Identification of target genes. The key words “acute lung injury” and “paraquat” were searched in the GeneCards database (http://www.genecards.org) to identify disease-related genes. The keyword “Luteolin” was searched in the CTD database (http://ctdbase.org/) to identify potential target genes. Then, the genes that were commonly associated with “acute lung injury”, “paraquat” and “Luteolin” were selected using VENNY version 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html).
Protein-protein interaction (PPI) network. The overlapping genes were imported into the STRING database (https://www.string-db.org/). Then, the resulting TSV file was downloaded and imported into Cytoscape version 3.7.1. The Centiscape 2.0 plugin was used to screen the key genes of the network by degree, betweenness, and closeness.
Enrichment analysis for target proteins. Gene ontology (GO; including enrichment of cellular components, molecular functions, and biological processes) and KEGG pathway enrichment analyses were performed using the Bioconductor data package in R version 3.6.0. The enrichment results with corrected P-values < 0.05 were obtained and considered to have high reliability.
Reagents. PQ standard (≥ 98%) was purchased from AccuStandard (cat. no. Cas 1910-42-5); Lut standard (≥ 98%) was purchased from Macklin (cat. no. Cas 491-70-3); 3-Methyladenine (3-MA) was obtained from MedChemExpress (cat. no. Cas 5142-23-4); The reagent containing 20% PQ was purchased from Henan Shangqiu Pesticide Factory. Hematoxylin and eosin (H&E) staining reagent was purchased from Beyotime Institute of Biotechnology. The superoxide dismutase (SOD) assay kit was obtained from Nanjing Jiancheng Engineering Institute; malondialdehyde (MDA), reactive oxygen species (ROS), and Cell Counting Kit-8 (CCK-8) assay kits were purchased from Biosharp Life Sciences; bicinchoninic acid (BCA) kit, cell apoptosis analysis kit and ECL plus kit were purchased from Shanghai Yeasen Biotechnology Co., Ltd.; PVDF membranes were purchased from MerckMillipore; the SDS-PAGE gel preparation kit was purchased from Yamei Biotechnology Institute.
Experimental animals. Adult male Sprague-Dawley rats (6–8 weeks old, weighing 220 ± 20 g) were purchased from Changsha Tianqun Biotechnology Co. Ltd. All the rats were maintained in an SPF-grade environment with a 12 h light/dark cycle, and the rats were given free access to food and water. All the animal experiments were approved by the Animal Care and Use Committee of Hainan Medical University. The study was also carried out in compliance with the ARRIVE guidelines.
Experimental protocol. The experimental rats were randomly selected and divided into three groups (15 rats per group): the normal control group (Ctrl), the PQ group (PQ) and the PQ + Lut group (Lut).
Lut (10 mg/kg) was dissolved in DMSO and intraperitoneally injected into rats in the Lut group 2 h before model establishment, and rats in the Ctrl and PQ groups received equal volumes of DMSO. A model of PQ-induced ALI was established by administering PQ (100 mg/kg) by gavage to rats in the PQ group and Lut group. Rats in the Ctrl group received an equal volume of saline. Reasonable doses of Lut (10 mg/kg) and PQ (100 mg/kg) were selected according to published literature (13, 14). A total of 72 h after model establishment, Approximately a total of 50% of the rats in the PQ group and 40% of the rats in the Lut group died. These dead rats will not participate in specimen collection and subsequent experiments. The remaining rats were euthanized by intraperitoneal injection of an overdose of sodium pentobarbital (≥ 150 mg/kg), and their serum and lung tissues were harvested for further analysis.
For survival analysis, another 10 rats were included in each group, were treated as above, and the death of the rats in the three groups was monitored for 72 h after model establishment.
The experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of the First Affiliated Hospital of Hainan Medical University [approval no. 2020 (Research) No. (97); Haikou, China].
Lung wet/dry ratio (W/D). The right lungs were harvested and weighed to determine the wet weight, and then the lung tissues were incubated at 60˚C for 24 h and weighed to determine the dry weight. The W/D ratio was calculated as an indicator of pulmonary edema. Three samples in each group.
H&E staining. Lung tissues were collected from rats in each group and fixed in 4% paraformaldehyde fixing solution (10–22°C, about 24h), embedded in paraffin, and cut into 5 µm sections. Subsequently, these sections were processed for dewaxing, as well as hematoxylin and eosin staining (at room tempreture, for 2min). After H&E staining, the pathological changes in the lung tissues were observed using a light microscope and scored. The scoring criteria were: i) alveolar congestion, ii) hemorrhage, iii) neutrophil infiltration or aggregation in the airspace or vessel wall; and iv) alveolar wall thickness/hyaline membrane formation. Each item was scored on a 5-point scale as follows: 0, minimal damage; 1, mild damage; 2, moderate damage; 3, severe damage; and 4, maximal damage. Five samples in each group
Immunohistochemistry. Lung tissue sections (5 µm) were deparaffinized and rehydrated. The sections were subjected to antigen retrieval and quenched with 3% H2O2. After blocking with 5% BSA for 1.5 h at 37˚C, the sections were incubated with an anti-P62 antibody (Proteintech, 18420-1-AP, 1:500) at 4˚C overnight. Then, the sections were incubated with HRP-conjugated secondary antibodies, stained with diaminobenzidine, counterstained with H&E, and visualized under a microscope. Images were analyzed using ImageJ (v1.8.0.345). Three samples in each group.
Evans blue (EB) staining. A total of 72 h after the model was established, rats were anesthetized by intraperitoneal injection of 2% pentobarbital (60 mg/kg) and then injected with 2% EB solution (30 mg/kg) through their tail veins. After 30 min, PBS was injected into the right ventricle of the heart to eliminate the EB solution from the pulmonary vascular system. A portion of lung tissue was collected and weighed, and the lung tissue was treated with 100% formamide at 60˚C for 24 h. The samples were then centrifuged at 1,000 x g for 30 min at room temperature, and 200 µl supernatants were collected to measure the absorbance at 620 nm using a spectrophotometer. The EB content of each sample was calculated using a standard curve. Lung microvascular permeability was then expressed as the ratio of EB content to the wet lung weight. Three samples in each group.
Cell culture and treatment. Immortalized Human pulmonary microvascular endothelial cells (HPMECs) were purchased from Otwo Biotechnology Development Co., Ltd. (cat. no. HTX2255). Cells were grown in DMEM (Gibco; Thermo Fisher Scientific, Inc.) medium supplemented with 10% FBS (Life Science & Technology Co., Ltd.) in an incubator at 37˚C supplied with 5% CO2.
The cells were treated with different concentrations of PQ (0, 1, 10, 100, 200, 400, 800, or 1,000 µM) for 24 and 72 h. The results showed that cell viability decreased in a dose-dependent manner, and the decrease in cell viability was more pronounced after 72 h of treatment than at 24 h (Fig. S1C). Therefore, treatment with 200 µM PQ for 72 h was selected for subsequent experiments. Next, Lut was dissolved in 0.1% DMSO and the cells were pretreated with different concentrations of Lut (0, 1, 10, 20, 40, 80, 100, or 200 µM). Subsequently, pretreatment with 20 µM Lut for 2 h was selected for further mechanistic studies (Fig. S1D and E).
Four experimental groups were continually treated for 24 h as follows: i) Ctrl, HPMECs were incubated under normal conditions; ii) PQ, cells were treated with 200 µM PQ to establish a cell injury model; iii) Lut, cells were pretreated with 20 µM Lut for 2 h and then exposed to 200 µM PQ; and iv) 3-MA + Lut, cells were cocultured with 5 mM 3-MA, based on a previous study (15), and 20 µM Lut, followed by treatment with 200 µM PQ.
CCK-8 assay. HPMECs in the logarithmic phase of growth were uniformly seeded in 96-well plates at a density of 3–5 x 103 per cell culture (100 µl/well). After administration, 10 µl CCK-8 solution was added to each well, and cells were further incubated for 1–2 h. The absorbance was then measured at 450 nm using a microplate reader. The cell viability of different groups was assessed in terms of the percentage of the control group using three independent repeats. Three samples in each group.
Cell migration assay. Cell migration assays were performed using Transwell plates with 5 µm pores to assess cell migration. Briefly, 3x104 HPMECs were seeded into the upper chamber in serum-free media. The lower chamber was filled with supplemented media, which served as a chemoattractant. After 72 h of incubation, the cells that had not migrated and remained in the upper chamber were removed using a cotton swab. The cells that had migrated were stained using 0.1% crystal violet (Biosharp) for 10 min and then imaged and counted under a light microscope. Three samples in each group.
Apoptosis analysis using flow cytometry. To detect cell apoptosis, after the cell model had been established, the cells were washed twice with PBS and centrifuged at 300 x g at 4˚C for 5 min; then, the cells were incubated in 5 µl Annexin V-FITC and 10 µl PI staining solution. After incubation for 15 min in the dark at room temperature, after which the cells were examined using flow cytometry. Three samples in each group.
Confocal microscopy analysis. Cells were fixed using 4% paraformaldehyde at room temperature, washed with PBS, blocked with QuikBlock immunostaining blocking solution (Beyotime Institute of Biotechnology) at room temperature, and then incubated overnight at 4˚C with 1% BSA containing an anti-LC3 antibody (ProteinTech, 14600-1-AP, 1:300). Then, a fluorescent secondary antibody (1:250) was prepared in 1% BSA, added to the cells, and incubated for 1 h at room temperature in the dark. Finally, a fluorescent anti-quencher containing DAPI was added. Fluorescent images were acquired using a laser scanning confocal microscope (Olympus FV3000, Olympus Corporation). Three samples in each group.
Transmission electron microscopy (TEM). Autophagosomes in HPMECs were observed using TEM. After the establishment of the cell model, the cells were collected and fixed with 2.5% glutaraldehyde at 4˚C. Subsequently, cell clusters were fixed using 1% osmium tetroxide, dehydrated in a gradient of alcohol solutions, embedded in resin, and then ultrathin sections were stained using uranyl acetate and citric acid. Typical autophagosomes were observed using a Transmission Electron Microscope (Hitachi HT7700; Hitachi, Ltd.). Three samples in each group.
ROS level detection. According to the instructions of the ROS detection kit, H2DCFDA was diluted to 10 µM with serum-free media. The cells were washed with PBS twice, and incubated at 37˚C for 30 min in the dark. Subsequently, the cells were observed and imaged using a fluorescence microscope. Finally, changes in the fluorescence intensity of each group were analyzed using ImageJ. Three samples in each group.
MDA and SOD level detection. After the establishment of the cell model, the cells were lysed using RIPA lysis buffer and centrifuged at 7500 x g at 4˚C for 10 min; then, the supernatants were collected. According to the instructions of the MDA and SOD detection kit, the relevant reagents were added to the samples, and the absorbance values were measured at 532 and 450 nm, respectively, using an enzyme labeling instrument. Three samples in each group.
Western blotting analysis. Cell or lung tissue samples were lysed in RIPA buffer containing protease and phosphatase inhibitors. The protein concentrations were determined using a BCA kit. Protein samples (30 µg) were loaded on 8–10% SDS gels, resolved using SDS-PAGE, and transferred to PVDF membranes. The membranes were blocked using 5% skimmed milk for 2 h at room temperature, followed by incubation with a primary antibody at 4˚C overnight. The primary antibodies were diluted as follows: Anti-LC3 antibody (ProteinTech, 14600-1-AP, 1:2,500), anti-P62 antibody (ProteinTech, 18420-1-AP, 1:5,000), anti-IL-6 antibody (BIOSS, bs-0782R, 1:1,000), anti-IL-1β antibody (BIOSS, bs-20449R, 1:1,000), anti-TNF-α antibody (Boster, BA0131,1:1,000), anti-VE-cadherin antibody (Invitrogen, XF354429, 1:250), anti-ZO-1 antibody (ProteinTech, 21773-1-AP, 1:10,000), anti-Bcl-2 antibody (Affinity, AF6139, 1:2,000), anti-Caspase-3 antibody (BIOSS, bs-0081R, 1:2,000), anti-PI3K antibody (ProteinTech, 21739-1-AP, 1:5,000), anti-p-PI3K antibody (Invitrogen, PA5-104853, 1:1,000), anti-Akt antibody (ProteinTech, 10176-2-AP, 1:1,000), anti-p-Akt antibody (ProteinTech, 80455-1-RR, 1:1,000), anti-mTOR antibody (ProteinTech, 66888-1-Ig, 1:10,000), and anti-p-mTOR antibody (ProteinTech, 67778-1-Ig, 1:5,000). Then, the membranes were washed at least three times with TBST and incubated with HRP-conjugated secondary antibodies (Biosharp, BL003A, 1:10,000) at room temperature for 2 h. Finally, the protein bands were visualized using an enhanced chemiluminescence reagent. Three samples in each group.
Statistical analysis. Data are presented as the mean ± the standard error of at least three independent experiments. All statistical analyses were performed using GraphPad Prism version 8.02 (GraphPad Software, Inc.). Comparisons between two groups were performed using a Student’s t-test and comparisons between multiple groups were performed using a one-way ANOVA. P < 0.05 was considered to indicate a statistically significant difference.