2.1 Animals Studies
PLD2−/− mice on a C57BL/6 background were purchased from Gempharmatech Co., Ltd. (Jiangsu, China), and Male C57BL/6 mice (WT) (18–23 g; 8–10 weeks old) were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (Shangdong, China) and housed in a room maintained at 22–25°C and 40–50% humidity. Food and water were available ad libitum. All animal experiments and feeding methods complied with the guidelines for the Care and Use of Laboratory Animals established by the US National Institutes of Health and were approved by the Binzhou Medical University Institutional Review Board.
Experimental mice were randomly allocated into four groups: Wild Type mice normal group (WT), Wild Type mice model group (WT + LPS), PLD2 gene knockout mice normal group (PLD2−/−), PLD2 knockout mice model group (PLD2−/− + LPS). To establish an ALI animal model, WT + LPS and PLD2−/− + LPS mice were intraperitoneally injected 20 mg/kg LPS (L2880, Escherichia coli O55:B5, Sigma-Aldrich St. Louis, MO, USA). WT and PLD2−/− mice were intraperitoneally injected with the same amount of sterile normal saline. After LPS was administered in mice for 6h, all mice were intraperitoneally injected with 4% chloral hydrate. Lung tissues of mice were obtained after full anesthesia.
2.2 Histopathology and Lung Injury Score
Mouse lung tissues were collected and fixed with 4% paraformaldehyde for 48 h. The tissues were processed, embedded in paraffin, sectioned into 5-µm-thick slices, stained with hematoxylin and eosin (H&E) (Novland, Shanghai, China), and visualized under an optical microscope (Olympus Optical, Tokyo, Japan) for histological analysis. For each sample, six high-magnification fields were randomly scored for each feature, and the obtained mean was the pathological score of each sample following the lung injury score criteria .
2.3 Wet/dry (W/D) lung weight ratio
The lung was excised, blotted dry, weighed to obtain the wet weight, and then placed in an oven at 60◦C for 48 h to obtain the dry weight. The W/D ratio was used to evaluate the degree of pulmonary edema.
2.4 Cell culture
HUVECs were purchased from the Shanghai cell bank and cultured in a 5% CO2 incubator at 37ºC in a complete culture medium (AllCells, Shanghai, China). Cells were cultured to 85–90% confluency and passaged in 0.25% trypsin (containing 0.02% EDTA; Procell, Wuhan, China). The cells were also separated into four groups: control, LPS (10µg/ml), LPS + CAY10594 (PLD2 inhibitor) (10µmol/L; 1130067-34-3, Cayman chemical, Michigan, USA), and CAY10594 group. In the experiment of adding exogenous PA, HUVECs were divided into four groups: control, PA (50µmol/L), PA + STAT3-IN-1 (STAT3 inhibitor) (5uM; Cat. No.Hy-100753, MedChemExpress, Shanghai, China), and STAT3-IN-1 group. The LPS, CAY10594 and STAT3-IN-1 group concentrations used were based on the results of preliminary experiments.
2.5 Assessment of HUVECs permeability
HUVECs were seeded at 15,000 cells per insert well and cultured for 1 to 3 d to allow for growth of a confluent monolayer. Cells were pretreated with CAY10594 (10µmol/L) for 1 h, followed by LPS treatment (10µg/ml) for 6 h. After treatment, Fluorescein isothiocyanate-dextran (FITC-dextran) (40842-46-8, Sigma-Aldrich St. Louis, MO, USA) with a final concentration of 2mg/ ml was added to the upper layer of the transwell chamber (3470, 6.5 mm Transwell® with 0.4 um Pore Polyester Membrane Insert, Costar, Massachusetts, USA ) and incubated at 37℃ for 30min. 100 µl of medium was withdrawn from the lower well and upper well, respectively. Measurements were taken with a microplate reader using excitation and emission wavelengths of 490 and 525 nm, respectively. The permeability coefficient of endothelial cells was =[A]/ T × L /A× V /[L]. Where [A] is the fluorescence protein concentration in the lower chamber, t is the time, expressed in seconds, A is the filtration area, expressed in cm2; V is the volume of liquid in the lower chamber; [L] is the concentration of fluorescent protein in the upper compartment .
2.6 Transendothelial Electrical Resistance
Briefly, Before the experiment begins, 300 µL of culture medium was added to the upper of transwell inserts, and 1ml of culture medium was added to the lower of transwell inserts. Two Millicell® ERS-2 (MERS00002, USA) resistance meter electrodes were placed on the surface and under the filter membrane. The basic resistance values of each compartment (blank resistance) were measured and HUVECs were seeded into each compartment at a density of 15,000 cells per well. After the cells fuse, cells were pretreated with CAY10594 (10µmol/l) for 1 h, followed by LPS treatment (10µg/ml) for 6 h. The resistance values of each compartment were then measured: (Ω/㎝2) = (measured resistance - blank resistance) × effective membrane area [27, 28].
2.7 The content of PA in cell supernatant
After conducting different treatments and pretreatments of cells, a cell culture medium was collected, and the supernatant of each sample was collected after 3000 rpm under 4°C for 20 min. Thus, PA levels in the supernatant were evaluated with the ELISA kits (ml675014, Shanghai Enzymelinked Biotechnology Co., Ltd, Shanghai, China ) by the manufacturer’s instructions.
2.8 Western Blot Analysis
After treatment, HUVECs and lung tissue proteins were extracted according to the requirements of the protein extraction kit (Cat#R0020 and Cat.No.P0100, Solarbio, Beijing, China), and protein concentrations in the supernatant were determined using a BCA Protein Assay kit (Cat.No.P0010S, Beyotime Biotechnology, Beijing, China). According to the different molecular weights of the target protein, the appropriate gel was prepared. Proteins were separated by SDS-PAGE (Cat.No.P0012A, Beyotime Biotechnology, Beijing, China) and transferred to polyvinylidene fluoride (PVDF) (Cat. No.ISEQ00010, Merck Millipore, USA) membranes which were subsequently blocked with 5–7% skimmed milk for 2 h. Then the Membranes were incubated with primary antibodies against occludin (1:1000; 33-1500, Invitrogen, Carlsbad, CA, USA), ZO-1 (1:1000; ab276131, Abcam, Cambridge, UK), P-STAT3 (1:1000; ab32143, Abcam, Cambridge, UK), STAT3 (1:1500; ab68153, Abcam, Cambridge, UK), and β-actin (1:1000; 20536-1-AP, Proteintech, USA) overnight at 4℃, followed by incubation with horseradish peroxidase-conjugated antibodies (1:2000; Cat. No.SA00001-1 and Cat. No.SA00001-2, Proteintech, Chicago, USA) for 60 minutes at room temperature and protein bands were visualized by an electrochemiluminescence kit (MAO0186-1, Meilunbio, Dalian, China). Densitometry analysis was performed in the Image-J software.
2.9 Quantitative real-time PCR
Total RNA was isolated from HUVECs using Trizol Reagent (Ambion, Thermo, MA, USA) according to the manufacturer's protocol and the concentration and purity were detected on a nucleic acid analyzer. Total RNA was reverse transcribed and amplified by PCR using the Takara PrimeScript RT Reagent Kit ( RR037A, Takara, Shiga, Japan ). PCR reactions consisted of 12µL of PCR mix, 2µL cDNA, 1µL upstream primer, 1µL downstream primer, and qs to a final volume of 20 µL using ultrapure water. The reaction solution is prepared on ice. A two-step PCR procedure was used for detection and analysis by fluorescence quantitative PCR. mRNA expression was calculated using formula 2- (∆Ct sample–∆Ct control). The primer sequences are listed as follows. ZO-1 (Forward, 5’-ATAAAGTGCTGGCTTGGTCTGTTTG-3’ and Reverse, 5’-GCACTGCCCACCCATCTGTA-3’); occludin (Forward, 5’-ACTGGGTCAGGGAATATCCA-3’ and Reverse, 5’-TCAGCAGCAGCCATGTACTC-3’); GAPDH (Forward, 5’-GCACCGTCAAGGCTGAGAAC-3’ and Reverse, 5’-GCACTGCCCACCCATCTGTA-3’).
The cells were inoculated on 24 well plates to detect the contents of occludin, and ZO-1 in HUVECs. HUVECs were fixed with 4% paraformaldehyde for 15 minutes and then blocked with goat serum for 1 h. After processing, the HUVECs were incubated with occludin (1:200) and ZO-1 (1:200) antibodies for 6 h. Next, a fluorescence secondary antibody (1:200; Cat #: A23220 and Cat #: A23420, Abbkine, California, USA) was added to the stain for 1h at room temperature. The samples were imaged by a fluorescence microscope, and the fluorescence intensity was quantitatively evaluated by image-J software.
To detect the contents of occludin and ZO-1 in lung tissues, lung tissues were sectioned into 5-µm-thick slices, lung specimens were sealed with goat serum after dewaxing and antigen repair. After processing, lung specimens were incubated with primary and secondary antibodies. the samples were imaged by fluorescence microscope. Finally, the sample is imaged with a fluorescence microscope.
2.11 Statistical analysis
All data were expressed as mean ± standard deviation (SD). One-way ANOVA was used to calculate differences between groups, and then the SNK test was performed. p < 0.05 was considered Statistically significant. P-value༜0.05 was considered Statistically significant. All statistical analyses were performed using IBM SPSS 25 software. All mathematical calculations and graphing were performed using GraphPad Prism 5.