Reagents
LPS (Escherichia coli serotype 055:B5), formyl methionyl leucyl phenylalanine (fMLP), interleukin-8 (IL-8), phorbol ester (PMA), cell chromatography C (Cytochrome C), superoxide dismutase (SOD), Elastase, Hydroxyethylpiperazine Ethylsulfonic Acid (HEPES) and Emo were obtained from Sigma-Aldrich (St Louis, MO, USA). Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) ELISA kits were obtained from R&D Systems (Minneapolis, MN). Anti-Myeloperoxidase (MPO) antibody (abcam, ab65871), MNase, RP-1 antibody (BD 550002), SYTOX Green, and Annexin V-FITC were obtained from eBioscience (San Diego, CA). pHrodo red E. coli (Cat.No.4615), pHrodo green S.aureus (Cat. No. 4620) were obtained from Sartorius (Göttingen, Germany). RPMI 1640, fetal bovine serum (FBS), trypsin, and enzyme-free cell dissociation buffer were purchased from Gibco (Grand Island, NY, USA). Penicillin and streptomycin in saline citrate buffer were from Invitrogen (Carlsbad, CA, USA). Other chemical reagents are of analytical grade.
Animals, experimental procedure, and treatments
Experiments were performed on adult male Sprague Dawley rats (250–300 g; Shanghai Experimental Animal Center of China). Rats were provided with water and food ad libitum. The use of animals in this study was approved by Animal Studies Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University.
Rats were randomized into five groups (n = 6): control group, LPS group, LPS + Emo 5mg/kg group, LPS+Emo10 mg/kg group and LPS + Emo 20mg/kg group. In LPS group, rats received LPS (20mg/kg) through the tail vein. LPS was dissolved in 0.9% normal saline. In the Emo group, rats received Emo (5mg/kg, 10mg/kg and 20mg/kg) via intraperitoneal injection 30 min before LPS exposure. Emo was dissolved in 100% DMSO at a concentration of 200mg/mL and diluted in saline to the final concentration of 1 mg/ml. The action time of LPS-induced acute lung injury in rats was 4 hours.
Pathological studies
Rats were anesthetized with chloral hydrate (7 ml/kg, intraperitoneally), intubated and connected to the animal ventilator (respiratory parameters are tidal volume: 10ml / kg and respiratory rate: 40-60bpm) 4 hours after injection of LPS. After anesthesia and mechanical ventilation with pure oxygen for 1 hour, rats were killed by cutting off the abdominal aorta and bloodletting. Rats were subjected to thoracotomy and PBS (25ml/min) was injected into the right ventricle to flush the pulmonary vessels. Finally, the right lower lung lobe of rats was cut and fixed in 4% paraformaldehyde for 24 hours at room temperature, and 4μ m sections were embedded in paraffin and stained with hematoxylin and eosin (H&E) for light microscopy analysis. The rest of the lung tissue was frozen in liquid nitrogen for 48 hours and stored in refrigerator at-80 ℃.
A semi-quantitative scoring system was adopted to evaluate lung injury, which included alveolar congestion, alveolar hemorrhaging, neutrophil infiltration or aggregation in the airspace or vessel wall, and alveolar wall/hyaline membrane thickness and inflammatory cell infiltration. The grading scale for the light microscopy pathologic findings was as follows: 0 = no injury; 1 = slight injury (25%); 2 = moderate injury (50%); 3 = severe injury (75%); and 4 = very severe injury (almost 100%). The results were graded from 0 to 4 for each item, as described previously. The four variables were summed to represent the lung injury score (total score: 0–16).
Determination of inflammatory cytokines in lung homogenate by enzyme-linked immunosorbent assay (ELISA)
Part of the right lung from individual rats was homogenized and centrifuged, and the levels of TNF-α and IL-1β in the resulting tissue supernatants were determined using TNF-α and IL-1β ELISA kits.
After ultrasonic lysis of lung homogenate, the supernatant was obtained by centrifugation at 4 ℃ for 5000 r/min for 15 minutes. Follow the reagent instructions. 100 μ l of standard or sample to be tested was added to each hole, and the reaction plate was fully mixed and placed at 37 ℃ for 30 minutes. Wash the reaction plate fully with washing solution for 4 times and print it on the filter paper for 6 times. 100 μ l of enzyme-labeled antibody working solution was added to each well. Put the reaction plate at 37 ℃ for 30 minutes. The washing board is the same as before. 100 μ l of substrate working solution was added to each hole and reacted in the dark at 37 ℃ for 15 minutes. Add 100 μ l terminating liquid to each hole and mix well. The absorbance value of 450nm was measured by enzyme labeling instrument within 30 minutes.
Separation and of rat neutrophil
20ml of heparinized fresh rat blood was treated with dextran to induce sedimentation of the red blood cells. Prepare a Percol gradient in a 15ml Falcon Tube by first pipetting 5ml 56% Percol, then put the sucker to the bottom of the tube, and slowly pipetting 2.5ml 80% Percol to the bottom. Then carefully draw up the plasma and white blood cell suspension from the blood sample with a pipette and slowly layer them on top of the Percol gradient.4 ℃, 220g, centrifuge for 20 minutes, accelerate to 1, decelerate to 0, remove the top layer of serum, suck out the neutrophil layer, add PBS to wash twice, the cells were resuscitated with RPMI-1640 medium containing 5% FBS and then counted so that the cell concentration was 1 × 106/ml.
Neutrophils were divided into five groups: control group, LPS group(100ng/ml), LPS+ Emo 5μM group, LPS+ Emo 10μM group and LPS+ Emo 20μM group. Stimulation with Emo was performed for 30min prior to LPS treatment.
Immunofluorescence staining
Neutrophils were isolated and adjusted concentration to 5×105/ml. They were centrifuged for 5 minutes in order to fix the cells on glass slides and dried. Neutrophils were fixed with 4% paraformaldehyde at room temperature for 15 minutes. Then they were permeabilize with 0.5%TritonX-100PBS for 20 minutes and blocked with blocking solution buffer for 30 minutes. Neutrophils were incubated overnight at 4 ℃ with primary antibody (MPO antibody 1: 500), and then incubated the secondary antibody (1:200) onto glass slides for 1 h at room temperature in moist environmental box. DAPI was diluted into PBS in accordance with the instructions and added onto glass slides for 5 minutes at room temperature. 20μL of mounting medium was promptly added onto the slide, then put a cover slip on it, followed by mounting the coverslip with nail enamel. Finally, the slides were observed under a fluorescence microscope.
Cell counting kit-8 (CCK8 assay)
Neutrophils were isolated and adjusted concentration to 1×106/ml. Neutrophils were added to 96-well plate (100 μ l per hole). Three multiple holes and blank control holes were set up at the same time (no cells). Emo was added into test wells as ainhibitor , which was divided into five concentration gradients of 5 μ M, 10 μ M, 20 μ M, 40 μ M and 80 μ M. PBS was used as negative control. After 4 hours of Emo intervention, 10 μ l of CCK-8 reagent was added into each well. The plate was cultured in 5%CO2 incubator at 37 ℃ for 3 hours, the OD value of each well of wavelength 450nm was detected by enzyme labeling instrument. Cell inhibition rate (IC)can be calculated according to the formula:
Cell inhibition rate (IC) = [(control group OD value-experimental group OD value) / (control group OD value-zeroing group OD value) ×] 100%
Respiratory burst detection
The reactive oxygen species released by the activated inflammatory cells can reduce the membrane non-penetrating cytochrome C. The reduced cytochrome C has an absorption peak at 550 nm. Therefore, the amount of reduced cytochrome C is measured using a spectrophotometer. The amount of active oxygen produced can be inferred from this data. ①Set control group, LPS group and 3 Emo groups, and the appropriate amount of Emo was added in each group, 100 μ l of cytochrome C (1.5 mg/ml) and 100 μl neutrophils (2×107/ml) was then added; ②10μl SOD (5000 U/ml) was added, and the corresponding dose was added to the test group, equilibrated in a 5% CO2 incubator at 37 °C for 10min; ③10 μ l cytochalasin B (1mmol/L) was added to each group and after 3min, 10 μ l fMLP (0.1mmol/L) was added for a total of 1 ml and each group was incubated in a 5% CO2 incubator at 37 °C for 30min; ④ Each group was removed and centrifuged at 2000r/min for 10 min;⑤ Supernatant was collected and the OD value was measured with a spectrophotometer. Since the production of O2- and the decrease in cytochrome C is in a 1:1 mole stoichiometric relationship, the yield of O2- is easily calculated. The millimolar extinction coefficient of the 1 cm optical path is 21.1, and the amount of O2- produced by 2 × 106 / ml of cells in 1 ml of the solution with a diameter of 1 cm can be directly calculated according to the formula:
OD×47.4=nmol O2-/2×106cells/time unit test group O2- inhibition rate = (control O2- content-test group O2- content)/control group O2- content×100%.
Elastase release assay
The detection of neutrophil elastase release was mainly carried out by Neutrophil Elastase Activity Assay kit (ab204730). Five elastase solution test groups were prepared: Control group, LPS group, and Emo group (reaction solution concentration: 5μ M, 10μ M, 20μ M). ①The isolated neutrophils were rinsed twice with PBS (pH 7.4), the number of cells was adjusted to 1×107/ml, 500μl of solution was added to each group, and the corresponding drugs were added. Finally, each group was supplemented with PBS to 600μl. The groups were pre-incubated for 30 min at 37 °C in a 5% CO 2 incubator. In addition to Control group, 6μl of cytochalasin B (1 mmol/L) and LPS (100ng/ml) was added to each group and cultured at 37 °C in a 5% CO 2 incubator for 20 min. The tubes were then placed in an ice-water bath to terminate the reaction. They were centrifuged at 1500 r/min for 5 min, and the supernatant was dispensed and stored at -80 °C until use. ②Elastase determination. Elastase standard was diluted with PBS: 50, 37.5, 25, 18.75, 12.5, 9.38, 6.25, 4.69, 3.125 series concentrations (μ g /ml) and a PBS blank were used to generate a standard curve. Using a 96-well microtiter plate containing a standard or a 50 μ l sample to be tested, 100 μ l buffer was added (containing elastase substrate 1 mmol/L, HEPES 0.1 mol/L, NaCl 0.5 mol/L, pH 7.5). The OD value was read at 405 nm by a microplate reader (the emodin absorption is at 405 nm), and then cultured at 37 °C in a 5% CO 2 incubator for 60 min. The OD value at 405 nm was then read again, and the difference between the two OD values was recorded. The OD value of the substrate decomposes, and the elastase content is calculated according to the standard curve.
Measuring neutrophil NETs production
Clear 96-well flat-bottomed plates were prepared, and 100 µl of neutrophils were added to the relevant wells. Lipopolysaccharide (LPS, 100 ng/ml), interleukin-8 (IL-8, 100 ng/ml), phorbol ester (PMA, 1.5 ng/ml) and N-formylthionyl-leucyl-phenylalanine (fMLP, 1000 ng/ml) were used to treat the cells respectively. The control group was treated with an equal volume of medium. They were incubated for 3 h at 37 °C in a 5% CO2 incubator. SYTOX Green was diluted 1:500 (5mM Stock; 1ul SYTOX Green into 499ul PBS), and then stored in the dark. 20 µl of diluted SYTOX green was added to each well using a fresh tip for each well. 1 µl of MNase was added to each well using a fresh tip for each well. They were then incubated at room temp for 10 min in the dark. Samples were transferred to 0.5 ml micro-centrifuge tubes without any pipetting of the liquid up and down. They were immediately centrifuged at 5000 rpm for 10 min in the micro-centrifuge before 160 µl of the supernatant was removed and transferred to a black 96-well flat-bottomed plate. Fluorescence was measured immediately (programme: Gen5; excitation 485nm, emission 528nm with optics position in top 50% of well with a 10-second ‘medium’ shake immediately prior to read).
Measuring ROS production by isolated neutrophils
Following isolation, cells were resuspended at 1x106/ml in HBSS (with Ca2+ and Mg2+) (4.5 ml total) in 15 ml Falcon. 100 μl of neutrophils were added to each well of a 96-well plate. Cells were stimulated with Luminol(0.5mM),IL-8(1.25nM), fMLP(2.5μM), and PMA(25nM) for 1 hour. The luminometer was set up and the ROS level was tested on the instrument.
Measuring the phagocytosis of neutrophils
Neutrophils were isolated and adjusted concentration to 1×106/ml. Following LPS and Emo treatment, neutrophils were inoculated into 96-well plates at 100 µl/well. pHrodo red E. coli and pHrodo green S. aureus were added to neutrophils respectively to stimulate neutrophils for 30 min, 45 min, and 60 min. Neutrophils were incubated at 37℃ in a 5% CO2 incubator in the dark, and then centrifuged at 250g and 4℃ for 5 min to remove the supernatant. The cells were resuspended with 100ul of 2% PBS/BSA, and this was repeated twice before the cell suspension from each well was transferred into flow tubes. 100μl of 2% PBS/BSA was added to each tube, gently mixed and placed on ice. Finally, the phagocytosis of neutrophils was measured using flow cytometry.
Measuring the rate of apoptotic neutrophils
Neutrophils were isolated and inoculated into six-well plates at an adjusted concentration of 1 ×106/ml. The groups were divided into groups and treated for 4 h and 24 h. Cells were harvested as normal and cells were transferred to the appropriate FACS tubes. They were centrifuged at 600 g for 4 min before the supernatant was poured off. Cells were resuspended in 200 µl Annexin V buffer to wash the cells and then pelleted again. The cells were incubated in 100 µl Annexin V-FITC diluted 1:100 in Annexin V buffer for 15-20 min on ice and protected from the light. 200 µl Annexin V buffer was added to each tube. SYTOX was removed from the freezer and defrosted while being protected from the light. A SYTOX stock diluted 1:500 in Annexin V buffer was prepared. Immediately prior to running the sample on the CyAN, 30 µl of the SYTOX solution was added to each tube and they were vortexed well to mix. The FITC and Violet 1 channels on the FACS machine were used to measure.
To establish a rat model of acute lung injury with neutrophil deficiency
Cyclophosphamide (CTX) is a kind of non-specific chemotherapeutic drug in cell cycle, which is widely used in clinic. It can kill the cells in each phase of the proliferation cycle and inhibit the number of leukocytes in bone marrow.
In this experiment, rats were injected intraperitoneally with cyclophosphamide (75 mg /kg )4 days before and 1 day before the acute lung injury induced by LPS. And one day before the acute lung injury model was prepared and one day after the model was prepared, the number of neutrophils in rat tail vein blood was less than 2×105/ml by using the blood cell count version technology, Therefore, the rat model of acute lung injury with neutrophil deficiency was successfully prepared.
Statistical analysis
The data represent the mean ± SD. There were no missing, lost, or excluded data. Based on previous experience, no prior power analysis was conducted; all data were analyzed by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. All tests were two-sided, and significance was determined at the p < 0.05 level. Statistical analyses were performed using Prism 6.0 software (GraphPad Software, San Diego, CA).