Reagents
Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (St.Louis, MO, USA). Antibodies for P38, p-P38, JNK, p-JNK, ERK, p-ERK, p-c-jun, c-jun, GAPDH, and IκB were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-F4/80 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-LY6G was obtained from Abcam (Cambridge, UK). Antibodies for CD11B, CD45 and LY6Cwere obtained from BD Pharmingen (New York, USA). The mouse TNF-α and IL-6 ELISA kits were purchased from eBioscience (San Diego, CA, USA). Compound 34# was synthesized in our lab and structurally identified using MS and 1H-NMR analyses. For in vivo studies, 34# was dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) in water solution and 0.5% CMCNa alone was used as vehicle control. For cell culture studies, 34# was dissolved in dimethyl sulfoxide (DMSO), and the same volume of DMSO alone was used as the vehicle control.
Cell culture
Mouse primary peritoneal macrophage (MPMs) were prepared from male C57BL/6 mice. C57BL/6 mice were intraperitoneal (i.p.) injected of 2.5 mL starch broth which was constituted with 1 g tryptone, 0.5 g NaCl, 6 g soluble starch and 0.3 g beef extract, and boiled in 100 mL double-distilled H2O. After 2 days, peritoneal macrophages were collected by washing the peritoneal cavity with 10 mL RPMI-1640 medium (Gibco, Eggenstein, Germany) per mouse. The cells were centrifuged and resuspended in RPMI-1640 medium with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA). The macrophages were cultured at 37 °C in 5% CO2-humidified air. Four hours later, the non-adherent cells were removed by washing with PBS. Firmly adhered macrophages were used for experiments.
Animal care and ALI mouse model
Male C57BL/6 mice (18–20 g) were obtained from the Animal Centre of Wenzhou Medical University (Wenzhou, China). Animals were housed at a constant room temperature with a 12:12 h light-dark cycle and fed with a standard rodent diet for at least 7 days before used in the Animal Centre of Wenzhou Medical University. All animal care and experimental procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee (Approval Document No. wydw2019-0438). All animals received humane care according to the National Institutes of Health (USA) guidelines.
The C57BL/6 mice were randomly divided into four groups (n=8 per group). The groups consisted of vehicle control (CON), LPS-induced ALI group (LPS), 34# treatment of LPS-induced ALI (LPS+34# 10 mg/kg), 34# treatment-only group (34# 10 mg/kg). Mice were given 10 mg/kg/day 34# for 3 continuous days by gavage, then challenged with intratracheal instillation of 5 mg/kg LPS or equal volume 0.9% saline. Mice were euthanized with chloral hydrate 6 h after LPS injection, lung tissues, broncho alveolar lavage fluid (BALF) and blood samples were collected.
MTT assay
MPMs were seeded into 96-well plates at a density of 5 x 104 cells per well in RPMI-1640 medium with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Cells were incubated with different concentrations of 34# (2.5, 5, 10, 20 and 40 μM) for 24 h. Then, 20 μL MTT (5 mg/mL) was added to all wells, and the plate was incubated in 5% CO2 at 37 °C for another 4 h. Cells were dissolved with 150 μL DMSO and were then analyzed in a multi-well-plate reader at 490 nm.
Determination of cytokines
After treatment of cells with 34# and LPS, TNF-α and IL-6 content in culture medium and serum were determined by ELISA according to manufacturer's instructions (Bioscience, San Diego, CA). The total amount of TNF-α or IL-6 in medium was normalized to the protein concentration of lysates.
RNA extraction and Real-time quantitative PCR
MPMs and lung tissues (10-20 mg) were homogenized in TRIZOL (Invitrogen, Carlsbad, CA, USA) for total RNA extraction. The purity of the sample was estimated by calculating the OD ratio (A260/A280, ranging from 1.8 to 2.2). Both reverse transcription and Quantitative PCR (qPCR) was carried out using IQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). QuantStudio®3 Real Time PCR Systems (ABI, CA, USA) was used for qPCR analysis. The primers of the target genes are listed in the Table 1 and were obtained from Invitrogen (Shanghai, China).
Western blot assay
MPMs were pretreated with DMSO (vehicle) or 10 μM 34# for 30 min, which was followed by 0.5 μg/mL LPS for 15 min. Collected cells or homogenized lung tissues were then lysed. Concentration of total proteins was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA).
Lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electro-transferred to a nitrocellulose membrane. The membranes were then blocked for 1.5 h at room temperature in Tris-buffered saline (pH 7.6) containing 0.05% Tween 20 and 5% non-fat dry milk. Primary antibody incubations were carried out at 4 °C overnight. Secondary antibodies were then introduced for 1 h at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence reagents (BI, Beit Haemek, Israel) and was quantified using Image J analysis software version 1.38e (NIH, Bethesda, MD, USA). Values were normalized to their respective protein controls.
BALF analysis
A tracheal cannula was inserted into the primary bronchus, and BALF was performed through the cannula using Ca2+ / Mg2+-free PBS. Approximately 0.8 mL BALF was acquired. The collected BALF was centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was then immediately stored at -80 °C for protein concentration and cytokine determination. The sediment was resuspended in 50 μL physiological saline in order to determine the number of total cells using a cell counting instrument (Count star, Shanghai, China).
Flow cytometric analysis
The cells were resuspended in the collected BALF in PBS without Ca2+/Mg2+. The cells were mixed in the BALF of each group of mice together in pairs, after which 100 μL cell suspension was taken from each tube. Next, CD11B, CD45 and LY6C antibodies were used to stain the cells for 30 min. The cells were then washed with 1 mL PBS, centrifuged at 5000 rpm for 5 min, and resuspended with 500 μL PBS. ACCURI C6 PLUS (488 nm) flow cytometer and Flow Jo software were then used to analyze the cell subpopulations.
Histopathology and immunohistochemistry
Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 5 μm thickness. After dehydration, sections were stained with hematoxylin and eosin (H&E) and were evaluated for general histopathological damage using light microscopy (Nikon, Japan).
Paraffin sections were also used to perform immunohistochemistry for CD68 and LY6G using routine techniques. Sections were then deparaffinized, rehydrated, treated with 3% H2O2 for 30 min to block endogenous peroxidase activity, and blocked with 1% BSA for 30 min. Slides were incubated overnight at 4 °C with primary antibodies, and immunoreactivity was detected by diaminobenzidine (DAB). Slides were counterstained with hematoxylin for 5 min, dehydrated, and mounted for viewing by bright field microscopy (Nikon, Japan). The percentage expression was measured using Image J software (NIH, Bethesda, MD, USA).
MPO
MPO test kit was used to measure myeloperoxidase (MPO) activity in lung tissue. The lung tissue was homogenized according to the kit’s instructions, and was centrifuged at 12000 rpm at 4°C for 10 min. The supernatant was then taken for MPO activity detection and indicated in the form of U/g protein. BCA assay was used to determine the total protein content in the sample.
Statistical analysis
All experiments were randomized and blinded. In vitro experiments were repeated at least three times. All data are expressed as Mean ± SEM. All statistical analyses were performed using GraphPad Pro Prism 8.0 (GraphPad, San Diego, CA). We used one-way ANOVA, followed by Dunn's post hoc test when comparing multiple independent groups. Differences between group means were considered statistically significant at p < 0.05.