2.1 Animal
Adult male C57BL/6J mice (20 ± 2 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). All animal experiments were approved by the Animal Care and Use Committee at Central South University and strictly followed guidelines regarding the care of laboratory animals.
2.2 ALI model and animal treatment
C57BL/6J mice were randomly divided into four groups: (a) the Control group, (b) ATA group, (c) ALI group, and (d) ALI+ATA group. Mice in the Control and ATA groups received 50 µL sterile saline intratracheally. Mice in the ALI and ALI+ATA groups were given intrabronchial injection of LPS (E. coli O111:B4; Sigma-Aldrich; USA) at 5 mg/kg dissolved in 50 µL sterile saline. After intratracheal instillation, the mice were kept vertical for 5 min to ensure the distribution of the LPS or sterile in the lungs. In the ATA and ALI+ATA groups, ATA (20 mg/kg) was intraperitoneally injected 1 h prior to the LPS instillation. All mice were sacrificed 12 h after LPS instillation and the lungs were collected and weighted. For survival study, the mice were intratracheally injected with LPS at a lethal dose (25 mg/kg). Saline or ATA (20 mg/kg) was intraperitoneally injected into mice 1 h before or after LPS administration. The survival rate was monitored every 6 h. All surgical procedures were performed under 1.0% pentobarbital in sterile (80 mg/kg).
2.3 Hematoxylin-eosin (HE) and inflammatory injury analysis
The lung tissue samples were collected, fixed in 10% formalin, embedded in paraffin, sectioned at 4 µm, and stained with HE. Histological scoring was performed blindly in line with four parts: alveolar congestion, hemorrhage, infiltration, and thickness of the alveolar wall/hyaline membrane formation. The lung injury score was measured as previously described [18]. The severity of the injury was graded from 0 to 4, according to 5 variables: hyaline membranes, neutrophils in the alveolar space, hemorrhage, septal thickening, and pertinacious debris filling the airspaces.
2.4 Lactate dehydrogenase (LDH) assay
The activity of LDH in the serum of mice was determined by Lactate Dehydrogenase Activity Assay Kit (Jincheng Bioengineering Institute, Nanjing, China). Briefly, the serum samples were incubated with NADH and pyruvate for 15 min at 37 ℃. Then, the enzymatic reaction was stopped by 0.4 mol/L NaOH. Then, absorbance was measured at 450 nm.
2.5 Bronchoalveolar lavage fluid (BALF)
BALF was collected with 0.8 mL of ice-cold PBS as previously described [19]. The BALF was centrifuged at 1500 rpm for 5 min. The pelleted cells were resuspended in PBS, and the cell numbers were counted. Protein concentration in BALF, an indicator of alveolar-capillary permeability, was measured using a BCA kit (Thermo Fisher Scientific). The number of total cells, macrophages, and neutrophils were counted with a hemocytometer and Wright-Giemsa staining.
2.6 Measurement of myeloperoxidase (MPO)
The lung tissue was weighed and homogenized. Then, according to the manufacturer's instructions, MPO activity in the lungs was measured by the MPO assay kit (Nanjing Jiancheng Bioengineering Institute, China).
2.7 Measurement of superoxide dismutase (SOD) and malondialdehyde (MDA)
About 40 mg of left upper lung tissue was homogenated in cold PBS at a ratio of 1:10 (weight: volume). Protein concentration was determined using the BCA kit. The MDA level was measured by the thiobarbituric acid (TBA) method using a lipid peroxidation MDA assay kit (Jiancheng Biotech, Nanjing, Jiangsu, China) according to the manufacturer’s instructions. Absorbance at 530 nm was measured by the microplate reader. SOD activity was evaluated by the xanthine oxidase method using a SOD activity assay kit (Jiancheng Biotech, Nanjing, Jiangsu, China) according to the manufacturer's instructions. Absorbance was measured at 450 nm by the microplate reader.
2.8 Measurements of cytokine levels in the lungs
The contents of tumor necrosis factor-alpha (TNF-α), interlerkin-1beta (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) in lung tissue, BALF, and cell culture supernatant were determined by specific enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen; Thermo Fisher Scientific) according to the manufacturer’s instructions.
2.9 Real-time polymerase chain reaction (PCR)
Total RNA was collected from lung tissue using RNAiso Plus (TaKaRa Clontech, Kusatsu, Japan) based on the manufacturer’s instructions. RNA was quantified by measuring absorption at 260 nm. The generation of cDNA from the total RNA (1 µg) was performed with Prime Script RT reagent Kit with gDNA eraser (TaKaRa). Real-time PCR was performed with SYBR Fast qPCR mix (TaKaRa) on a Bio-Rad real-time PCR system (CFX96 Touch™, Bio-Rad, USA). The expression levels (relative values) of the objective genes were calculated using the 2−ΔΔCt method [20]. β-actin was used as the internal control.
2.10 Western blot analysis
Total protein was extracted from mouse lungs and cells by RIPA. Protein concentration was detected by the bicinchoninic acid assay (BCA, Thermo Fisher Scientific). Equal amounts of protein (30 µg) were loaded on SDS-PAGE and transferred to a polyvinylidene fluoride membrane [21]. After blocking with skim milk (5%) in Tris-buffered saline (TBST) for 1 h at room temperature, the membrane was probed with the primary antibodies at 4°C overnight: rabbit anti-NLRP3 antibody (1:2000; CST, Danvers, MA), rabbit anti-Nrf2 antibody (1:1000, CST), goat anti‐IL‐1β antibody (1:2000; R&D, Minneapolis, MN), rabbit anti‐caspase‐1 antibody (1:1000; Abcam, Eugene, OR), rabbit anti‐ASC antibody (1:1000, CST), rabbit anti-Fn14 antibody (1:1000, Abcam), rabbit anti-β-actin antibody (1:7500, Signal way Antibody, College Park, MD, USA), and rabbit anti‐α‐tubulin antibody (1:7500, Servicebio, China). After incubation with peroxidase‐conjugated secondary antibodies (1:7500, Signal way Antibody) at room temperature for 1 h, the signal was developed using an ECL chemiluminescence kit (Millipore, USA). The band intensities were quantitated using Image Lab software and normalized to internal reference values accordingly.
2.11 Isolation and treatment of primary murine peritoneal macrophages
The isolation and culture of primary murine peritoneal macrophages were conducted as described [22]. Four days after the intraperitoneal injection of 3 mL 3% thioglycolate (Sigma-Aldrich) into C57BL/6J mice, peritoneal macrophages were harvested by peritoneal lavage with precooled Roswell Park Memorial Institute RPMI 1640 (Gibco, Life Technologies, Carlsbad, CA). The cells were collected by centrifuging at 1000 rpm for 8 min and washed with cooled phosphate‐buffered saline. The cells were resuspended in cell culture medium and counted on the counting plate after sufficient mixing. Macrophages were plated into 6-well or 12‐well plates (2×106 cells/well or 1×106 cells/well). To investigate the effect of rTWEAK on the macrophages, the cells were treated with rTWEAK (100 ng/mL) and LPS (100 ng/mL) for 12 h. To investigate the activation of NLRP3 inflammasome, the cells were primed with rTWEAK (100 ng/mL) and LPS (100 ng/mL) for 135 min. Cells were then stimulated with ATP (2.5 mM, Solarbio, China) for 45 min.
2.12 Measurement of ROS
The primary peritoneal macrophages (2×106 cells/well) were seeded into the 6-well plates. Dilute DCFH-DA (Nanjing Jiancheng Bioengineering Institute, China) with RPMI 1640 at the ratio of 1:1000 to a final concentration of 10 µM. Add 1 mL diluted DCFH-DA solution to each well and incubate for 20 min at 37 ℃. The cell suspension was obtained by adding 1 mL RPMI 1640, then was centrifuged at 1500 rpm for 5 min, followed by resuspension with 400 µL PBS for flow cytometry assay (BD, USA).
2.13. Silenced the expression of Fn14 by shRNA
The short hairpin RNA (shRNA) of Fn14 was purchased from Gene Chen (Shanghai, China). Fn14 shRNA sequences were gattcggcttggtgttgatgc. Macrophages were infected with concentrated lentivirus. The supernatant was replaced with complete culture medium after 16 h. After treated with shRNA, the cells were primed with LPS (100 ng/mL) for 135 min. Cells were then stimulated with ATP (2.5 mM, Solarbio, China) for 45 min.
2.14 Statistical analysis
All experiments were independently repeated three times. Data were presented as means ± SEM and conducted using SPSS 21. Differences between the two groups were analyzed with a t-test. Differences between multiple groups were analyzed using ANOVA, followed by Tukey's post hoc test. The survival rate was assayed by the log-rank test. A value of P < 0.05 was considered significant.