Twenty-four male C57BL/6 mice were obtained from the Shanghai SLAC animal Company (SLAC, Shanghai, China PRC) and kept in a pathogen-free condition. The mice had free access to food and water. In the present study, mice were randomly divided into the control group, LPS stimulation group, TCs treated group in mice challenged with LPS, and TCs silencing miRNA-146-5p in mice challenged with LPS. The mouse ARDS model was set up by instilling LPS (5 mg/kg) via the trachea. TCs (1x106 cells) transfected with antagomir-146a-5p or not were administrated via the tail vein two hours before the LPS stimulation. The animals were sacrificed, and the lungs and bronchoalveolar lavage fluid were collected twenty-four hours after the establishment of the mouse ARDS model.
All animal procedures were performed by bioethics guidelines and were approved by the animal committee of Zhongshan Hospital, Fudan University, Shanghai, China PRC.
Assessment of lung injury
To collect white blood cells in bronchoalveolar lavage fluid, mouse lungs were perfused with ice-cold phosphate-buffered saline. After centrifugation, white blood cells were counted under microscopy. The left lung was fixed in 10% formalin solution and embedded in parafﬁn for hematoxylin and eosin staining（H&E stain）. The right lung was collected to assess edema by the ratio of wet weight/dry weight (W/D) of the lung after incubating in a 60℃ oven for 48 hours. The lung injury was evaluated by a modified lung injury histological scoring system(21). In brief, lung injury was scored on a scale of 0-4 using the average score of the following items: (I) alveolar capillary congestion, (II) hemorrhage, (III) infiltration of neutrophils into the airspace or the vessel wall, and thickness of the alveolar wall, and (IV) alveolar wall thickness/hyaline membrane formation. A score of 0 represented normal findings, and scores of 1, 2, 3, and 4 represented mild (<25% lung involvement), moderate (25-50% lung involvement), severe (50-75% lung involvement), and very severe (>75% lung involvement) lung injury. The cytokines levels of IL-1, IL-6, TNF-α in bronchoalveolar lavage fluid were detected by Bio-Rad Bio-Plex Pro Mouse Cytokine 23-Plex Assay on the Bio-Rad MAGPIX Multiplex Reader
Mouse lung TCs were kindly provided by Dr. Wang X (Biomedical Research Center, Zhongshan Hospital, Fudan University, Shanghai, China). Mouse RAW264.7 and MLE-12 cells were bought from Zhongqiao Tech (Zhongqiao, Shanghai, China PRC). All the cells were cultured in Dulbecco's modified Eagle's medium/F12 (DMEM/F12, Hyclon, Gaithersburg, USA).
HEK293T cells were cultured in DMEM high glucose medium (Hyclon, Gaithersburg, USA) for the Dual‑luciferase reporter assay.
Reverse transcription-polymerase chain reaction (RT-PCR)
Cultured TCs were collected in TRIzol reagent (Takara, Shiga Japan). MicoRNAs were reversely transcripted with a Bulge-Loop miRNA qRT-PCR Starter Kit (Ribobio, Guangzhou, China), and mRNAs were reversely transcripted to complementary DNA (cDNA) with a PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan). Both miRNA and mRNA were amplified on an ABI 7500 sequence detection system (Applied Biosystems, Inc., Foster, USA). The expression levels of miR-21a-3p and miR-146a-5p were normalized with U6. and mRNAs expressions were normalized with Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). Primer set of miR-21a-3p and miR-146a-5p were provided by Ribobio (Ribobio, Guangzhou, China). Primers of mRNA used in the present study were synthesized by Sangon Biotech Co., Ltd (Shanghai, China) (Table 1).
Cultured cells were lysed in RIPA buffer containing protease inhibitors (Roche, Indianapolis, IN, USA), phosphatase inhibitors (Beyotime Shanghai, China), and phenylmethanesulfonyl fluoride (PMFS) (Beyotime Shanghai, China). Total protein concentration was determined using the BCA assay (Beyotime, Shanghai, China) Samples (30 µg) were loaded for protein electrophoreses. Polyvinylidene Fluoridewere (PVDF) membranes were blocked with 5% bovine serum albumin in tris-buffered saline mixed with 0.1% tween 20. The membranes were then incubated with primary antibody against JAK1 (1:1000), phosphorylated JAK1(1:500), STAT1 (1:1000), phosphorylated STAT1(1:500) CREB (1:1000), phosphorylated CREB (1:500), DUOX2 (1:1000), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:10000) at 4°C for overnight. On the second day, the membranes were further incubated with an HRP-conjugated anti-rabbit IgG secondary antibody (1:2000) for one hour. Target protein signals were visualized with Omni-ECL Femto light chemiluminescence kit (EpiZyme, Shanghai, China) in a fluorescent image detection system (Tanon-52Multi, Tanon, Shanghai, China). The related signals were quantified using ImageJ software and normalized with GAPDH.
Antagomir-146a-5p was bought from Ribo Biotech (Ribo, Guanzhou, China) and delivered by Lipofectamine RNAiMAX (Thermo Fisher, Carlsbad, MA, USA) according to the manufactory's protocol. In brief, 50 nM antagomiR-146a-5p was prepared in a serum-free and antibiotic-free medium. After transfection, TCs were supplied with full growth medium until confluence. For animal experiments, TCs (1x106) were freshly prepared in 100 µl phosphate-buffered saline.
Dual‑luciferase reporter assay
The PHY-881 reporter vector (Hanyin Biotechnology, Shanghai, China) was used to construct the luciferase reporter vector plasmids PHY-881-WT-CREB1-3'-UTR and PHY-881-Mut-CREB1-3'-UTR. The plasmid CREB1-WT or CREB1-Mut were co-transfected with miR-146a-5p mimic or negative control into human embryonic kidney (HEK293T) cells using Lipofectamine 2000 reagent (ThermoFisher, Carlsbad, MA, USA). A dual-luciferase reporter assay system (Promega, Madison, WI, USA) was used to assess luciferase activity after 24 h of transfection. Relative firefly luciferase activity was normalized to renilla luciferase activity.
Malondialdehyde levels in TCs and culture medium were measured according to the manufacturer's instructions. Briefly, culture medium or cell lysates were incubated in MDA working solution at boiling temperature for 15 min. The MDA concentration was measured at absorbance 532 nm and normalized with protein concentration in culture medium or cell lysates, respectively.
Reagents and antibodies
LPS was purchased from Sigma (Sigma-Aldrich, St. Louis, MO). Antibodies against JAK1 (CST-3344), phosphorylated JAK1(CST-74129), STAT1 (CST-14994), phosphorylated STAT1(CST-7649), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, CST-5558) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Antibodies against CREB1 (ab31387) and phosphorylated CREB1 (ab32096) were purchased from Abcam (Abcam, Cambridge, MA, USA). Antibody against DUOX2 (NB110-61576ss) was purchased from NOVUS (NOVUS, Centennial, CO, USA). Specific inhibitors of CREB1, KG-501(HY-103299), and 666-15(HY-101120) were purchased from MCE (MedChem Express, Monmouth Junction, NJ, USA), Lipofectamine RNAiMAX was purchased from Thermo Scientific (Thermo, Rockford, MA, USA). Bicinchoninic acid assay (BCA) kit, RIPA lysis buffer, and loading buffer were purchased from Beyotime Biotechnology (Beyotime Shanghai, China PRC).
Data were presented as mean ± standard error of the deviation. The data were analyzed using the GraphPad Prism software (GraphPad Software, LaJolla, CA). One-way analysis of variance (ANOVA) was used for comparison. P < 0.05 was considered statistically significant.