Inhibition of importin-7 attenuates ventilator-induced lung injury by targeting nuclear translocation of p38

Animal care

Animal handling and care complied with the National Guide for Care and Use of Laboratory Animals and was approved by the ethics committee of Shandong Provincial Third Hospital affiliated to Shandong University. Animals were housed in laminar flow cages in a specific pathogen-free facility and maintained under a 12-h light–dark cycle with free access to standard laboratory chow and water. All operations were performed under anesthesia and every effort was made to minimize suffering.

VILI model

VILI was generated in C57BL/6 mice (8–12 weeks old, 20–30 g body weight) using tidal volume MV as described previously [27]. Briefly, mice received an intraperitoneal injection of an anesthesia mixture containing ketamine (80 mg/kg) and xylazine (10 mg/kg) and were positioned supine on a heating pad. A tracheostomy was performed, after which a sterile 20-gauge catheter was inserted into the trachea and sutured to avoid air leakage. Mice were then connected to a small animal ventilator (Inspira; Harvard Apparatus, Holliston, MA, USA) and ventilated for 4 h in volume control mode with a tidal volume of 15 ml/kg and a respiratory rate of 100 breaths/min. The positive end air pressure was zero and the inspired oxygen fraction was 0.21. The ventilation parameters were chosen based on results from preliminary experiments showing that these conditions lead to VILI in mice with reproducible alterations in lung inflammation. Control animals were anesthetized and underwent tracheostomy but breathed spontaneously. During the experiment, mice were injected intraperitoneally with ketamine and xylazine at 45-min intervals at one-third of the initial dose to maintain anesthesia, and intraperitoneal injection of saline (0.01 ml/g body weight) to maintain intravascular volume status. After the experiment was completed, the mice were euthanized with 150 mg/kg ketamine intraperitoneally. Lung tissue was removed for wet/dry weight ratio assessment, Western blotting, and myeloperoxidase (MPO) activity assay, and bronchoalveolar lavage fluid (BALF) was collected for cytokine analysis. Separate animals were used for histology and immunofluorescence (n = 3).

Evaluation of lung inflammation and histological injury

The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in BALF were determined by ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). As a marker for neutrophil infiltration, MPO activity in lung tissue lysates was determined with a mouse MPO ELISA kit (jiancheng, Nanjing, China) according to the manufacturer’s protocol. Lung wet weight was determined immediately after removal, then the lungs were placed in an oven at 75 °C for 48 h and reweighed. The wet/dry weight ratio was calculated as the ratio of wet weight to dry weight. For histological examination, lung tissue was serially sectioned, embedded in paraffin, and stained with hematoxylin and eosin (H&E).

Primary AECs isolation

Primary type II AECs were isolated from male Sprague–Dawley rats and cultured as previously described [28, 29]. Briefly, rat lungs were isolated in a sterile site. Protease solution (300 U/ml collagenase type I, 4 U/ml elastase, 5 U/ml dispase, and 100 μg/ml DNase I in Hanks’ balanced salt solution) was used for the digestion of the lungs for 25 min at 37 °C. Next, a continuous digestion with 0.1% trypsin–EDTA and 100 μg/ml DNase I was performed for 20 min at 37 °C. Digested lungs were dissociated and made into a single cell suspension. Purity was assessed using flow cytometry. AECs were seeded onto culture dishes at 1 × 106/cm2 and cultured in 5% CO2 and 95% air in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Experiments were conducted the day after isolation.

Cyclic stretch

AECs were seeded on collagen I-coated BioFlex culture plates (Flexercell International, McKeesport, PA, USA) at a density of 1.2 × 105 cells/well. Plates were mounted on a Flexercell Tension Plus system (FX-4000T; Flexercell International, McKeesport, PA, USA). This system provides uniform radial and circumferential cyclic strain on the membrane surface along all radii, mimicking the lung breathing mechanisms [30]. According to preliminary data, cells undergo 15% elongation at 24 cycles per minute for various time points with the stretch/relax ratio of 1:1, which mimics the mechanical stress induced by high tidal volume MV [30, 31]. Control BioFlex plates with static cell culture were placed in the same cell culture incubator. When necessary, cells were preincubated for 1 h before CS with either gossypetin (60 µM; ChromaDex Inc., St. Santa Ana, CA, USA), SB203580 (5 µM; Sigma-Aldrich, St. Louis, MO, USA), nocodazole (1 µM; Calbiochem, San Diego, CA, USA), paclitaxel (100 nM; Sigma-Aldrich, St. Louis, MO, USA), cytochalasin D (1 µM; Sigma-Aldrich, St. Louis, MO, USA), phalloidin (10 µM; Sigma-Aldrich, St. Louis, MO, USA), ciliobrevin D (20 µM; EMD Millipore, Billerica, MA), ML-7 (10 µM; Sigma-Aldrich, St. Louis, MO, USA), or vehicle control DMSO (Sigma-Aldrich, St. Louis, MO, USA).

Western blotting

Cells were washed twice in phosphate-buffered saline (PBS) and lysed with 1 × radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) for 10 min on ice. Lung samples were homogenized in 1 × RIPA lysis buffer. The protein content of cell lysates was determined by bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). For Western blotting, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes followed by immunostaining with primary antibody specific for p38 (1:1000), phosphorylated p38 (p-p38) (1:1000), ATF-2 (1:1000), p-ATF-2 (1:1000), p-MK2 (1:1000), p-Elk1 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and Imp7 (1:1000, Abcam, Cambridge, MA, USA). A horseradish peroxidase-conjugated secondary antibody (1:2000, Pierce, Rockford, IL, USA) was then used in a standard enhanced chemiluminescence reaction according to the manufacturer’s instructions. In some experiments, Western blotting was used to detect the Imp7 proteins extracted from the cytoplasm and nucleus with NE-PER nuclear and cytoplasmic extraction reagents (Pierce Thermo Scientific, Rockford, IL, USA).

Immunofluorescence

Cells were fixed with 2% (w/v) paraformaldehyde, permeabilized with 0.1% Triton X, and blocked with 2% bovine serum albumin (BSA) in PBS. Lung tissue sections were deparaffinized, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA. The samples were then incubated with primary antibody specific for p38 (1:500), p-p38 (1:200), p-MK2 (1:200), and p-Elk1 (1:200) (Cell Signaling Technology, Danvers, MA, USA), Imp7 (1:200, Thermo Fisher Scientific, Rockford, IL, USA), and F-actin (1:500, Invitrogen, San Diego, CA, USA). Samples were washed with PBS containing 0.5% BSA followed by incubation in appropriate Cy3 (1:1000, Invitrogen, Carlsbad, CA, USA) and Cy5 (1:1000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) conjugated secondary antibodies, and then stained with 100 ng/ml DAPI (Sigma-Aldrich, St. Louis, MO, USA) to visualize nuclei. Positively stained cells in 6 random fields were imaged with a confocal microscope (FluoView 1000; Olympus, Melville, NY, USA). For quantification, RGB images were adjusted to the maximum entropy threshold and analyzed using Image J software (NIH Image, Bethesda, MD, USA).

Real-time quantitative PCR

Total RNA was extracted from cell samples using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). For each sample, 1 μg RNA was reverse transcribed using the iScript reverse transcription supermix kit (Bio-Rad, Hercules, CA, USA). PCR amplification mixtures were prepared using iTaqTM Fast SYBR Green Supermix with ROX (Bio-Rad, Hercules, CA, USA) and Real-time PCR was performed with MX3000p (Stratagene, La Jolla, CA, USA). Quantification of TNF-α, IL-1β, and IL-6 gene expression was normalized to endogenous β-actin.

Pull-down assay

CS-stimulated cells and static controls were lysed as described above. The samples (200 μg protein) were then incubated with Sepharose beads-conjugated p-p38 antibody (Cell Signaling Technology, Danvers, MA, USA) in reaction buffer (50 mM Tris–HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40, 2 μg/mL BSA, 0.02 mM PMSF, 1 × protease inhibitor mixture). After overnight incubation at 4 °C with gentle shaking, the beads were washed 5 times with buffer (50 mM Tris–HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40, 0.02 mM PMSF), and proteins bound to the beads were analyzed by Western blotting.

In vitro binding assay

GST and GST-Imp7 were purified with Glutathione Sepharose 4B beads (GE Healthcare, Pittsburg, PA, USA) and His-p38 protein was purified with Ni2+-NTA agarose (Qiagen, Chatsworth, CA, USA) following the manufacturers’ protocols. His-p-p38, for the in vitro Imp7 pull-down assay, was obtained by incubating His-p38 with an ATP regeneration system [Ojala PM, Sodeik B, Ebersold MW, Kutay U, Helenius A. Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro. Mol Cell Biol. 2000;20(13):4922–4931.]. Equal amounts of GST and GST-Imp7 were incubated with His-p38 or His-p-p38 in protein binding buffer (50 mM Tris–HCl pH 8.0, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 5 mM DTT) for 3 h on a rotary shaker at 4 °C. After 3 washes with binding buffer, the beads were boiled in 1 × SDS loading buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 100 mM DTT) and then proceeded to SDS-PAGE.

Co-immunoprecipitation

Cells were washed with ice-cold PBS and then lysed with IP lysis buffer (Pierce, Rockford, IL, USA). Whole cell lysates were collected and centrifuged at 10,000×g for 10 min at 4 °C. The resulting supernatant was quantified using the BCA assay. For co-immunoprecipitation with transfected cells, 500 μg of total protein was used to incubate with anti-FLAG conjugated Sepharose beads (Sigma-Aldrich, St. Louis, MO, USA) for 4 h on a rotating rocker at 4 °C. For endogenous co-immunoprecipitation with anti-Imp7 antibody, 1 mg of total protein was used. Immunocomplexes bound to the Sepharose beads were recovered by brief centrifugation followed by 3 washes with cold PBS. The harvested beads were suspended in 20 μl of 5 × SDS-PAGE sample buffer and boiled for 5 min. A 50-μg cell lysate was used as an input control. Samples were analyzed by Western blotting.

Proximity ligation assay (PLA)

In situ PLA was used to simultaneously detect p38 and Imp7 upon spatial proximity in CS-stimulated AECs. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Nonidet P (NP)-40. PLA was performed using the Duolink PLA kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, fixed cells were incubated overnight with anti-p-p38 and anti-Imp7 antibodies. After multiple washes, cells were sequentially incubated with PLA probe, ligation solution, and amplification solution at 37 °C. The pictures were taken with a confocal microscope (FluoView 1000; Olympus, Melville, NY, USA).

siRNA transfections and rescue experiments

siRNAs targeting Imp7, Imp-α1, Imp-α5, Imp-β1, Imp3, Imp9, p150Glued (the main component of the dynein-dynactin complex), MLCK or a scramble control (control siRNA) were obtained from Dharmacon (Lafayette, CO, USA). siRNAs were transfected at 20 nM with Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA). For rescue experiments, Imp7 siRNA was mixed with HA-Imp7 plasmid and transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). siRNA/plasmid complexes were suspended in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) and incubated with transfection reagent for 15 min at room temperature. The mixture was then incubated with AECs in serum- and antibiotic-free conditions for 12 h at 37 °C. After the cells were washed twice with sterile PBS, they were incubated for an additional 12 h before exposure to CS.

Preparation of Imp7 siRNA-loaded nanoparticles

Imp7 siRNA-encapsulated nanoparticles were prepared as previous reported [32]. Briefly, Imp7 siRNA was dissolved in citrate buffer (10 mM, pH 3.0) and quickly mixed with the lipid mixture by vortexing. The lipid mixture consisted of C12-200, cholesterol, DSPC, and mPEG-DMG dissolved in ethanol at a molar ratio of 50:38.5:10:1.5. Unentrapped siRNA was removed by ultrafiltration centrifugation. Entrapment efficiency was measured by RiboGreen assay (Molecular Probes, Eugene, OR, USA). The siRNA-loaded nanoparticles were diluted in PBS for in vivo therapeutic studies. For the biodistribution study, DiR-labeled Imp7 siRNA nanoparticles were prepared by dissolving DiR at 1% w/w of total lipids in an alcoholic aqueous phase. Mice were anesthetized and injected intratracheally with 500 µL of DiR-labeled nanoparticles in PBS. Whole body images of animals were captured by the IVIS Lumina XR in vivo imaging system (Caliper Life Sciences, Hopkinton, MA, USA) using excitation/emission wavelengths of 740/790 nm. Untreated animals were used as controls.

Statistical analysis

All statistical analyses were performed using SigmaPlot 14.0 (Systat Software, Point Richmond, CA, USA). Data are expressed as mean ± SEM. Comparisons between two groups under the same treatment were performed by Student’s t test. We applied Welch’s test when statistical heterogeneity was evident. Significance was established at P < 0.05. P values were indicated by asterisks as followed: *P < 0.05, **P < 0.01, and ***P < 0.001.

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