Drugs and Reagents
LPS (Escherichia coli 055:B5), Dexmedetomidine, Evans blue, CCK8 was obtained from Sigma (St. Louis, MO, USA). LY294002 was obtained from MCE (Monmouth Junction, NJ,USA).Cytokines (TNF-α, IL-1β, IL-6, IL-10) and myeloperoxidase (MPO) enzyme-linked immunosorbent assay (ELISA) detection kits were obtained from Jianglai Biotechnology (Shanghai, China). A549 cells were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China). LDH assay kit, anti- αENaC antibody, anti-β ENaC antibody, anti-γ ENaC, HRP labelled goat anti-rabbit IgG antibody were purchased from Abcam (Cambridge, UK). Anti-p‐Akt antibody , anti-Akt antibody , Anti-Nedd4-2, and anti-β-actin antibody were purchased from CST (Boston, MA, USA).
Animals
The Medical Faculty Ethics Committee of Shenzhen People's Hospital , Shenzhen, China, approved all the animal procedures and care. The animal experiments complied with the Guidelines for the Care and Use of Laboratory Animals from the NIH. SPF grade male Wistar rats (6 weeks old, weighing 180 ~ 220 g) were purchased from Guangdong Medical Animal Experiment Center (Guangzhou, China). Animal handling was conducted according to the requirements of the animal protection committee of the Second Clinical Medical College Of Jinan University. All animals were housed in an air-conditioned room under a 12-hours dark/light cycle and were granted free access to water and food.
Experimental protocols
The rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p. injection). The skin of the left thigh was disinfected with ethanol, the skin was cut open, the femoral vein was exposed, a 24# trocar was inserted and fixed, and then LPS (20mg/kg) was injected ( approximately 10 minutes) to induce ALI. After LPS or saline injection, the neck skin was disinfected, the trachea was exposed and a homemade tracheal catheter was inserted. At 8 h after LPS administration, all the rats were sacrifificed. The study consisted of two parts. The fifirst part was the survival study, for which 10 animals were used in each group.The survival rate of rat was recorded every 2 h for 3 days after LPS administration, and determine the dosage of Dex.
In the second part, the rats were divided into four groups (n=6 per group): the control group, LPS group, LPS+Dex group, and LPS+Dex+ LY294002 group. The rats in the control group were intravenously administered 0.9% normal saline (5 ml/kg). The rats in the LPS group were intravenously administered 20 mg/kg LPS. The rats in the LPS+Dex group were intravenously administered 20 mg/kg LPS and then intraperitoneally administered 100 μg/kg Dex. The rats in the LPS+Dex+LY294002 group were intravenously administered 3mg/kg LY294002 30 min prior to LPS injection and then administered 100 μg/kg Dex.
Histopathological studies
The lower lobe of the right lung was taken and fixed with 10% neutral formaldehyde for 24h, embedded in paraffin and stained with H&E for light microscopy analysis. Histological lung injury was scored based on alveolar edema, pulmonary capillary congestion, neutrophil infiltration, and the thickness of the alveolar septum in five random fields in a blind manner using light microscopy. Lung sections were scored as 1 (no or very slight pathological changes), 2 (slight pathological changes), 3 (moderate pathological changes), or 4 (severe pathological changes). Evaluation scores were added to the total injury score.
Measurement of cytokines
After the rats were sacrificed, the main bronchus were exposed. The right bronchus was ligated, and a homemade tracheal catheter was inserted into the main bronchus. Then, 2 mL cold phosphate‐buffered saline (PBS) was infused into the left lung and extracted three times. The bronchoalveolar lavage fluid ( BALF) was centrifuged at 1200×g for 10 min at 4°C. The supernatant was separated into aliquots and stored at -70°C. An aliquot of BALF supernatant was used to assay the levels of TNF-α, IL-1β, IL-6, IL-10 by ELISA according to the manufacturer’s instructions.
MPO activity assay in lung tissues
The middle lobe of the right lung was removed immediately after the animals were exsanguinated. Then, the activity of MPO in the lung tissue was determined by ELISA according to the manufacturer’s instructions.
Arterial oxygen tension (PaO2) assay
Arterial blood (0.5 ml) was extracted from the right common carotid artery before the rat was sacrificed for blood gas analysis, and the PaO2 was measured with a blood gas analyzer.
Measurement of lung wet/dry(W/D) weight ratio
At the end of the experiments, we immediately removed the upper lobe of the right lung, and precisely measured the lung wet weight. After that, we placed the lung tissues in a 75°C constant temperature oven for 24 h, and measured the lung dry weight. Finally, the W/D ratio was calculated to evaluate the extent of pulmonary edema.
Measurement of alveolar fluid clearance (AFC)
The AFC was determined by Evans blue- tagged albumin concentration .The 5% bovine serum albumin perfusion solution labeled with Evans blue was injected (5ml/kg) into the left lung via the trachea, and 2 ml oxygen was injected to facilitate distribution. Rats were ventilated with 100% oxygen, positive end expiratory pressure was kept at 2~3 cm H2O during the baseline period to maintain lung tension. These tissue units were wrapped with plastic wrap and then incubated in a 37°C water bath for 1 hour. The alveolar fluid was immediately aspirated, and labeled albumin was measured by a spectrophotometer at a 620nm. AFC was calculated based on the following formula: AFC (%) = [(Cf -Ci) / Cf]×100%, where Ci represents the concentration of injected Evans blue-labeled 5% albumin and Cf represents the final concentration of Evans blue-labeled 5% albumin.
Cells culture and treatment
A549 cells were seeded in culture dishes at a density of 1× 106 cells/cm2 and cultured in a 5% CO2 and 95% air atmosphere in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum, 0.1 mg/ml streptomycin, and 100 U/ml penicillin. The culture medium was changed every two days. For all experiments, cells were subcultured in six-well plates. Once the cells reached 80% confluence, they were serum-starved for 24 h. Following starvation, the cells were treated as follows: (1) the cells in the control group were treated with PBS for 12 hours; (2) the cells in the LPS group were treated with LPS (1 μg/ml) for 12 hours; (3) the cells in the LPS+Dex group were treated with LPS(1 μg/ml) and the Dex (10 μΜ) for 12 hours; (4) the cells in the LPS+Dex+ LY294002 group were treated with LY294002 (10 μΜ) 30 min prior to LPS (10 μΜ) administration and were then treated with Dex (10 μΜ) for 12 hours .
Cell viability assay
CCK8 assay was performed to measure cell viability.The cells (100µl/well) were cultured in a 96-well plate for 24 hours. Then the cells were incubated with 1 µg/ml LPS for 12 h in the absence or presence of Dex, following the 10µl CCK8 solution was added to each well, the culture plate was incubated in the incubator for 2 hours under 5% CO2 at 37˚C, and the absorbance at 490nm was measured with a microplate reader.
LDH activity assay
The cells(100 µl/ well) were cultured in a 96-well plate for 24 hours. Then the cells were incubated with 1 µg/ml LPS for 12 h in the absence or presence of Dex,The supernatant was collected to measure lactate dehydrogenase (LDH) activity by using LDH Assay kit ,according to the manufacturer's instructions. The absorbance at 490nm was measured with a microplate reader
Immunohistochemistry analyses
Lung tissues were fixed in 10℅ neutral formaldehyde solution, and paraffin tissue sections were produced. Paraffin sections were then baked overnight in a 60°C oven, dewaxed with dimethyl benzene, dehydrated with in gradient ethanol solutions, repaired with 500 ml EDTA antigen repair solution, treated with a drop of 50 μl 3% hydrogen peroxide solution at room temperature for 20 min to block the activity of endogenous peroxidase and rinsed with TBS 3 times (3 min each time). Then, 5% normal goat serum solution was added at room temperature for 20 min, and the superfluous liquid was discarded without washing. Diluted primary antibody was added, and the tissues were incubated at room temperature for 30 min and washed with TBS 3 times (3 min each time). Secondary antibody (biotinylated goat anti-rabbit IgG) was added, and the tissues were incubated at room temperature for 30 min and washed with TBS 3 times (3 min each time). After that, the cells were stained with 3,3́-diaminobenzidine for 3~5 min. PBS was used instead of primary antibody for the negative control group. The average optical densities (AODs) of α-, β-, γ-ENaC were measured by an imaging analysis system .
Western blotting analysis in rat lung tissues and A549cells
Proteins were obtained with RIPA lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, and leupeptin) and PMSF. The protein concentrations of the supernatants were determined by using a BCA protein assay kit . The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane. Tris buffer solution containing 5% skim milk powder was used to seal the membrane for 1 h at room temperature. The membrane was blocked with PBS containing 0.05% Tween 20 and washed with PBST 5 times (5 min each time). Then, the membrane was incubated overnight at 4°C with the following primary antibodies: α-, β-, and γ-ENaC, Akt, p‐Akt, Need4-2 and β-actin.The membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti- rabbit antibody at room temperature for 3h, and the membrane was washed with PBST 5 times(5min each time). Finally, the bands were visualized using an enhanced chemiluminescence kit (ECL) by UVP gel imaging system. The band intensities were analyzed by Image J Software .
Statistical analysis
All data are expressed as the mean ± standard deviation (SD). The significance of the differe- nces among the four groups was tested using one-way ANOVA followed by a least significant difference (LSD) multiple comparison test. Survival analysis was done using the Kaplan-Mei- er method, and comparisons between groups were made using the log rank test. A two-side p value less than 0.05 was considered statistically significant.