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 (superoxide dismutase, SOD), Elastase, Hydroxyethylpiperazine Ethylsulfonic Acid (HEPES) and Emodin were obtained from Sigma-Aldrich (St Louis, MO, USA). Tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) ELISA kits were obtained from R&D Systems (Minneapolis, MN). 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
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 the Animal Studies Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University.
Rats were randomized into five groups (n = 10): control group, LPS group, LPS+Emo 5 mg/kg group, LPS+Emo 10 mg/kg group and LPS+Emo 20 mg/kg group. The LPS-induced ALI model was produced by injecting 20 mg/kg LPS via the caudal vein. In the Emo group, rats received Emo (5 mg/kg, 10 mg/kg and 20 mg/kg) via intraperitoneal injection 30 min before LPS exposure. Emo was dissolved in 100% DMSO at a concentration of 200 mg/mL and diluted in saline to the final concentration of 1 mg/mL. Animals were anesthetized with an intraperitoneal (IP) injection of 5% chloral hydrate (7 ml/kg), after which a tracheostomy tube was placed. Rats were sacrificed after 60 min of mechanical ventilation and lungs were harvested for further analyses.
Pathological studies
The right lower lung lobes were harvested, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and stained with hematoxylin and eosin (H&E) for light microscopy analysis. 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[24-25]. The four variables were summed to represent the lung injury score (total score: 0–16). Part of the right lung from individual rats was homogenized and centrifuged, and the levels of TNF-α and IL-1 in the resulting tissue supernatants were determined using TNF-α and IL-1 ELISA kits.
Separation and of rat neutrophils
20ml of heparinized fresh rat blood was treated with dextran to induce sedimentation of the red blood cells. Lymphocyte separation solution was used to remove lymphocytes and monocytes and distilled water was used to solubilize residual red blood cells. A two-step Histopaque gradient technique was then used to obtain high purity neutrophils. The purity of the rat neutrophils was further evaluated using flowcytometry (Attune, ABI) after staining with RP-1 antibody (BD 550002).
Neutrophils were divided into five groups: control group, LPS group(100ng/ml), LPS+Emo 5μM group, LPS+Emo 10 μM group and LPS+Emo 20 μM group. Stimulation with Emo was performed for 30min prior to LPS treatment.
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. The determination of neutrophil respiratory burst was slightly modified by reference[26]. ① Set blank control group C1, control group C2 and 5 test groups, and the appropriate amount of KRB was added in each group, 100 μl 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; ③ 10 μl cytochalasin B (1mmol/L) was added to each group and after 3min, 10 μl 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 are 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). 5 elastase solution test groups were prepared: No fMLP control group C1, fMLP control group C2; rolipram group(reaction solution concentration: 1.38 μg/ml), Shuanghuanglian powder injection group (reaction solution concentration: 50 mg crude drug/ml), and Emo group (reaction solution concentration: 10, 30, 50 mg crude drug/ml). ① The isolated neutrophils were rinsed twice with PBS (pH 7.4), the number of cells was adjusted to 1×107/ml, 500 μl of solution was added to each group, and the corresponding drugs were added. Finally, each group was supplemented with PBS to 600 μl. The groups were pre-incubated for 30 min at 37 °C in a 5% CO 2 incubator. In addition to the control group C1, 6 μl of cytochalasin B (1 mmol/L) and fMLP (0.1 mmol/L) 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. ② Elastase 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 (μg/ml) and a PBS blank were used to generate a standard curve. Using a 96-well microtiter plate containing a standard or a 50 μl sample to be tested, 100 μl 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. Test group inhibition rate of elastase release = (control group C2 elastase content - test group elastase content) / control group C2 elastase content × 100%
Measuring Neutrophil NETs Production
Clear 96-well flat-bottomed plates were prepared, and 100 µl of cells 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 IL-8, fMLP, and PMA. The concentration of IL-8, fMLP, and PMA was as shown in the following table. The Luminometer was set up and the ROS level was tested on the instrument.
|
Dilution
|
Concentrations
|
|
|
Factor
|
Volumes
|
Stock
|
Working
|
Final (in cells)
|
Luminol
|
|
1:10
|
1 ml into 9 ml; pH to 7.3
|
30 mM
|
3 mM
|
0.5 mM
|
IL8
|
|
1:625
|
1 μl into 624 μl
|
6.25 μM
|
10 nM
|
1.25 nM
|
fMLP
|
|
1:500
|
12 μl into 5922 μl
|
10 mM
|
20 μM
|
2.5 μM
|
PMA
|
A
|
1:800
|
1 μl into 799 μl
|
1620 μM (1 mg/ml)
|
200 nM
|
25 nM
|
|
B
|
1:10
|
55 μl into 495 μl
|
|
Measuring the phagocytosis of Neutrophils
Neutrophils were isolated and the cell concentration was adjusted 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 250 g and 4℃ for 5 min to remove the supernatant. The cells were resuspended with 100 ul of 2% PBS/BSA, and this was repeated twice before the cell suspension from each well was transferred into flow tubes. 100 μl 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.
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).