Chemical and drugs

CU06-1004 was synthesized as previously described [27]. Briefly, CU06-1004 was synthesized via tetrahydropyran deprotection and subsequent glycosidation with 4,6-di-O-acetyl-2,3-didieoxyhex-2-enopyran in the presence of acid. LPS and dexamethasone were purchased from Sigma Aldrich (St. Louis, MO, USA).

Animals and ethical statement

Eight-week-old male mice (C57BL/6J, 21–23 g body weight) were purchased from DBL (Eumseong, Chungcheongbuk-do, Republic of Korea) and were used for the entire study. All mice were housed in standard cages at constant temperature (22 ± 1 °C) and humidity (55 ± 5%), with a 12-hour light/dark cycle and free access to food and water. All experiments involving animals were approved in advance by the Animal Care and Use Committee of Yonsei University (Seoul, Republic of Korea) and were performed in accordance with approved guidelines (IACUC-A-202010-1151-01).

LPS challenge and drugs treatment

All mice were acclimated for 1 week and randomly divided four groups: PBS group, LPS group, LPS + CU06-1004 group, and LPS + dexamethasone group. LPS (Sigma Aldrich, St. Louis, USA, L2880) was dissolved in PBS (45 mg/kg) and was injected via the intraperitoneal route. CU06-1004 was dissolved in olive oil (O1514, Sigma Aldrich, St. Louis, USA) and was injected via the oral route (10 mg/kg) 4 h after the LPS challenge. Dexamethasone (Sigma Aldrich, St. Louis, USA, D2915) was dissolved in PBS and injected via the intraperitoneal route (5 mg/kg). The mice in the PBS groups received PBS only, instead of LPS, and an equal volume of olive oil (per os) or PBS (intraperitoneal injection), instead of CU06-1004 or dexamethasone, was given.

Survival test

To assess survival rates (percent survival) during the entire study period, 75 mice were randomly assigned to one of four groups: PBS, LPS, LPS + CU06-1004 (10 mg/kg), or LPS + dexamethasone (5 mg/kg). Mortality of the mice was recorded every 12 h for 4 days.

Pulse oximetry

To assess arterial oxygen content, mice were monitored for the percentage of hemoglobin saturated with oxygen. Mice were anesthetized using avertin (2,2,2-tribromoethanol, Sigma Aldrich, USA, T48402) at 240 mg/kg, and the fur on the neck was removed by shaving. The sensor was placed on the skin, and three consecutive sustained readings were averaged using MouseOx Plus oximeter (Starr Life Sciences, Oakmont, PA, USA).

Tissue wet/dry ratio

All mice were euthanized a day after the LPS challenge. The tissue wet weights were recorded after tissues were excised. The tissues were then placed in an oven at 60 ℃ for 48 h, and then the dried tissue weights were recorded. Wet/dry ratios were calculated by dividing dry weight by wet weight.

Tissue collection

At the end of the experiment, all mice were anesthetized using avertin (2,2,2-tribromoethanol, Sigma Aldrich, USA, T48402) at 240 mg/kg, and a CO2 chamber was used to euthanize the animals. The lungs were dissected, transferred to 4% PFA for 1–2 days, and then used for the subsequent experiments.

Histopathological analysis

Fixed lung tissues were paraffinized using a tissue processor (Leica, Wetzlar, Germany, 0422). The tissues then were sectioned at 5 μm using a microtome (Leica, Wetzlar, Germany, 9224) and were stained using an H&E staining kit (Abcam, Dawinbio Inc., Hanam, Gyeonggi-do, Republic of Korea, ab245880). The stained samples were examined under an optical microscope (Nikon, Tokyo, Japan, Eclipse 80i), and the images were graded from 0 to 4. The grades used were: 0, no injury (normal); 1, minimal (injury up to 25% of the field); 2, mild (injury between 25 and 50% of the field); 3, moderate (injury between 50 and 75% of the field); and 4, severe (injury over 75% of the field), for thickening of the alveolar-capillary walls, the numbers of infiltrated cells, hemorrhage, and the vascular congestion.

Immunohistochemistry analysis

Each tissue section was de-paraffinized using xylene (three times) and serial alcohol (100%, 90%, 80%, 70%, and 50%) and then exposed to citrate butter (10mM sodium citrate acid, 0.05% Tween20, pH 6.0) for antigen retrieval. The section was blocked with blocking solution (DAKO, Santa Clara, CA, USA, X0909) for 1 hour at room temperature and was incubated overnight at 4°C with primary antibodies. The primary antibody was NF-κB p65 antibody (Thermo Fisher Scientific, IL, USA, 33-9900). An EnVision Detection Kit (DAKO, Santa Clara, CA, USA, K5007) was used for secondary-peroxidase complex and 3,3’-diaminobenzidine tetrahydrochloride salt staining. Stained samples were examined under an optical microscope (Nikon, Tokyo, Japan, Eclipse 80i).

Scanning electron microscopy

Lung tissue from mice was cut into longitudinally through the lumen of the mediastinal airways extending from the lobar bronchus to the distal airways. Specimens were fixed for 24 h in Karnovsky’s fixative (2% Glutaraldehyde, 2% Paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) and washing two times for 30 min in 0.1 M PBS. They were postfixed with 1% osmium tetroxide for 2 h and dehydrated in ascending gradual series (50, 60, 70, 80, 90, 100, and 100%) of ethanol and used a Critical Point Dryer (EM CPD300, LEICA, Germany). They were coated with platinum by ion sputter (EM ACE600, LEICA, Germany) and observed with a field emission Scanning electron microscopy (MERLIN, ZEISS, Germany).

Transmission electron microscopy

For transmission electron microscopy (TEM) lung tissue was obtained from ALI mice and cut into small pieces (1 mm3). Specimens were fixed for 12 h in 2% glutaraldehyde at 4 °C, rinsed in 0.1 M phosphate buffer (pH 7.4), followed by post fixation in 1% osmium tetroxide for 2 h, and dehydrated with an ascending ethanol series (50, 60, 70, 80, 90, 95, 100, and 100%) for 10 min each. Specimens were embedded with a Poly/Bed 812 kit (08792-1, Polysciences, PA, USA), polymerized in an electron microscope oven (TD-700, DOSAKA, Japan) at 65℃ for 12 h. The block is equipped with a diamond knife in the ultramicrotome and is cut into 200 nm semi–thin section and stained toluidine blue for observation of optical microscope. The region of interest was then cut into 80 nm thin sections using the ultramicrotome, placed on copper grids, double stained with 3% uranyl acetate for 30 min and 3% Lead citrate for 7 min staining, and imaged with a transmission electron microscopy (JEM-1011, JEOL, Tokyo, Japan) at the acceleration voltage of 80 kV equipped with a Megaview III CCD camera (Soft imaging system-Germany).

Myeloperoxidase activity

MPO activity was evaluated to assess neutrophilic infiltration to the lung tissue [ ]. The MPO activity was measured using an MPO activity assay kit (Abcam, Dawinbio Inc., Hanam, Gyeonggi-do, Republic of Korea, ab105136), according to the manufacturer’s instructions.

Collection of bronchoalveolar lavage fluid and serum

BALF and serum samples were collected 32 h after the LPS challenge. Briefly, each mouse was anesthetized using avertin (240 mg/kg) and the middle of the trachea was carefully punctured using a 26-gauge needle. A catheter was then inserted into the trachea and the lungs were washed three times with PBS (0.7 ml each time). For serum collection, whole blood was collected from the heart using a 1 ml syringe. The collected blood was allowed to clot at room temperature for 30 min. The clot was then separated from the supernatant using centrifugation at 1000 × g for 10 min at 4 ℃, and the supernatant was collected.

Wright-Giemsa staining

Each BALF sample was centrifuged at 300 × g for 10 min in a cytocentrifuge with a slide for attaching the cells. Then, the slide was stained using a Wright-Giemsa Stain Kit (Abcam, Dawinbio Inc., Hanam, Gyeonggi-do, Republic of Korea, ab245888), according to the manufacturer’s instructions.

Measurement of cytokine levels in bronchoalveolar lavage fluid and serum

The levels of inflammatory cytokines were measured in the BALF and serum samples. TNF-α, IL-1β, and IL-6 were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA, #MTA00B, #M6000B, and #MLB00C), following the manufacturer’s instructions.

Western blot analysis

Proteins from lung tissues were extracted in radioimmunoprecipitation assay buffer (100 mM Tris-Cl, 5 mM EDTA, 50 mM NaCl, 50 mM β-glycerophosphate, 50 mM NaF, 0.1 mM Na3VO4, 0.5% NP-40, 1% Triton X-100, and 0.5% sodium deoxycholate). Sample protein concentrations were quantified using a SMART BCA Protein Assay kit (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea). Next, tissue lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin in 0.1% tris-buffered saline with Tween 20 (TBST) and then probed with primary antibodies. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Thermo Fisher Scientific, IL, USA, #31,460, #31,430) secondary antibodies. β-Actin was used as a loading control. The primary antibodies used (1:1000 dilution) were: TNF-α (Cell signaling Technology, Danvers, USA, #3707), IL-6 (Cell signaling Technology, Danvers, USA, #12,912), IL-1β (Abcam, Dawinbio Inc., Hanam, Gyeonggi-do, Republic of Korea, ab234437), NF-κB p65 (Cell Signaling Technology, Danvers, USA, #8242), Phospho-NF-κB p65 (Cell Signaling Technology; Danvers, USA, #3031), and β-actin (Thermo Fisher Scientific, IL, USA, MA5-15739).

Vascular permeability assay

Evans-blue (1% in PBS; Sigma Aldrich, St. Louis, USA, E2129) dye was used to evaluate the pulmonary leakage. Evans-blue dye was injected via the intravenous route 30 min before the lung tissue was collected. Evans-blue leakage was quantified in the lung tissue after the lung was homogenized and incubated in formamide (24 h, 55 °C). The Evans-blue assay result was measured in the supernatant from each sample (absorbance, 620 nm). Results were calculated using a standard curve for Evans-blue in formamide and were presented as micrograms per gram of lung tissue.

Cryo-section and immunofluorescence staining

Lung tissue was exposed to paraformaldehyde (4% in PBS) for 1 or 2 days (4 °C) for fixation. The tissue was then rinsed with PBS at room temperature, incubated overnight (4 °C) in sucrose (15%), and then transferred to sucrose (30%) at 4 °C until the tissue sank. Tissue-Tek optimum cutting temperature (OCT) embedding medium was then used to infiltrate the fixed lung for 30 min at room temperature. The samples were stored at -80 °C after transfer to an OCT-filled embedding mold and freezing with dry ice. While frozen, Sect. (10 μm-thick) were cut onto slides at -20 °C for immunostaining. The slides were stored at -80 °C until use for this procedure. Briefly, the sections were prefixed in acetone for 30 min at -80 °C and air dried. Flowing water was used to rinse the OCT. The sections were then incubated overnight in primary antibody (1:200, 4 °C), washed three times (5 min per wash) with Triton X-100 (0.1%) in PBS, and were incubated for 2 h in secondary antibody (1:300, room temperature). The sections were then counterstained using DAPI (4′,6-diamidino-2-phenylindole, 1 µg/ml) and were washed three times with Triton X-100 (0.1%) in PBS (5 min per wash). Antibody diluent (Dako, Agilent Technologies, Santa Clara, CA) was used to dissolve each antibody. A confocal microscope (LSM 880, Carl Zeiss) was used to examine each section.

Statistical analysis

GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for the data analysis. The results were presented as mean ± standard deviation or standard error of the mean values. The data were analyzed using one-way analysis of variance, followed by Tukey’s multiple comparison tests. Kaplan-Meier survival curves were compared using log-rank tests. A P < 0.05 was considered to indicate a statistically significant difference.