Reagents

CAP (HY-10448), SIRT3 selective inhibitor 3-TYP (3-(1H-1, 2, 3-triazol-4-yl) pyridine, HY-108331) and Phen Green™ SK (PGSK,) diacetate were procured from MedChemExpress Company (Shanghai, China). The ferroptosis selective inhibitor ferrostain-1 (Fer-1) was sourced from Targetmol (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for quantifying tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) were obtained from Multisciences (Lianke) Biotech Company (Hangzhou, China). The tissue iron content assay kit and the mitochondrial membrane potential (MMP) fluorescent probe (JC-10) were acquired from Solarbio (Beijing, China). Kits for detecting total protein, malondialdehyde (MDA), glutathione (GSH), manganese superoxide dismutase (Mn-SOD), ATP, and ROS levels were purchased from Beyotime Biotechnology (Shanghai, China). C11- BODIPY581/591 and the cell counting kit-8 (CCK8) were procured from Glpbio (Montclair, CA, USA). SLC7A11 (12509; Affinity), GPX4 (sc-166570; Santa), SIRT3 (10099; Proteintech), and β-actin (4970; Cell Signaling Technology) were employed as primary antibodies. Goat anti-rabbit IgG H&L (ab216773) was sourced from Abcam (Cambridge, MA, USA), and the goat anti-mouse IgG H&L (92632210) from LI-COR (NE, USA). Cy3-labeled goat anti-rabbit IgG (GB21303) and Cy3-labeled goat anti-mouse IgG (GB21301) were purchased from Servicebio Technology Co., Ltd (Wuhan, China). Specific primers for SLC7A11, GPX4, and SIRT3 were synthesized and provided by Sangon Biotech (Shanghai, China).

Establishment of the VILI mouse model

Male C57BL/6 J (wild-type) mice, aged 6–8 weeks and weighing approximately 25 ± 2 g, were procured from the Animal Center of Guangxi Medical University (Nanning, China). They were placed in controlled situations at indoor temperature of 22 °C ± 2 °C, a dark/light period of twelve hours, and free obtain standard water, food. Animals were randomly assigned to five groups: the control group (CON group, spontaneous breathing after tracheal intubation for 4 h), HTV group (HTV group, MV with HTV of 20 ml/kg for 4 h), HTV + Fer-1 group (Fer-1 [1 mg/kg] administered intraperitoneally for 14 consecutive days before MV [20 ml/kg for 4 h]) (Ling et al. 2023), CAP + HTV group (CAP administered intraperitoneally for 3 consecutive days at various concentrations [0.5, 1, or 2 ml/kg] before MV [20 ml/kg for 4 h]) (Chen et al. 2021b), and 3-TYP + CAP + HTV group (3-TYP [50 ml/kg] administered intraperitoneally every 2 days for a total of three times (Liu et al. 2022a; Zhai, et al. 2017), with CAP [1 ml/kg] administered on the final 3 days preceding MV [20 ml/kg for 4 h]). The VILI animal mode was developed according to our prior papers (Ye et al. 2020; Liao et al. 2021).

After concluding MV or spontaneous breathing, mice were euthanized through the intraperitoneal administration of a lethal anesthetic dose. Bronchoalveolar lavage fluid (BALF), Lung tissue, Blood serum, were then harvested for subsequent experimental analysis. It is crucial to highlight that all animal procedures must be executed with the utmost care to minimize inflammation induction.

Histopathological analysis

The lung tissue was put in a 4% formaldehyde solution before being put in paraffin. These tissue parts were assigned to 4-µm-thick pieces and mounted onto slides. Since dewaxing, these slices were carefully stained with H&E, next, scrutinized under a light microscope. The extent of lung damage was assessed using standardized ALI protocols, which was demonstrated before (Matute-Bello et al. 2011).

Immunofluorescence staining

Immunofluorescence was conducted as described previously (Zhang et al. 2017). Put simply, the lung tissue Sects. (4 µm) underwent immunofluorescent staining following a series of dewaxing and hydration. Primary antibodies against SLC7A11 (1:200 dilution), GPX4 (1:200 dilution), and SIRT3 (1:200 dilution) were applied. Cy3-labeled goat anti-rabbit IgG (1:300 dilution) or Cy3-labeled goat anti-mouse IgG (1:300 dilution) served as fluorophore-conjugated secondary antibodies. Nuclei were stained using DAPI. The stained lung tissue specimens were subsequently observed under a fluorescence microscope (Nikon EclipseC1, Nikon). The mean fluorescence intensity was analyzed with ImageJ 1.53.

Inflammatory responses

A crude method to gauge lung exudation during VILI involves computing the wet/dry (W/D) ratio. The wet lung, specifically the middle lobe of the right lung, next, dried in a 60 °C oven for 48 h. Pulmonary permeability was assessed by quantifying total protein levels in the BALF supernatant using a bicinchoninic acid (BCA) assay. Cells in BALF were enumerated using a hemocytometer to gauge inflammatory infiltration. Concentrations of IL-1β, IL-6, and TNF-α in lung tissue were identified with ELISA kits, adhering to the producer's guidelines.

Assessment of the iron level in vivo

To ascertain ferroptosis levels, the iron content in the lung tissue was examined using an iron assay kit according to the manufacturer’s recommendations.

Establishment of an in vitro model of VILI

Mouse lung epithelial cells (MLE12) were propagated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, USA), and maintained at a temperature of 37 °C with 5% CO2 in a humidified air environment. For a stable knockdown of the SIRT3 gene in MLE12 cells, lentiviral vectors GV248 and GV358 offered by Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China) were employed. Following this, an in vitro VILI model was developed based on previously reported studies (Ling et al. 2023; Wang et al. 2011; Jing, et al. 2020; Carta et al. 2011). Briefly, cells of logarithmic growth phase were inoculated in standard density onto six-well BioFlex plate (Flexcell International). After 24 h of cultivation, cells underwent cyclic stretching (CS) using the FX 4000 T Flexercell Tension Plus system, which is equipped with a 25-mm loading station (Flexcell International, McKeesport, PA, USA), the frequency is 30 cycles/min (0.5 Hz), maintaining a 1:1 stretch/relaxation rate and a sine wave pattern. Pathological CS in our study was set at a 20% alteration in the basement membrane surface area, equating to 80% of the total lung capacity. Cells were stretched for 4 h at 37 °C in a 5% CO2 humidified incubator. The entire operation was rigorously managed using computer software.

To investigate whether CAP could curtail ferroptosis in cells experiencing pathological CS, cells in the 20% group were pre-treated with various concentrations of CAP (1, 10, and 100 μM) for 1 h (Zhou et al. 2020). After the cells were stretched for 4 h, cell pellets were harvested for next tests.

Cell viability measurement

Cell viability was ascertained with the CCK-8 assay kit. Post respective treatments, cells were settled in 96-well plates for 12 h. The culture medium was replaced with fresh medium supplemented with 10% CCK-8 reagent and incubated at 37 °C for 1 h. The absorbance (optical density) was recorded with a microplate reader at 450 nm.

Evaluation of lipid oxidation in vitro

Lipid oxidation in MLE12 cells was evaluated using C11-BODIPY581/591. Cells were treated with C11-BODIPY581/591 (20 μM) at 37 ℃ for 10 min and then washed with phosphate-buffered saline (PBS). Nuclei were stained with DAPI. Similarly, intracellular chelating iron was quantified using PGSK diacetate. Cells were treated with 20 μM PGSK at 37 °C for 15 min, and outcomes observed using fluorescence microscopy (Leica, Germany) and analyzed with ImageJ 1.53.

Transmission electron microscopy

The procedures utilized were consistent with those described in in our past paper (Wu et al. 2013). Briefly, the lung tissue was procured within 1–3 min after modeling and then sectioned into 1–2 mm cubes. In 0.1 M sodium acetate buffer (pH 7.4), fixed at 4 ℃ for at least 2 h with 2.5% glutaraldehyde and 2.5% paraformaldehyde. The cubes were subsequently treated with the same buffer containing 1% osmium tetroxide for 2 h at room temperature, followed by dehydration in graded alcohols and propylene oxide. The specimens were put in resin, cut to ultrathin sections, stained with uranyl acetate and lead citrate, and identified under an HT7800 transmission electron microscope (Hitachi, Japan). Likewise, MLE12 cells were gathered for transmission electron microscopy (TEM) to inspect cell damage and mitochondrial ultrastructure.

Single-cell suspensions

For ROS measurements, lung tissue was processed to prepare a monoplast suspension (Koppula et al. 2018). The whitening regions in the lungs were ground and subjected to digestion with 5000 U/mL collagenase type IV and 20 U/mL DNase at 37 °C for 40 min on a shaking bed. The resultant mixture was then filtered through a 100-mm cell strainer to secure single-cell suspensions.

Evaluation of mitochondrial damage

ROS assay kit was utilized to detect ROS extents. Cells were incubated with 10 mM DCFH-DA at 37 °C for 30 min. After two washes with PBS, the fluorescence was quantified via flow cytometry. MMP was gauged using JC-10 staining, adhering to the producer's structure, and visualized utilizing a fluorescence microscope. Moreover, ATP concentrations were ascertained through firefly luciferase-associated chemiluminescence, in compliance with the producers' guidelines.

Measurement of oxidative damage markers in mitochondria

Relative concentrations of MDA, Mn-SOD, and GSH in tissue or cell lysates were evaluated using the appropriate commercial kits. Notably, all experimental procedures were rigorously followed as per the manufacturer’s guidelines.

Real-time polymerase chain reaction

mRNA expression levels for SLC7A11, GPX4, and SIRT3 were detected using real-time polymerase chain reaction (PCR) and specific primers on a real-time PCR system, as previously detailed (Wu et al. 2013). The primer sequences were as follows (5′ to 3′):

Mouse SLC7A11-F GCTGACACTCGTGCTATT.

Mouse SLC7A11-R ATTCTGGAGGTCTTTGGT.

Mouse Gpx4-F GCCTGGATAAGTACAGGGGTT.

Mouse Gpx4-R CATGCAGATCGACTAGCTGAG.

Mouse SIRT3-F CCACGACAAGGAGCTGCTTCTG.

Mouse SIRT3-R ACCCTGTCCGCCATCACATCA.

Mouse β-actin-F CCACGACAAGGAGCTGCTTCTG.

Mouse β-actin-R ACCCTGTCCGCCATCACATCA.

β-Actin was used as an internal standard for normalization, applying the 2−△△Ct quantification method.

Western blot analysis

Total proteins were isolated from lung tissue and MLE12 cells utilizing radio-immunoprecipitation assay buffer enriched with protease inhibitors. Concentrations were identified with the BCA protein assay kit. Equal amount of protein, along with the molecular weight marker, were loaded onto sodium dodecyl sulfate–polyacrylamide gels, next shifted to polyvinylidene fluoride film. Since blocking with a western blot rapid blocking buffer for 15 min at indoor temperature, the membranes were treated with primary antibodies for SLC7A11 (1:700 dilution), GPX4 (1:100 dilution), SIRT3 (1:1000 dilution), β-actin (1:1000 dilution). This was succeeded by incubation with secondary antibodies: either goat anti-rabbit IgG H&L (1:15,000 dilution) or goat anti-mouse IgG H&L (1:20,000 dilution). Finally, western blotting strips were visualized utilizing Odyssey two-color infrared laser imaging system (LICOR, America).

Statistical analyses

SPSS 26.0 software (IBM, USA) was utilized for statistical analysis. All data were presented as mean ± standard deviation. Differences were assessed using analysis of variance, with subsequent least significant difference-t test and the Student–Newman–Keuls test for pairwise comparisons. A p-value of below 0.05 was deemed statistically significant.