Animals

C57BL/6 mice (6-week-old and 8-week-old, male) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. The mice were housed under conditions of the temperature of 22 ± 1 °C, the humidity of 45–55%, and a 12 h day/night circle. The animal experiments in this study were conducted per the guideline for the care and use of experimental animals. The Animal Ethics Committee of Henan Provincial Institute for Food and Drug Control approved the experiments involving animals.

LPS-induced ALI murine model and C75 treatment

Six-week-old mice were randomly divided into a regular chow diet (Beijing Keao Xieli Feed Co., Ltd, 12% kcal fat) group (lean) or a high-fat diet (Research Diets, D12492, 60% kcal fat) group [diet-induced obese (DIO)]. After feeding for 24 weeks, the mouse model of ALI was established according to the previous literature [21]. Briefly, mice were anesthetized with pentobarbital and LPS (O55:B5, L2880, Sigma, 100 μg dissolved in 50 μL of saline) or vehicle (50 μL of saline) was instilled intratracheally into both lean and DIO mice. For FASN inhibition, DIO mice were intraperitoneally injected with C75 [MedChemExpress, HY-12364, dissolved in 10% dimethyl sulfoxide (DMSO)] at 10 mg/kg body weight at 30 min before LPS administration, and mice in other group were intraperitoneally injected with DMSO (vehicle) at the same concentration. Mice were euthanized and sacrificed at 6 h or 8 h after LPS administration, and their lung tissues were harvested for subsequent procedures.

Isolation of lung endothelial cells

The lung endothelial cells were isolated from whole lung tissues using CD45 (Miltenyi Biotec, 130-052-301) and CD31 Microbeads (Miltenyi Biotec, 130-097-418) as previously described [22]. In brief, the harvested lungs were minced and digested with collagenase II (Gibco, 17101015) and filtered through a 100-μm nylon mesh. The cell suspension was resuspended in bovine serum albumin and incubated with anti-CD45-conjugated magnetic beads for negative selection, followed by positive selection with anti-CD31-conjugated magnetic beads. The CD31-positive cells were collected with an MACS separator (Miltenyi Biotec) following the manufacturer’s protocols. The purity of the endothelial cell population was confirmed by immunofluorescence of anti-CD31 (Abcam, ab7388) and anti-VE-cadherin (Affinity, AF6265). The primary mouse lung endothelial cells (MLECs) from 8-week-old male mice were cultured for in vitro experiments. Isolated MLECs were cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing endothelial growth supplement, and the cells between passages 2 and 6 were used for in vitro experiments. The freshly isolated lung endothelial cells from lean and DIO mice were collected immediately to detect the protein levels by western blot.

Palmitic acid treatment

For palmitic acid (PA, Sigma) treatment, PA combined with bovine serum albumin (BSA, Sigma) was added to the culture medium of MLECs. Briefly, sodium salt PA was dissolved in ethanol at 65 °C for 15 min and then allowed to combine with fatty acid free-BSA at final concentrations of 0.3 mM. After 24 h of PA treatment, MLECs were stimulated with 1 μg/mL LPS for 12 h and C75 (25 μM, 50 μM) was added 2 h prior to LPS. The solvent of ethanol with BSA and DMSO at the same concentration as the PA and C75, respectively, was used to treat control cells.

Differentiation of 3T3-L1 cells into adipocytes and Transwell co-culture system

The 3T3‐L1 cells (iCell Bioscience Inc, iCell-m066) were cultured in DMEM supplemented with 10% FBS, 50 units/mL penicillin, and 50 μg/mL streptomycin in a humidified atmosphere of 5% CO2. The 3T3‐L1 cells were cultured for 2 days to confluence, and adipogenic differentiation was induced by treatment with DMEM containing 10% FBS, 0.5 mM IBMX, 5 µg/mL insulin, and 1 µM dexamethasone for 2 days. After differentiation induction, the medium was replaced with a differentiation–maintenance medium containing DMEM supplemented with 10% FBS and 5 µg/mL insulin and cultured for 4 days. Then, the cells were cultured in DMEM supplemented with 10% FBS, and the medium was replaced every 2 days. The differentiation efficiency of 3T3-L1 cells to adipocytes were assessed by Oil Red O staining. After differentiation into mature adipocytes, the medium was changed to the conditioned medium as performed with MLECs.

MLECs and mature adipocytes were co-cultured in a six-well Transwell system (Corning Inc., 3450) with a 0.4-μm porous membrane to separate the upper and lower chambers. The adipocytes were cultured in the lower chamber, while the MLECs were seeded and cultured in the upper chamber. After co-culture for 48 h, MLECs were removed from the upper chamber to a new culture chamber. Then, 1 μg/mL LPS was added into the medium and incubated for 12 h. C75 (25 μM, 50 μM) was added 2 h prior to LPS treatment. MLECs cultured without LPS or with DMSO at the same concentration as the C75 solvent were used as controls.

shRNA-induced FASN gene silencing

For silencing of FASN, the MLECs were transfected with shRNA using the Lipofectamine 3000 Transfection Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The sequences of FASN shRNA were as follows: sense, 5′-CTTTCTTCTTCGACTTCAAAG-3′, antisense, 5′-CTTTGAAGTCGAAGAAGAAAG-3′; the lentiviral control vector contained a non-sense FASN sequence [negative control (NC) shRNA]: sense, 5′-TTCTCCGAACGTGTCACGT-3′, antisense, 5′-ACGTGACACGTTCGGAGAA-3′. At 48 h post-transfection, validation of FASN knockdown was performed at the mRNA level using relative quantitative real-time polymerase chain reaction (qPCR) and at the protein level using western blot. After 24 h of PA treatment or 48 h of co-culture with adipocytes, the MLECs with FASN shRNA or NC shRNA were challenged by 1 μg/mL LPS for another 12 h.

FASN activity assay

The lung homogenates and sonicated MLECs were centrifuged at 12,000×g for 20 min at 4 °C to obtain particle-free supernatants. FASN activity was estimated by a commercially available kit (Solarbio, BC0550), according to the manufacturer’s protocol. FASN activity was determined spectrophotometrically by measuring the decrease of absorption at 340 nm due to the oxidation of NADPH.

Transmission electron microscopy

For mitochondrial ultrastructure observations, the treated MLECs were fixed overnight at 4 °C in 2.5% glutaraldehyde. The cells were rinsed in 0.1 M phosphate buffer (PH 7.4), and then fixed for 2 h in 1% osmic acid in buffer. Then the fixed cell mass were dehydrated in gradient ethanol solutions for dehydration, embedded into 812 embedding agent and polymerized at 60 °C for 48 h. Ultrathin sections of 70 nm were cut and post-stained with uranyl acetate and lead citrate. Mitochondrial morphology was observed under a transmission electron microscope (H7650, Hitachi, Japan).

Analysis of mitochondrial membrane potential

The mitochondrial membrane potential (MMP) of MLECs was determined using the JC-1 fluorescent dye (Beyotime, C2006). MLECs were washed with ice‑cold phosphate-buffered saline (PBS) and incubated with JC-1 staining working solution at 37 °C for 20 min in the dark. Subsequently, the cells were washed twice with PBS, and the fluorescence intensity of the JC-1 polymer and JC-1 monomer was analyzed by a Novocyte flow cytometer. The mitochondrial membrane potential was shown as a ratio of the fluorescence intensity of polymers to monomers.

Mitochondrial ROS detection

The mitochondrial reactive oxygen species (mtROS) contents were measured using MitoSOX (Maokang Biotech, M36008), a mitochondrial superoxide indicator. MLECs were washed with PBS three times and incubated with 5 μM MitoSOX at 37 °C for 15 min. After washing three times with PBS, the nuclei were stained with DAPI (1 μM) for 15 min. The photographs were captured using a microscope (BX53, Olympus, Japan) and the red fluorescence intensity was measured. The measurement was also performed using a Novocyte flow cytometer and analyzed using the FlowJo software.

Western blot analysis

The lung tissues/cells were homogenized in RIPA buffer containing protease and phosphatase inhibitors. The lysates were centrifuged at 14,000 rpm (15 min, 4 °C), and the supernatant was collected for further analysis. The proteins were separated with SDS-polyacrylamide gels and then transferred to a polyvinyl difluoride (PVDF) membrane (Millipore, IPVH00010). Immunoblotting was performed at 4 °C overnight using primary antibodies directed against FASN (1:2000, Abcam, ab22759), p38 MAPK (1:500, Proteintech, 14064-1-AP), phospho (p)-p38 MAPK (Thr180/Tyr182) (1:1000, Proteintech, 28796-1-AP), NLRP3 (1:500, wanleibio, WL02635), VE-cadherin (1:1000, Affinity, AF6265), Drp1 (1:2000, Proteintech, 12957-1-AP), phospho (p)-Drp1 (Ser616) (1:500, Affinity, AF8470), Mfn2 (1:1000, abclonal, A19678) and β-actin (1:2000, Proteintech, 60,008–1-Ig). The membranes were then incubated with the secondary antibody (1:10,000, SA00001-1, SA00001-2, Proteintech) at room temperature for 1 h. The protein bands were detected using ECL solution (E003, 7 Sea Biotech, China) and visualized using the ImageJ software (v.1.53c; NIH).

Quantitative real-time PCR

Total RNA was isolated from lung tissues or MLECs using the TRIzol Reagent (Invitrogen). For the quantitative real-time PCR (qPCR), cDNA was synthesized using PrimeScript RT Master Mix (TaKaRa, Japan) following the manufacturer’s protocols. qPCR was performed on cDNA using TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa) and ROX Reference Dye (TaKaRa) on a ABI 7900HT Real-Time PCR System (Thermo Fisher Scientific Inc.). The mRNA levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1β (IL-1β) were calculated using the comparative threshold method (ΔΔCt), and normalized to endogenous control β-actin. The primers and probes were as follows:

mouse IL-6 forward, 5′-ATGGCAATTCTGATTGTATG-3′;

mouse IL-6 reverse, 5′-GACTCTGGCTTTGTCTTTCT-3′;

mouse TNF-α forward, 5′-CAGGCGGTGCCTATGTCTCA-3′;

mouse TNF-α reverse, 5′-GCTCCTCCACTTGGTGGTTT-3′;

mouse IL-1β forward, 5′-CTCAACTGTGAAATGCCACC-3′;

mouse IL-1β reverse, 5′-GAGTGATACTGCCTGCCTGA-3′;

mouse β-actin forward, 5′-GGCACCCAGCACAATGAA-3′;

mouse β-actin reverse, 5′-TAGAAGCATTTGCGGTGG-3′.

Immunofluorescence staining

For immunofluorescence assay, MLECs were grown on coverslips in 24-well plates. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. After blocking with serum blocking solution, the cells were incubated with rabbit antibody to VE-cadherin (Affinity, AF6265, 1:200) overnight at 4 °C and then incubated with secondary antibody fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (Abcam, ab6717, 1:200) for 1 h at room temperature followed by DAPI staining (Aladdin reagent, D106471). Representative images were further captured under a microscope (BX53, Olympus, Japan).

Measurements of endothelial cell permeability

For permeability studies, MLECs were grown on the apical side of the collagen-coated polyester membrane of a Transwell (0.4 μm pore size). After 24 h of PA treatment or 48 h of co-culture with adipocytes, the MLEC monolayer was treated with or without LPS (1 μg/mL, 12 h) in the presence or absence of C75 (25 μM, 50 μM). C75 was added 2 h prior to the addition of LPS. The solvent controls were added as above. Then, 1 mg/mL fluorescein isothiocyanate (FITC)-dextran was administered in the upper chamber. The fluorescence intensity of the medium from each chamber was measured using BioTek Synergy H1 (Biotek) 30 min after the stimulation of dextran. The flux was expressed as the percentage of dextran diffusion per hour per square centimeter [23].

Transendothelial electrical resistance

The cellular barrier properties were assessed by measuring the transendothelial electrical resistance (TEER) across MLECs using Millicell-ERS (MERS00002, Millipore). MLECs were grown to a confluent monolayer on the apical side of the polycarbonate membrane of a Transwell (0.4 μm pore size). After 24 h of PA treatment or 48 h of co-culture with adipocytes, the MLEC monolayer was treated with or without LPS (1 μg/mL, 12 h) in the presence or absence of C75 (25 μM, 50 μM). C75 was added 2 h prior to the addition of LPS. The solvent controls were added as above. The TEER across MLEC monolayer was measured with the electrode tips in the upper and lower compartments at various time points and expressed as Ω cm2 [24].

Enzyme-linked immunosorbent assay

The levels of TNF-α, IL-6, and IL-1β in mouse lung homogenates and cell culture supernatant were assayed using commercial ELISA kits following the manufacturer’s protocols (Cloud-Clone Corp, China).

Lung histology and lung injury score

The left upper lung lobes were collected from mice 8 h after LPS instillation and they were fixed with 4% paraformaldehyde for 24 h. The tissue sections were embedded in paraffin and cut into 5-μm-thick sections. The tissue sections were stained with hematoxylin and eosin (H&E), and the lung injury was evaluated based on the hemorrhage, hyperemia, edema of the alveolar wall, and inflammatory cell infiltration according to the previously reported protocol [25].

Lung vascular leak assessment

The lung vascular leak was measured in each experimental group using (1) Evans blue dye (EBD) extravasation assay in lung tissues, (2) lung wet-to-dry ratio measurement, and (3) total protein concentrations in bronchoalveolar lavage fluid (BALF). After 8 h of LPS instillation, EBD (20 mg/kg; Macklin, E6135) were injected into the mice via the tail vein and allowed to circulate in the blood vessels for 3 h. The anticoagulant blood was collected and centrifuged at 3000 rpm for 20 min. Then, the intravascular EBD in the lung was washed through the right ventricle with saline for 5 min. EBD was extracted from the lungs by incubation with formamide (60 °C for 24 h) and then centrifuged at 5000 rpm for 30 min. The absorbance of plasma and lung supernatants was measured by spectrophotometry at 620 nm and corrected for the presence of heme pigments as follows: A620 (corrected) = A620 − (1.426 × A740 + 0.030) [26]. Evans blue leaking index was calculated as the ratio of the dye concentration in the lung to the plasma.

The right lung lobes were collected and weighed immediately (wet weight) at 8 h following LPS instillation. The samples were reweighed after dried in a 60 °C oven for 48 h (dry weight). The ratio of wet weight to dry weight (W/D) was calculated.

BALF was collected by cannulating the trachea with a blunt 22-gauge needle and then lavaging the lungs four times with 0.75 mL of ice-cold PBS. The levels of total protein content in BALF was determined using a bicinchoninic acid protein assay kit (Solarbio, PC0020).

Statistical analysis

The values were expressed as the mean ± standard deviation (SD). Unpaired-sample Student t tests were used to analyze the differences between the two groups when appropriate. Multi-group comparisons were performed using two-tailed ANOVA with the post hoc analysis of least significant difference (LSD) or Games-Howell method. For all analyses, the two-tailed P < 0.05 indicated a statistically significant difference. SPSS version 26.0 was used for statistical analysis.