LPS-induced ALI rat model
All 6-week old male Wistar rats were obtained from Wenzhou Medical University and maintained in a pathogen-free environment. Nine rats were assigned to each treatment group and a control group. The ALI model was established following the well-established protocol. In brief, the rats were anesthetized with 3% sodium pentobarbital, followed by the instillation of 2 mg/kg LPS (Sigma-Aldrich, St. Louis, MO, USA) solution into the tracheas. Instead of LPS solution, an equal volume of normal saline was used in the control group. For neutralizing IL-33, the rats received an intratracheal instillation of anti-IL-33 antibodies (5 μg, ab187060, Abcam, Cambridge, UK) or isotype IgG (ab172730, Abcam) 1 h after the LPS treatment. Then the rats were allowed to recover and were euthanized 24 h later with pentobarbital. All animal procedures were approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University.
Lung wet/dry ratio analysis
After the rats were euthanized, their lungs were harvested and weighed immediately. Then the blood on the lung surface was washed away, and the lungs were dried at 60 °C for 72 h. The dried specimens were weighed again, and the wet/dry ratio was calculated accordingly.
BALF collection and inflammatory cell analysis
The BALF was collected at 24 h after LPS treatment. Using a tracheal cannula, the lung was washed with 2 ml of normal saline three times. All flushing fluid was collected. The lavaged sample was then centrifuged at 1500×g for 10 min at 4 °C and the supernatant was collected for protein concentration analysis. Total protein concentration in the supernatant was measured. Each cell pellet was re-suspended in PBS and the total cell number determined in an automatic blood cell analyzer (Sysmex). Macrophages were marked with F4/80 antibodies or FITC-secondary antibodies and then analyzed and sorted with flow cytometry.
Enzyme-linked immunosorbent assay (ELISA)
Concentrations of IL-33 (BMS2048), TNF-α (KRC3011), MMP2 (KHC3081), MMP9 (BMS2016–2), TIMP1 (ERTIMP1), IL-6 (BMS625), IL-10 (BMS629), and IFN-γ (BMS621) in BALF or cell culture medium were determined using their specific enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Cell cultures and treatment
NR8383 AMs (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were derived from Sprague Dawley rats and cultured with Ham’s F-12 K medium containing 15% FBS (Gibco). 1 × 106 NR8383 cells were stimulated with 1 μg/mL LPS (Sigma-Aldrich) or 100, 200, 400 pg/mL recombinant IL-33 (Novoprotein, Shanghai, China). For neutralizing IL-33, IL-33 antibody was added to the culture medium of NR8383 cells 1 h after the LPS treatment.
The whole cell protein was obtained with cold cell lysis buffer and the total protein concentration was measured using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on 8–12% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk and then incubated with primary antibodies (MMP2 (ab92536), MMP9 (ab38898), ST2 (ab228543), IL-1RAP (ab8110), and p65 (ab16502), Abcam, 1:1000; p-STAT3 (Y705, #4113), STAT3 (#12640), p-MAPK (#4511), and MAPK (#9212), Cell Signaling Technology, Beverly, MA, USA, 1:1000; H3 (sc-517576) and GAPDH(sc-32233), Santa Cruz, Dallas, TX, USA, 1:2000) at 4 °C overnight. Next, the membranes were incubated with appropriate secondary antibodies at room temperature for 1 h. IRDye 800CW- or IRDye 680-conjugated secondary antibodies (1:10000) were used for staining and then the proteins were detected using an Odyssey infrared imaging system (both from LI-COR, Lincoln, NE, USA).
Quantitative real-time PCR
Extraction of total RNA from NR8383 cells was performed with RNAiso Plus reagent and further reverse-transcribed using a PrimeScript RT reagent kit (both from Takara, Tokyo, Japan). SYBR-Green mix (Roche) was used to carry out quantitative PCR according to the manufacturer’s instructions. Target gene expression was normalized to β-actin levels in respective samples as an internal control and calculated using the 2−ΔΔCq method, and the relative mRNA expression was further calculated through normalizing to the control group.
The software SPSS 13.0 and GraphPad Prism 5 were used in the statistical analyses. Group distributions were performed with Student’s t-test or analysis of variance (ANOVA). P < 0.05 was considered statistically significant.