Male C57BL/6 mice aged 6–8 weeks with a body weight of 22 ± 5 g were purchased from the Experimental Animal Center of Guangxi Medical University. All mice were raised at 20–25°C and 30–70% relative humidity and provided with protein-containing feed and purified water. The study was approved by the Experimental Animal Committee of Guangxi Medical University, and all animal experiments were performed in accordance with local and international ethical guidelines. Age- and weight-matched controls were used in all experiments.

Before tracheal incubation, all mice were fasted for 8h and left without water for 4 h. Mice were then anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg); they were administered in 1/3 of the first dose to maintain anesthesia every 45 min. A sterile 20G intravenous trocar was used for tracheal intubation at a depth of 1.2 ± 0.5 cm. The catheter was fixed in the trachea to prevent one-lung ventilation and was connected to a small-animal ventilator (catalog no. SAR-1000, CWE) for mechanical ventilation with air. Non-ventilated mice were used as controls, while mechanically ventilated mice were ventilated with either normal tidal volume (NTV, 7 mL/kg) or high tidal volume (HTV, 20 mL/kg).

To explore the source of IL-17 in the mice treated with HTV, the mice were injected intraperitoneally with anti-Ly6G antibody (400 µg; BP0075, BioXCell) one day before ventilation to deplete neutrophils in vivo. To assess the role of IL-17 in the p38 MAPK/MCP-1 pathway after HTV ventilation, mice were randomly divided into four groups (H1-4) depending on whether ventilation lasted 1, 2, 3, or 4 h. One day before ventilation, the mice were injected intraperitoneally with anti-IL-17 antibody (400 µg; BE0173, BioXCell), and at 30 min before anesthesia 200 µg anti-IL-17 antibody was administered through intratracheal instillation. Recombinant mouse IL-17 (rmIL-17; 0.5 μg, catalog no. 576006, Biolegend) was administered through intratracheal instillation 30 min before anesthesia. HTV-ventilated mice administered with saline served as controls.

RAW264.7 mononuclear macrophage leukemia cells were purchased from the American Type Culture Collection. Cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM; catalog no. C11995500BT, Gibco), and the optimal infection conditions and multiplicity of infection were determined. In the optimized procedure, a 500 μL suspension of RAW246.7 cells and 20 μL of HitransG A infection-enhancing solution were added to each well of a 24-cell well plate. After cells reached 50% confluence, they were co-cultured with 20 μL of HitransG P infection-enhancing solution and lentivirus infection solution. After 16 h, the medium was replaced with fresh DMEM medium. At 72 h after transfection, the transfection efficiency was determined by fluorescence microscopy. Transfected cells were selected using 2 μg/mL puromycin, which was reduced to 1 μg/mL for further screening.

To clarify the mechanism of IL-17 in vitro, wild-type RAW264.7 cells were stimulated with rmIL-17(100 ng/mL) and cultured for 1, 2, 3, 4, or 5 h. Non-stimulated wild-type RAW264.7 cells served as controls. RAW264.7 cells transfected with negative-control lentivirus and stimulated with rmIL-17 for 4 h are referred to below as the CN group. RAW264.7 cells transfected with lentivirus encoding short hairpin RNA (shRNA) against p38 MAPK (Shanghai Jikai Gene Technology, Shanghai, China) and cultured with rmIL-17 for 4 h are referred to below as the shRNA group.

After the animal model was established, all mice were sacrificed by carotid artery bleeding, and their lungs were excised and weighed immediately (wet weight) and again after drying in an oven at 60°C for 72 h (dry weight). All operations were conducted carefully to minimize the risk of inflammatory activation.

The total protein concentration in BALF was estimated using the bicinchoninic acid (BCA) assay (P0012S, Beyotime) to assess pulmonary permeability. The levels of IL-17 and MCP-1were assayed in BALF and culture medium were detected using the ELISA kits (CSB-E04608m, Cusabio Biotech and EK0568, Boster) according to the manufacturers’ instructions.

The pathological damage in mouse lungs after HTV ventilation was assessed after hematoxylin–eosin (H&E) staining. Briefly, the collected mouse lungs were fixed with 3.7% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. After dewaxing, the sections were stained and the tissue morphology was observed with a microscope. The degree of lung injury was evaluated by applying a four-point scale (0, no injury; 1, minor injury; 2, moderate injury; 3, severe injury) to each of the following parameters (6, 23, 24): alveolar congestion, hemorrhage, inflammatory cell infiltration, and alveolar wall thickening. These individual scores were summed to give the total score of lung damage.

Lung tissues and RAW264.7 cells were lysed in RIPA lysis buffer (P0013B,Beyotime) supplemented with protease inhibitor (ST505, Beyotime) and phosphatase inhibitor (P1050, Beyotime), and the protein concentration in each sample was determined using the BCA assay. Samples were denatured, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The non-specific membrane binding sites were blocked with 5% bovine serum albumin (BSA) for 1 h. Membranes were then incubated at 4°C overnight with primary antibodies (all diluted 1:1000) against the following proteins: IL-17 (ab79056, Abcam), MCP-1 (catalog no. 2029, CST),p38 MAPK (catalog no. 9212, CST), and phosphorylated (p)-p38 MAPK (catalog no. 4511, CST). Blots were washed several times with PBS buffer, then incubated for 1 h with goat anti-rabbit IgG secondary antibody (diluted 1:15000; ab96899, Abcam). The bands were visualized using a fluorescent scanner. As an internal reference, GAPDH was immunostained using an antibody (1:1000; catalog no. 5174, CST).

Total RNA was isolated from lungs and RAW264.7 cells using the MiniBEST Universal RNA Extraction kit (catalog no. 9767, TaKaRa Bio, Otsu) following the manufacturer’s instructions. After determining RNA quality and quantity by spectrophotometry, cDNA was synthesized using the PrimeScript™ RT Master Mix kit(RR036A, TaKaRa) and amplified with the SYBR^® Premix Ex Taq™ II(TliRNaseH Plus) kit(RR820A, TaKaRa). The following primers were used: GAPDH forward, 5′-TGTGTCCGTCGTGGATCTGA-3′; GAPDH reverse,5′-TTGCTGTTGAAGTCGCAGGAG-3′; MCP-1 forward, 5′-CAGGTCCCTGTCATGCTTCT-3′; MCP-1 reverse, 5′-GTGGGGCGTTAACTGCATCT-3′; p38 forward, 5′-CTGTCGAGACCGTTTCAGTCCA-3′; p38 reverse,5′-GTGTGAACACATCCAACAGACCAA-3′. The relative expression of GAPDH, MCP-1, and p38 was determined using the 2^−ΔΔCt method and normalized to expression of GAPDH.

Flow cytometry was used to detect the accumulation of neutrophils in the lung tissues of mice and the expression of IL-17 in neutrophils, Th17 cells, and T cells. Lung sections were treated with 200 U/mL Type I collagenase (950 μL, 17018029, Gibco), DNaseII(20 U,4942078001, Merck), and FBS (50 μL,10099141, Gibco)to prepare single-cell suspensions. Red blood cell lysis solution (1mL) was then added to each cell suspension to remove red blood cells and adjust the density to 5 × 10⁶ mL⁻¹. Cells were labelled using antibodies against Ly6G (catalog no.REA256, Miltenyi Biotec), CD4 (catalog no. GK1.5, Miltenyi Biotec), and CD90 (catalog no. 105201, Biolegend). After washing with PBST buffer, cells were ruptured and intracellular proteins were labelled for 20 min using anti-IL-17 antibody (catalog no. 560522, BD Horizon) and analyzed at 4 °C using a flow cytometer.

Immunofluorescence was used to assess the expression of MCP-1 in RAW264.7 cells; the expression of IL-17 (catalog no.14-7179-82, Thermo Fisher Scientific), CD4 (catalog no.100401, Biolegend), CD90 (catalog no.105201, Biolegend), and Ly6G (catalog no.127601, Biolegend) in mouse lungs; and the source of IL-17 in mouse lung tissues. All sections were fixed with 3.7% paraformaldehyde, ruptured with 0.2% Triton X-100, and blocked with BSA and goat serum. After incubation with the above- mentioned primary antibodies at 4°C overnight, samples were incubated with goat anti-rabbit IgG secondary antibody for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole, then samples were observed using a confocal laser scanning microscope.

Statistical analysis was performed using SPSS 22.0 software (IBM, USA). All data were reported as mean ± standard deviation (SD). Differences in parametric data that showed a normal distribution were assessed for significance using one-way analysis of variance and the Bonferroni test. Differences associated with a two-tailed P < 0.05 were considered statistically significant.

To assess the effect of HTV ventilation on IL-17 expression, we determined the levels of IL-17 in non-ventilated, NTV, and HTV ventilated mice. The IL-17 levels in lungs were significantly higher in the HTV group than in the control and NTV groups ( Figures 1A–C ). Compared with CON group, the concentration of IL-17 in BALF also increased in HTV group ( Figure 1D ). These results suggest that IL-17 is closely associated with the development of VILI.

IL-17 is expressed mainly by Th17 cells and neutrophils (25, 26). Flow cytometry showed no significant difference in the numbers of CD4⁺IL-17⁺ cells among control, NTV and HTV groups ( Figure 2A ). IL-17 in the HTV group did not co-localize with the Th17 cell surface marker CD4 or the T cell surface marker CD90 ( Figure 2B ), suggesting that IL-17 is not produced by Th17 cells and T cells during VILI. In contrast, the number of Ly6G⁺neutrophils was significantly higher in the HTV group than in the other two groups ( Figure 3A ). Immunofluorescence shows that IL-17 co-localizes with the neutrophils surface marker Ly6G ( Figure 3B ). Furthermore, when neutrophils were depleted in mice ( Figures 3C, F ), compared with the HTV group, the levels of IL-17 in lung tissue ( Figure 3D ), BALF ( Figure 3E ) and plasma ( Figure 3G ) were significantly reduced in the HTV+anti-Ly6G group. These results indicate that IL-17 is produced mainly by Ly6G⁺neutrophils that are recruited to the lungs at the onset of VILI.

The wet weight to dry weight ratio (W/D) of mouse lungs reflects the degree of lung edema, as the fluid that accumulates in the lung interstitium, alveolar cavity, and small bronchi increases lung tissue wet weight. Moreover, lung inflammation leads to the secretion of several inflammatory molecules in the alveolar cavity. We took advantage of these phenomena to assess whether inhibiting the function of IL-17 using an anti-IL-17 antibody ( Figure 4A ) would reduce pulmonary edema and total protein levels in BALF. Indeed, treating the HTV group with anti-IL-17 antibody led to significantly lower pulmonary edema and total BALF protein ( Figures 4B, C ), as well as milder alveolar congestion, alveolar interstitial thickening, and neutrophil infiltration ( Figure 4D ). These results suggest that blocking IL-17 can mitigate inflammation caused by VILI.

The p38 MAPK, as a classic downstream molecule of TLR4, has been associated with the pathogenesis of VILI (6). RAW264.7 cells were stimulated with rmIL-17 to examine whether IL-17 may interact with p38 MAPK. RT-qPCR analysis revealed that rmIL-17 significantly upregulatedMCP-1 mRNA expression ( Figures 5A, B ), which p38 MAPK knockdown partially reversed ( Figures 5C–F ). Western blot and RT-qPCR analysis also showed that rmIL-17 promoted the phosphorylation of p38 MAPK and MCP-1, without affecting total levelsofp38-MAPK ( Figures 5A, E ). These results suggest that IL-17 upregulates the expression ofMCP-1 by promoting the phosphorylation of p38 MAPK in lung macrophages.

To explore whether the observed effects of IL-17 on the p38 MAPK/MCP-1 pathway in vitro also occur in vivo, HTV ventilated mice were injected with anti-IL-17 antibody orrmIL-17 before ventilation. Anti-IL-17 antibody significantly reduced MCP-1 and p-p38 MAPK expression ( Figures 6A–C ), whereas rmIL-17 exerted the opposite effects ( Figures 6D, E ). At the same time, neither treatment affected the total level of p38 MAPK in lung or BALF ( Figures 6C, E ). These results confirm that IL-17 can upregulate MCP-1 by promoting the phosphorylation of p38 MAPK.