Sequences, primer, antibodies, and ELISA kit

The shRNA targets Srg3 and Irf7 are exhibited in Table 1.

Table 1 shRNA ds-oligo sequences

The primer for qRT-PCR or ChIP-qPCR is exhibited in Table 2.

The antibodies utilized for western blot, immunohistochemistry, and immunofluorescence are exhibited in Table 3.

The ELISA kit’s detail is exhibited in Table 4.

The detail of commercial merchandise used in the research is exhibited in Table 5.

Table 5 Commercial merchandiseEthics declaration

All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Pulmonary Hospital (approval no. K23-179Y). The animals were housed in a facility with appropriate environmental conditions and received a standard diet and water ad libitum. We took measures to minimize the number of animals used in the study and to reduce their suffering, including the use of anesthesia during surgical procedures. We followed the principles of the three Rs (replacement, reduction, and refinement) in the design and implementation of this study.

Animal experiment

Forty-eight male Sprague–Dawley rats (6–8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). All rats were kept 2 rats per each cage in a specific pathogen-free animal laboratory with a temperature-controlled colony and 12/12 h light/dark cycle with free access to chow and water, and 42 rats were left until 20–21 months. The CLP-induced septic rate model was based on previous description [14]. Briefly, the rats were anesthetized by intraperitoneal injection of ketamine (K) and  xylazine (X) [55 mg/kg (K) + 10 mg/kg (X)]. Half of the cecum-free end was ligated and punctured twice with a 21-gauge needle. The cecum contents were squeezed out, the cecum was repositioned, and an incision was sutured in the muscle and skin layers. Subsequently, the rats were subcutaneously injected with saline (1 mL/100 g) at 37 °C, and returned to the cage to rewarm for 1 h. The control group had an abdominal incision, but their cecum was not ligated and punctured.

Bronchoalveolar lavage fluid (BALF)

The rat was anesthetized using an appropriate anesthetic agent and the anesthesia was maintained throughout the procedure. The rat was placed in a supine position on a surgical table and the limbs were secured to prevent movement. The skin was opened over the trachea using sterile surgical instruments to expose the trachea. Sterile scissors were used to make a small incision in the trachea and to insert a sterile cannula into the trachea. The lungs were infused with saline solution through the cannula and the fluid was withdrawn back into a sterile syringe. Saline solution was gently injected and aspirated several times to wash the lungs and collect the fluid. The fluid was collected in a sterile tube or container and centrifuged to remove cellular debris. The supernatant should then be stored at −80°C or processed immediately for analysis.

Wright–Giemsa staining

For each bronchoalveolar lavage fluid (BALF) sample, centrifugation was carried out at 300g for 10 min using a cytocentrifuge, utilizing a slide to immobilize the cells. Subsequently, the slide was subjected to staining using a Wright–Giemsa Stain Kit (ab245888, Abcam), following the instructions provided by the manufacturer.

Histological staining

After euthanizing the rats, the lung tissues were taken out and fixed in neutral formaldehyde solution (10%) for 24 h, and then dehydrated with gradient concentration ethanol (70%, 80%, 90%, 95%, and 100%). After xylene (Solarbio, China) was transparent, the lung tissues were embedded in paraffin and cut into 5-μm sections. The sections were treated with xylene for 4 min, differentiated in ethanol hydrochloric acid for 10 s, rinsed and stained with eosin (Solarbio, China) solution again for 2 min, fixed with neutral gum, and then the lung injury and inflammatory cell infiltration were observed under a DMM-300D microscope (Caikon, China). The experiment was repeated in triplicate.

PAS staining

PAS (Periodic acid–Schiff) staining is a special staining technique that can detect the presence of complex carbohydrates and other glycoproteins in tissue sections. Generally, after deparaffinizing and rehydrating them using xylene and graded ethanol solutions, subsequently the slides were washed in distilled water for 5 min. The sections were incubated with 0.5–1% periodic acid at room temperature for 5–10 min, followed by a rinsing of the sections with distilled water for 5 min. The sections were incubated with Schiff reagent at room temperature for 15–30 min and kept protected from light. The sections were rinsed with running tap water for 5–10 min. To counterstain the sections, they were incubated with hematoxylin for 1–2 min, followed by a rinsing with tap water for 5–10 min. The sections were dehydrated using graded ethanol solutions, and then cleared in xylene. Finally, the sections were mounted with a suitable mounting medium.

Immunohistochemical staining

The extracted rat lung tissue was dewaxed until hydration after routine inclusion and then dropped with 3% H2O2 at room temperature for 15 min, then with normal goat serum blocking solution at room temperature for 15 min. After cleaning, 50 μL iNOS, Arg1, Spc-1, phos-p65, and phos-Ikbkb (Abcam, UK) primary antibody were added and incubated at 4℃ overnight. After that, secondary antibody (1:500, ab199091, Abcam) was added and incubated at 37 ℃ for 15 min, and then 40 μL horseradase-labeled streptomycin working liquid was added and incubated at 37 ℃ for 15 min for DAB color reaction. After rinsing with distilled water, hematoxylin was used for restaining for 30 s, and then the tablets were dehydrated and sealed. Five nonoverlapping visual fields were selected for observation in each section under the microscope, and cells showing brown–yellow or brown–brown particles in the nucleus were positive cells. All experiments were conducted independently three times, and average values were taken for analysis.

TUNEL assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay is a commonly used technique to detect apoptotic cells in tissue sections. Briefly, 5-µm thick sections were mounted on glass slides. Next, the sections were deparaffinized using xylene or other deparaffinization agents and rehydrated through graded alcohol solutions. The TUNEL reaction mixture was prepared according to the manufacturer’s instructions and applied to the tissue sections, incubating for 1 h at 37 °C in a humidified chamber. After incubation, the sections were washed with PBS buffer to remove unbound TUNEL reagents. The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and then mounted using mounting medium and coverslip. Finally, the sections were observed under a fluorescence microscope or confocal microscope to detect apoptotic cells.

Lung permeation determination

Rats were utilized in these experiments and were anesthetized prior to insertion of a catheter into the jugular vein. Subsequently, Evans blue (EB) dye was administered intravenously, and it traveled through the blood vessels to reach the lung tissue. After 1–2 h, the animals were sacrificed, and the lungs were harvested, followed by a saline flush to eliminate any excess dye. The lung tissue was then homogenized in a buffer solution to extract the EB dye that had accumulated in the tissue. Finally, a spectrophotometer was used to measure the amount of EB dye in the tissue, which provided a measure of the lung tissue’s permeability.

RNA-seq

The lung tissue samples in three groups (n = 6 for each) for mRNA-sequencing (mRNA-seq) were collected as described below in the animal experiments section. These lung tissue samples were conducted using a CloudSeq mRNA enrichment kit and Illumina HiSeq sequencer (Thermo Fisher Scientific, Waltham, MA, USA) by Jgenebook Biotech Ltd. (Wuhan, China). Differentially expressed genes (DEGs) between the ALI model rats and sham rats were identified using Cuffdiff software (part of the Cufflinks software). The thresholds of the DEGs were set as fold change (FC) log |FC| ≥ 1.0 and P ≤ 0.05 with a fragments per kilobase million (FPKM) value ≥ 0.1, and at least one criterion was expected to be satisfied. Differentially expressed mRNA clustering was performed using FPKM values with the heat map function of R package. The RNA-seq data were deposited in the GEO database (the GEO no. is not available yet owing to the verification).

Immunofluorescence

To preserve protein structure and prevent degradation, the cells or tissue sample were fixed with paraformaldehyde. The cells or tissue were permeabilized using Triton X-100, and nonspecific binding sites on the sample were blocked with BSA to prevent the primary antibody from binding nonspecifically. Afterward, the sample was incubated with a primary antibody that specifically binds to the protein of interest. The primary antibody bound to the target protein, and any unbound primary antibody were removed through washing. Next, the sample was incubated with a fluorescent dye-conjugated secondary antibody, which bound to the primary antibody. Then, the sample was washed to remove any unbound secondary antibody and mounted on a slide with DAPI. Finally, a fluorescence microscope was used to analyze the sample.

qRT–PCR

Using the RNeasy Plus Mini kit (Tsingke, China), RNA in the cell was extracted from BEAS-2B cells and tissues. RNA expression was evaluated using the 2−ΔΔCT method, and GAPDH was used as an internal reference for genes.

Western blotting (WB)

First, the protein amount was quantified to extract proteins from cells or tissues. Next, the protein sample was mixed with a loading buffer, heated, and loaded onto a polyacrylamide gel that contained molecular weight markers. An electric current was applied to the gel to separate the proteins by their molecular weight. Then, the separated proteins were transferred from the gel to the polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with a blocking buffer to prevent nonspecific binding of the primary antibody. Afterward, the membrane was incubated with a primary antibody that specifically recognizes the protein of interest. Subsequently, the membrane was incubated with a secondary antibody that was conjugated with an enzyme, which bound to the primary antibody. A substrate was added to the membrane that reacted with the enzyme conjugated to the secondary antibody, producing a visible signal. Finally, a chemiluminescence imaging system was used to capture and analyze the signal intensity.

Coculture

THP-1 monocytes (no. GDC0100, CCTCC) were cultured in RPMI 1640 supplemented with 1% penicillin–streptomycin and 5% FBS. To induce THP-1 monocytes to become M0 macrophages, 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma–Aldrich, Shanghai) was added to the cells (106 cells/mL) and incubated for 24 h. THP-1 cells with a concentration of 106 cells/mL were inoculated into the upper chamber of the 24-well Transwell plate, and BEAS-2B (no. GDC0139, CCTCC) containing each group were inoculated into the lower chamber of a Transwell system (105 cells/mL). After coculture for 24 h, cells were collected for subsequent detection.

CCK-8

BEAS-2B were cultured on 96-well cell culture plates with 5 × 103 cells in each well. After 72 h of culture, CCK-8 solution (Thermofisher, USA) was added to the medium at the ratio of 10 mL/100 mL and incubated at 37℃ for 4 h. Optical density (OD) was measured at 450 nm using an iMark microplate reader (Bio-Rad, USA). Cell viability was determined by calculating absorbance values from reference standard curves. The analysis was repeated in triplicate.

Flow cytometry

To perform flow cytometric analysis to identify M1 or M2 macrophages, single-cell suspension was prepared from the BALF fluid. Following this, cells were incubated with antibodies that were specific to surface markers associated with M1 (CD86) or M2 (CD206) macrophages. The cells were washed and fixed with formaldehyde, and then permeabilized with a permeabilization buffer. Cells were incubated with FITC-labeled CD86 and PE-labeled CD206 antibodies. Then, the analysis was performed by BD FACS (BD, USA), and gating was done on the macrophage population based on size and granularity. The percentage of M1 or M2 macrophages was determined by analyzing the expression of CD86 or CD206.

EdU assay

First, cells of interest were plated in a culture dish and maintained under suitable culture conditions. Next, the cells were incubated with a medium containing EdU, which may vary based on the experimental conditions and cell type. Following this, the EdU-containing medium was removed and cells were washed with PBS buffer. It was essential that the cells were fixed using formaldehyde to retain the incorporated EdU. Then, the cells were permeabilized with a suitable permeabilization buffer. After that, a click chemistry reaction mixture was added containing a fluorescent probe to the fixed and permeabilized cells, and incubated for 30 min. Subsequently, the cells were washed with a suitable buffer to eliminate the unreacted click chemistry reagents. Finally, the cells were analyzed using a fluorescence microscope.

ELISA

The BALF fluid was taken and put into 4 ℃ for 30 min, and then centrifuged at 3000 rpm. An ELISA kit (Abcam, UK) was used to detect the concentration of inflammatory factors in BALF based on rat interleukin 4 (IL-4), IL-5, and IL-13.. A 100 μL standard working solution or sample was added into the corresponding plate hole and incubated at 37 ℃ for 90 min; 100 μL biotinated anti-TNF-α or IL-6 working solution was added and incubated at 37 ℃ for 60 min, and then 100 μL HRP enzyme conjugate working solution was added and incubated at 37 ℃ for 30 min. The 90 μL substrate solution was added and incubated at 37 ℃ for about 15 min. Finally, 50 μL termination solution was added. The iMark microplate reader (Bio-Rad, USA) was used to immediately take readings at a 450 nm wavelength.

ChIP–qPCR

BEAS-2B cells were directly fixed in the culture medium by adding formaldehyde (HCHO) to achieve a final concentration of 1%. The formaldehyde concentration was derived from a 37% HCHO–10% methanol stock solution (Calbiochem). Following a 20-min incubation with 125 mM glycine to neutralize excess formaldehyde, cells were washed with ice-cold phosphate-buffered saline (PBS) and then lysed in a solution containing 50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, and protease inhibitors. This mixture was incubated at 4 °C for 30 min. To induce chromatin fragmentation, a COVARIS S220 focused-ultrasonicator was employed, resulting in DNA fragments spanning from 200 to 1000 base pairs. Subsequently, the chromatin was diluted tenfold in a dilution buffer comprising 20 mM Tris pH 8.0, 2 mM EDTA, 1% triton X-100, 150 mM NaCl, and protease inhibitors. Immunoprecipitations were performed with antibody complexes for Irf7 and phos-p65, conducted overnight at 4°C. The resulting immunocomplexes were subjected to multiple washes. This included three washes with buffer TSEI (20 mM Tris pH 8.0, 2 mM EDTA, 1% triton X-100, 150 mM NaCl, 0.1% SDS, and protease inhibitors), followed by three washes with buffer TSEII (20 mM Tris pH 8.0, 2 mM EDTA, 1% triton X-100, 500 mM NaCl, 0.1% SDS, and protease inhibitors). Additionally, a single wash was performed with buffer TSEIII (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP40, 1% deoxycholate, and protease inhibitors), followed by a final wash with TE pH 8.0. The immunocomplexes were subsequently extracted in TE buffer containing 1% SDS, and protein–DNA cross-links were reversed by overnight heating at 65 °C. DNA was extracted using a PCR purification kit from Qiagen. For each PCR, one-tenth of the immunoprecipitated DNA was utilized, employing primers specific to the promoter region.

Luciferase reporter assay

Cells at subconfluent levels were transfected using Lipofectamine Plus (Invitrogen) with 100 ng of a SRG3 or Cox-2-luciferase reporter gene plasmid and 2 ng of the Renilla control reporter pRL-CMV (Promega). After a 48-h period, the cells were subjected with IRF7 expression vector or phos-p65 specific antagonists maslinic acid. To evaluate the promoter activity level, the firefly luciferase activity was quantified relative to the Renilla luciferase activity utilizing the Dual Luciferase Assay System (Promega), in accordance with the manufacturer’s instructions.

Statistical analysis

SPSS22.0 (IBM SPSS Statistics, USA) was used for statistical analysis. Measurement data were expressed as mean ± SD, and unpaired t test was used for comparison between the two groups. One-way ANOVA or two-way ANOVA and Tukey’s postmortem test were used for data comparison among multiple groups. Log rank test was used for post-statistical analysis, with P < 0.05 indicating statistically significant differences.