RNA was extracted from cultured cells using TRIzol (Invitrogen, USA). Normalized RNA was reverse transcribed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA), and cDNA was analyzed via qRT‒PCR with a 7500 Fast Real‒Time PCR System (Applied Biosystems, USA). Subsequently, relative mRNA levels were calculated using the comparative CT method and normalized to 18S rRNA mRNA. The average of the control group was set as one, and all the results are presented as the relative mRNA expression. The sequences of all the primers used are listed in Table 1.

Table 1 Primers SequencesApoptosis Determination by Flow Cytometry

For apoptosis determination, an Annexin V-FITC-PI kit (AP105-60-kit, Lianke, China) was used. NR8383 cells (1 × 106 cells/ml) were collected, suspended in 200 μL of 1 × binding buffer and subsequently stained with 5 μL of Annexin V-FITC and 10 μL of PI to surface markers at 4 °C for 20 min in the dark. Then, the stained single cells were analyzed using a BD Accuri C6 flow cytometer (BD Biosciences, USA). The data were analyzed with BD Accuri C6 Software (BD Biosciences, USA). Gates were constructed to identify target populations based on surface marker staining. Apoptotic cells were identified as Annexin V-FITC + and PI-, and necrotic cells were identified as Annexin V-FITC + and PI + .

Immunofluorescence Imaging

The NR8383 cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 at room temperature for 20 min, and blocked with 5% BSA at room temperature for 30 min. The cells were then incubated with an AT2R antibody (1:200, ab19134, abcam, USA) or a Giα antibody (1:200, ab20392, abcam, USA) overnight at 4 °C, followed by incubation with a secondary antibody (1:800) for 1 h at 4 °C. The nuclei were counterstained with 10 ng/ml DAPI (D9542, Sigma, USA) for 15 ~ 30 min at 4 °C. Images were scanned and analyzed using a fluorescence microscope (Zeiss 880, Germany).

Western Blot

For western blotting, proteins were extracted from the NR8383 cells in cold RIPA buffer (Beyotime Biotechnology, China). A total of 40 µg of protein was separated via SDS‒PAGE and subsequently transferred onto a PVDF membrane (0.22 µm, IPVH00010, Millipore, USA) via a Mini-Protein Tetra System (Bio‒Rad, USA). Then, the membranes were blocked with TBST (Tris-buffered saline plus 0.1% Tween-20) with 5% fat-free milk for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. The next day, the membranes were washed in TBST (3 × 10 min) and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. After the membranes were washed with TBST (4 × 10 min), ECL Plus Western blotting substrate (Beyotime, China) was added, and the samples were incubated for 2 min to develop the chemiluminescent signal. A ChemiDoc XRS + System (Bio-Rad, USA) was used to visualize the protein bands. pERK1/2 was normalized to total ERK1/2, pP65 was normalized to total P65, and the other proteins were normalized to GAPDH.

The following commercial antibodies were used: anti-p65 (4764s, Cell Signaling Technology, USA), anti-P50(1559–1, EPITOMICS, USA), anti-phospho-ERK1/2(43,703, Cell Signaling Technology, USA), anti-GAPDH(Mab5465, Lianke bio, China), anti- phospho-p65(sc-33020, Santa, USA), anti- phospho-IKBα(sc-101713, Santa, USA), Goat anti-Rabbit IgG(GAR0072, Liankebio, China), Goat anti-Mouse IgG(GAM007, Liankebio, China). All the primary antibodies used were diluted 1:1000 in primary antibody dilution buffer (Beyotime, China). Secondary antibodies were used at a 1:5000 dilution in secondary Antibody Dilution Buffer (Beyotime, China).

Animals and Modeling

Sixty-four male SD rats, SPF grade and weighing approximately 250–280 g, were purchased from SLAC Biotechnology. All animals were kept in rooms at a temperature of 23 ± 2°C, a 12-h light/dark cycle, and a relative humidity of 40%-80%. All the experimental protocols were approved by the Animal Ethics Committee.

Mechanical ventilation for 2, 4, 6, or 8 h was selected to establish the VILI model, and VILI rats were pretreated with C21 (1 mg/kg) in vivo. Sixty-four male SD rats were randomly divided into 8 groups: the normal control group, 4 VILI model groups (2 h, 4 h, 6 h, and 8 h), and 3 VILI model + AT2R agonist groups (4 h, 6 h, and 8 h), with 8 animals in each group. The rats were fasted for 12 h before the experiment and allowed to drink freely. The VILI model + AT2R agonist group received C21 (1 mg/kg) by intraperitoneal injection 30 min before surgery. The trachea was exposed during surgery (the control group underwent tracheotomy without mechanical ventilation), and a 16G needle was inserted into the trachea to serve as a tracheal intubation tube and connected to a small animal ventilator for mechanical ventilation. The parameters of the ventilator were adjusted as follows: RR = 40 times/min, I: E = I: 2, PEEP = 0, inhaled oxygen concentration (FiO2) = 21%, and tidal volume Vt = 40 mL/kg. Lung tissue, bronchoalveolar lavage fluid and peritoneal lavage fluid were collected after 2 h, 4 h, 6 h, and 8 h of ventilation.

Histopathological Analysis of the Lung

The right upper lobe lung tissue was fixed with 4% formaldehyde. After paraffin embedding, slicing, and hematoxylin and eosin staining, pathological changes were observed via light microscopy (CHX43; Olympus Corporation, Tokyo, Japan). The right lung middle lobe tissue of the rats was removed, the wet mass (W) was weighed, the tissue was baked in a drying oven at 80 °C to a constant mass, and the dry mass (D) was weighed to calculate the lung W/D ratio. The histopathological conditions of lung injury were scored following the Official American Thoracic Society Workshop Report [16, 17] (a) neutrophils in the alveolar space, (b) neutrophils in the interstitial space, (c) hyaline membranes, (d) proteinaceous debris filling the airspaces, and (d) alveolar septal thickening. The total score was calculated using the following formula: Score = [(20 × a) + (14 × b) + (7 × c) + (7 × d) + (2 × e)]/(number of fields × 100).

Analysis of Bronchoalveolar Lavage Fluid (BALF)

To collect BALF, left lung lavage was performed after recovery. The white blood cell (WBC) count was calculated via light microscopy. The extracted BALF was centrifuged at 2000 rpm for 6 min, and the supernatant was collected to determine the total protein and inflammatory cytokine levels. The total protein concentration in the BALF was determined using Coomassie brilliant blue staining. The inflammatory cytokines in the BALF were measured by an ELISA kit.

For alveolar macrophage polarization, M1-like macrophages were identified as CD68 + iNOS + , and M2-like macrophages were identified as CD68 + Arg-1 + . The detailed steps were as follows: the pellets were collected, 1 mL of DMEM was added to each tube for resuspension, and the mixture was transferred to a 1.5 mL EP tube and centrifuged at 1500 rpm for 5 min. The supernatant was removed, and the sediment was collected. Then, 500 μL of fixed membrane breaking solution was added to each tube and incubated overnight at 4°C. Afterwards, 1 × Permeabilization Buffer (1 mL per tube) was added to resuspend the sample on the second day. Centrifuged at 1500 rpm for 5 min, and the supernatant was removed; adding100 μL standing buffer to resuspend samples, adding CD68-PE antibody 1 μL, Arg1 antibody 0.1 μL, iNOS antibody 0.1 μL to each tube. A single staining tube and blank were set up and incubated at 37 °C in the dark for 30 min. Then, add 1 mL of DMEM culture medium to each tube in sequence, centrifuge at 1500rpm for 5 min, remove the supernatant, add 100 µL of DMEM for resuspension, and add secondary antibody 0.25 μL. Incubate in dark at 37 °C for 30 min; Add 1 mL of DMEM culture medium to each tube and centrifuge at 1500rpm for 5 min, 200 μL DMEM culture medium resuspended and tested on the flow cytometry. M1-type AMs were labeled with CD68 + /iNOS + , and M2-type AMs were labeled with CD68 + /arg-1 + .

The following commercial antibodies were used: CD68-PE(Miltenyi Biotec,130–103-363), iNOS(abcam, ab15323), Arg-1(Novus, NB100-59740), Donkey anti-rabbit IgG Secondary Antibody(Alexa Fluor® 488)(ThermoFisher, cat#A21206), Donkey anti-goat IgG Secondary Antibody(Alexa Fluor® 647)(ThermoFisher, cat#A21447). All the antibodies were diluted 1:500 in 1% BSA in PBS.

Analysis of Peritoneal Lavage Fluid (PLF)

To collect the PLF, peritoneal lavage was performed, and after recovery, the mixture was centrifuged at 2000 rpm/min for 10 min, after which the pellets were collected. Then, the cells were stained as described above for apoptosis.

Statistical Analysis

All the data are expressed as the mean ± SD. WB, PCR ELISA and flow cytometry data and fluorescence intensity data sets were analyzed by using two-way ANOVA with Tukey’s post hoc test to account for multiple comparisons in GraphPad Prism software. A value of p < 0.05 was considered to indicate statistical significance.