetized by intrapenitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and additional ketamine was used at about 20-30 minute intervals to maintain sedation. Harvard small rodent ventilator (model 683; Harvard apparatus, Holliston, MA)) was used for mechanical ventilation, and mice were ventilated with a high tidal volume of 24 ml/kg and a frequency of 100 breaths/min. For the NOX4 inhibitor during HTV group, 50 μL of DMSO with 50 μg of anti-GKT 137831 inhibitor (BioVision, cat # 9444-5) was injected intraperitoneally into mice at 120 min of ventilation. The mice were euthanized 5 h after the administration of HTV (by a lethal dose of ketamine and xylazine mixture), and their lung tissues were obtained for further analysis.
2.2. Bronchoalveolar lavage
For the BAL fluid (BALF) analysis, BAL was performed with a tracheal cannula and 1 cc of sterile saline. The BALF was centrifuged (4°C, 3000 rpm, 10 min), and the supernatant was stored at 80°C for further analysis. The cell pellet was reconstituted in 100 μL phosphate-buffered saline (PBS) and used for cell counts and cytospins. Total cell numbers in each sample were determined using a hemocytometer (Marienfield) according to the manufacturer’s protocol. A 90 μL aliquot of each sample was transferred to chamber slides, which were then inserted into a cytocentrifuge (Shandon Cytospin 4 cytocentrifuge, Thermo Scientific, Waltham, MA) facing outward. The slides were centrifuged at 800 rpm for 5 min, removed from the cytocentrifuge, and dried prior to staining with Diff-Quik Stain Set (Dade Behring, Newark, DE) to assess inflammation. Protein concentrations in the BAL supernatant were determined using a BCA assay (Thermo Fischer Scientific).
2.3. Histopathologic analysis
For the histopathology analysis, left lungs were inflated with low-melting point agarose (4%) in PBS through a tracheotomy incision at an H2O pressure of 25 cm until the pleural margins became sharp. The inflation-fixed left lungs of experimental mice were fixed in paraffin and cut to a thickness of 5 μm. After the slides were prepared, they were stained with hematoxylin and eosin, and lung injury scores were determined under light microscopy. These scores were calculated as described by Matute-Bello et al. by using neutrophils in alveolar spaces, neutrophils in interstitial spaces, hyaline membranes, proteinaceous debris filling airspaces, and alveolar septal thickening as a scoring parameters. Lung sections were processed for immunohistochemistry using an anti-NOX4 (UOTRIB492, Abcam) antibody.
2.4. Western blotting and enzyme-linked immunosorbent assay (ELISA)
Lung tissues were harvested and lysed in homogenization buffer (PRO-PREP Extraction solution, iNtRON Biotechnology). The samples were centrifuged at 13,000 × g for 30 min at 4°C. The concentrations of proteins in the supernatants were determined by BCA assay (Thermo Fischer Scientific). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were blocked with 5% skim milk in TBS-T (TBS [170-6435, Bio-Rad Laboratories] and 1% Tween-20 [170-6531, Bio-Rad Laboratories]) for 1 h at room temperature. Then, the membranes were incubated overnight with primary antibody diluted in 5% skim milk and TBS-T at 4°C. After washing with TBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies and 5% skim milk in TBS-T for 1 h at room temperature; they were then developed using a Super-Signal West Pico chemiluminescence detection kit (Pierce). The antibodies used in the present study included NOX4 (ab155071, Abcam), EphA2 (PA5-14574, Thermo Fisher Scientific), rabbit PI3 kinase 110γ (Cell Signaling Technologies), and rabbit α-tubulin (PA5-16891, Cell Signaling Technologies). Proteins resolved on western blots were quantified using ImageJ (Image Processing and Analysis in Java, NIH, USA) software.
Interleukin-1β (IL-1ß), interleukin-6 (IL-6), and interleukin-8 (IL-8) levels in lung lysates were measured using ELISA kits (Millipore) according to the manufacturer’s directions.
2.5. Real-time polymerase chain reaction (PCR)
Total RNA was isolated from homogenized lungs using a GenElute Mammalian Total RNA Miniprep Kit with DNase treatment. A High Capacity cDNA Reverse Transcription Kit was used to reverse transcribe RNA into cDNA. A real-time PCR analysis of ~25 ng cDNA was performed using TaqMan Universal PCR Master Mix for NOX4 (Mm 00479246_m1), GAPDH (Mm 99999915_g1), and pre-designed TaqMan Gene Expression Assays. The reaction was performed on a 7300 Real-Time PCR System (Applied Biosystems, Vienna). Data were analyzed using cyclophilin (Mm 00835365_g1) as a reference gene. No extraneous amplification was confirmed by inclusion of a no-template control.
2.6. Bronchoalveolar lavage fluid from patients with pneumonia
BAL fluid was obtained from 38 patients through bronchoscopy, to examine NOX4 levels in patients with pneumonia. To acquire BALF from patients, a bronchoscope was inserted and wedged through the mouth or nasal route and about 10 cc of BALF was acquired from the patient using 30 ml sterile 0.9% saline. BALF obtained from the opposite site of lung cancer was used as the control group (n = 10), and the NOX4 levels of these patients were compared with the NOX4 levels of BALFs obtained from patients with pneumonia (n = 28). The pneumonia patient group was divided into a group that did not receive ventilator care (n = 10) and a group who received ventilator care (n = 18) in the ICU due to high severity of pneumonia. NOX4 quantification was performed with an ELISA kit (Nori® Human NOX4 ELISA Kit) according to the manufacturer’s instructions.
2.7. Statistical analysis
A statistical analysis was performed in Prism version 5.0 (GraphPad Software, Durham, NC), and the data are expressed as mean ± standard deviation. Comparisons between groups were made using a two-way analysis of variance (ANOVA) and corrected using the Bonferroni correction method. P < 0.05 was considered significant.