Cell culture and treatment
Mouse lung epithelial cells (MLE-12) were purchased from American Type Culture Collection (Manassas, VA, USA). MLE-12 cells were seeded on collagen I-coated flexible-bottom BioFlex plates with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37 °C in a humidified atmosphere containing 5% CO2. Elk1 siRNA (si-Elk1), MMP-9 siRNA (si-MMP-9) and Negative control siRNA (si-Nc) were constructed and synthesized by Gene Pharma Corporation, and transfected into 50% confluent MLE-12 cells with the Lipofectamine 3000 transfection reagent. After transfection for 48h, MLE-12 cells were treated with cyclic stretch. For the cyclic stretch, we used the FX-5000 T Flexercell Tension Plus system, and its parameters (VILI model in vitro) were set as follows: 20% stretch amplitude, a frequency of 30 cycles/min, a stretch-to-relaxation ratio of 1:1 applied in a cyclic manner, time of cyclic stretch for 2h or 4h.
Animal model and experimental protocol
All animal procedures were performed in accordance with the established guidelines, reviewed and approved by the Laboratory Animal Ethics Committee of Qianfoshan Hospital of Shandong University. C57BL/6 mice that weighed 20-25 g (6-8 weeks of age) were purchased from Vital River Laboratory and housed under specific pathogen-free conditions. For si-Elk1 or si-MMP-9 in vivo transfection, mice were injected by caudal vein with si-Elk1 (2.5μg/g) or si-MMP-9 (2.5μg/g), 10% glucose solution, DEPC water, and the EntransterTM In Vivo Transfection reagent (Engreen, 18668-11-1, Beijing, China) at the dosage of 200 µL according to the manufacturer’s instructions. Animals were fasted for 24 h before experiments and given free access to water. After 72 h of transfection, the mice were treated with mechanical ventilation.
The ventilation parameters (VILI model in vivo) were set as follows: tidal volumes of 20mL/kg, a respiratory rate of 40 times/min, PEEP of 0 cm H2O, an inspiratory-to-expiratory (I/E) ratio of 1:2, a fraction of inspired oxygen of 21% and a time of mechanical ventilation of 2h or 4h.
All mice received the same standard diet during the experimental period. The mice were anesthetized via an intraperitoneal injection of pentobarbital and ketamine. Pancuronium was used to maintain muscle relaxation.
Bronchoalveolar lavage fluid (BALF)
After mechanical ventilation, the mice were treated with precooled saline (0.3 mL) into the lungs injected by tracheal intubation, and then pumped back after three seconds. The same operation was repeated three times. The number of cells was measured in the BALF.
Histopathological analysis
After mechanical ventilation, the mice were euthanized, and the lung injury scores were recorded. The upper lobe of the right lung was fixed in 4% paraformaldehyde for 72 h and embedded in paraffin. Tissue blocks were cut into 5-μm slices, stained with hematoxylin for 5 min and eosin for 2 min (HE). The HE staining sections were observed under a light microscope at a magnification of 400×.
Lung wet/dry (W/D) weight ratio
After mechanical ventilation, the left lung was collected after heparin was injected through the postcava vein. The lung was weighed to determine the wet lung weight, then dried at 65 °C for 48 h and weighed again to determine the dry weight. The lung W/D ratio was calculated.
Evans blue dye extravasation
The Evans blue dye was injected by the tail vein 1h before mice were euthanized. The lungs were transferred, weighed, incubated with formamide (500 μL) at 55 °C for 24 h to extract the dye. The formamide/Evans Blue mixture was centrifuged to pellet any remaining tissue fragments. The optical density was determined spectrophotometrically at 620 nm. The results were expressed as nanograms of dye per microgram of wet tissue.
Immunofluorescences
MLE-12 cells and lung tissue sections were permeabilized with immunostaining permeabilization buffer containing saponin for 5 min, then blocked with 5% BSA for 30 min at room temperature, and incubated with anti-E-cadherin (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-occludin (1:100, Abcam, Cambridge, MA, USA) antibodies diluted in 5% BSA overnight at 4 °C. The specimens were incubated with green-fluorescent Alexa Fluor 488 donkey anti-rabbit IgG or red-fluorescent Alexa Fluor 594 rabbit anti-mouse IgG (Invitrogen, Grand Island, NY, USA) at room temperature for 1h after primary antibodies were washed off. The nuclei were stained with 4′,6-diamidino-2-phenyl indole dihydrochloride (DAPI) for 5 min. A high-sensitivity laser confocal microscope was used to observe the changes of E-cadherin and occludin.
Western blotting and immunoprecipitation
After mechanical ventilation or cyclic stretch, the remaining right lung tissue or MLE-12 cells respectively were lysed on ice in a mixture of RIPA and PMSF (Beyotime, China). Equal amounts of protein were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, blocked in 5% nonfat milk for 2h at temperature, and incubated with primary antibodies at 4°C overnight. The primary antibodies were as follows: E-cadhrein (1:500), occludin (1:1000), Elk1 (1:500, Abcam, Cambridge, MA, USA), MMP-9 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (1:1000, Abcam, Cambridge, MA, USA). The membranes were incubated with goat anti-mouse or goat anti-rabbit secondary antibodies for 2 h at room temperature. The ECL SuperSignal reagent (Millipore, Billerica, CA, USA) was used to detect the protein bands by FluorChem E (ProteinSimple, CA, USA). The AlphaView software (ProteinSimple, CA, USA) was used to analyze the relative densities of the proteins. All experiments were performed in triplicates.
After treatment with cyclic stretch for 0h or 4h, MLE-12 cells were lysed in buffer with a protease cocktail for 30 min. The lysate was separated by centrifugation for 30 min. A small amount of supernatant was used for input, and the other part was precleared using isotype control IgG and protein A/G plus-agarose beads (20 μL) for 4 h, and then incubated with anti-Elk1 antibody and protein A/G plus-agarose beads (20 μL) at 4 °C overnight. The immunoprecipitated proteins were dissolved in 2× loading buffer for immunoblot analysis.
Statistical analysis
All data are expressed as mean ± SD. Significant differences were assessed using Student’s t-tests or Tukey and least significance difference (LSD, L) with one-way analysis of variance (ANOVA), when appropriate. Statistical analysis was performed using the SPSS 26.0 statistics package for windows. A P-value <0.05 was considered statistically significant.