Mouse models

A total of sixteen female C57BL/6 strain mice, aged 6–8 weeks and weighing 20–25 g, were acquired from the Shanghai Animal Laboratory Center (Shanghai, China). These mice were housed in a specific-pathogen-free animal facility and provided ad libitum access to food and water. The mice underwent adaptive feeding for a week, after which they were assigned randomly into normal and COPD model groups with 8 mice per group. A smoking chamber (measuring 70 cm × 50 cm × 40 cm) was custom-made to establish the COPD model group, as previously described in the literature [20, 21]. The mice in COPD group were placed inside the chamber and subjected to whole-body exposure to 10 cigarette smokes (each cigarette contains 1.2 mg of nicotine and 10 mg of tar) for 2 h once a day for six months. Following the final exposure, the mice were euthanized under anesthesia, and a ligation was performed on the trachea and one of the lung lobes. A puncture needle was then inserted into the upper part of the trachea. After unilateral lung perfusion (3–5 times) using 0.3 ml of PBS, the bronchoalveolar lavage fluid was obtained and stored temporarily at 4 °C. Lung tissues that were not irrigated were preserved using a combination of partial freezing at -80 °C and fixation with neutral-buffered formalin overnight.

Biological analysis of lavage solution

A sterile container was used to collect bronchoalveolar lavage fluid to count neutrophils, macrophages, and lymphocytes. The collected cells were stained using the May-Grunwald Giemsa method and analyzed through standard microscopy. The resulting cell counts were recorded.

Histology and immunohistochemistry

The lung tissues obtained from COPD and normal mice were subjected to ethanol dehydration, followed by embedding in paraffin. Subsequently, the tissues were sliced into sections measuring 5 μm in thickness. Following the process of deparaffinization and rehydration, specific sections were subjected to staining with hematoxylin (Solarbio, H8070, Beijing, China) and eosin (Sangon, A600190, Shanghai, China) for histological evaluation. Then, the alveolar wall thickness and mean alveolar septa under HE staining were measured by ImageJ software. For immunohistochemistry, selected sections of lung tissues from COPD and normal mice were deparaffinized, rehydrated, and placed in sodium citrate buffer to retrieve antigens. Subsequently, the sections were blocked with serum and subjected to incubation with primary antibodies targeting Bax (ab32503; Abcam), GPX4 (ab219592; Abcam) and GNPAT (14931-1-AP, Proteintech), which were diluted in PBS at 4 °C overnight. Afterward, the sections were incubated with an HRP-conjugated secondary antibody for 1 h at room temperature and visualized using a microscope imaging system (Nikon, Tokyo, Japan).

CSE preparation

To generate a solution of CSE for this study, a single unfiltered cigarette containing 1.2 mg of nicotine and 10 mg of tar was lit, and its smoke was continuously drawn into a sterile 10mL solution of phosphate-buffered saline (PBS) using a negative pressure suction device, resulting in a solution known as CSE. This CSE solution was purified through a 0.22 μm filter to remove any remaining cigarette residue and bacteria. Upon completion, the liquid was characterized as a CSE solution with a purity of 100%. CSE solution was diluted with DMEM medium to 0.1%, 0.5%, 2%, and 5% CSE concentration and used in subsequent experiments within 15 min after preparation.

Cell culture and intervention

The human type II alveolar epithelial cell line A549 was obtained from Procell Life Science and Technology Co., Ltd (Wuhan, China) and cultured in DMEM medium (Gibco) supplemented with 10% FBS (Gibco), penicillin (100 U/ml), and streptomycin (100 U/ml) at 37 °C with 5% CO2. According to the different experimental requirements, A549 cells were classified into different groups as follows: (1) Blank group (without treatment), 0.1%, 0.5%, 2% and 5% CSE groups; (2) Blank group, 5% CSE group, 5% CSE + DMSO group, 5% CSE + Erastin group that cells were co-treated with 5% CSE and 2.5 mM erastin (Selleck Chemicals, USA) and 5% CSE + Fer-1 group that cells were co-intervened with 5% CSE and 1 mM Fer-1 (Med Chem Express, USA); (3) A549 cells were transfected with sh-negative control (sh-NC) and sh-GNPAT (Ruibio Biotech Co., Ltd., Beijing, China) for 48 h, followed by 5% CSE treatment and divided into corresponding two groups; (4) A549 cells were transfected with overexpression plasmid SIRT4 alone or GNPAT together (GenePharma Co. Ltd., Shanghai, China), followed by 5% CSE treatment. Four groups were obtained, including blank + vector, 5% CSE + vector, 5% CSE + SIRT4, and 5% CSE + SIRT4 + GNPAT. All cell transfections were carried out using Lipofectamine 3000 as per the manufacturer’s instructions.

CCK-8 assay

Cell viability was determined using a Cell Counting Kit-8 (CCK-8) kit (CK04, Dojindo Laboratories, Japan). In brief, A549 cells from various groups (2 × 103 cells/well) were plated in 96-well plates and incubated at 37 °C with 5% CO2 for 6, 12, 24, and 36 h, respectively. Subsequently, the cells in each well were treated with a complete medium containing 10 µl of CCK-8 solution and incubated for an additional 2 h. The optical density of each well was then determined at 450 nm using a microplate reader.

LDH activity assay

The supernatants (100 µL) from different groups were collected, and lactate dehydrogenase (LDH) release was measured using an LDH Cytotoxicity Assay kit (C0016, Beyotime Institute of Biotechnology) according to the manufacturer’s instructions.

Measurement of reactive oxygen species (ROS)

The level of total ROS was determined using a 2’,7’-dichlorofluorescein diacetate (DCFH-DA) kit (Beyotime, Shanghai, China). Cells from different groups (5 × 105 cells/well) were placed into six-well plates and incubated with 10 µmol/L DCFH-DA diluted with serum-free medium for 20 min at 37 °C. After washing thrice with a serum-free medium, ROS levels were analyzed by flow cytometry.

Lipid oxidation detection

The level of lipid oxidation was detected using the C11-BODIPY (581/591) probe (Servicebio Technology, Wuhan, China), where non-oxidized lipids were stained in red and oxidized lipids in green. Briefly, cells from different groups were washed twice with PBS and incubated with 10 µmol/L BODIPYTM 581/591 C11 at 37 °C in the dark for 30 min. The fluorescence intensity was observed under an inverted fluorescence microscope (×500 magnification).

Enzyme-linked immunoassay (ELISA) assay

In short, the study involved collecting bronchoalveolar lavage fluid and cell supernatant in PBS. The levels of GPX4, IL-33, and IL-1α in both the bronchoalveolar lavage fluid and cell supernatant were measured using ELISA kits from Shanghai Enzyme-linked Biotechnology Co., Ltd (ml057982/ ml060706, ml063153/ml063084, ml002273/ml058010), following the manufacturer’s instructions.

Detection of MDA and GSH content

The MDA and GSH concentrations in lung tissue samples dissolved in extraction buffer or cellular supernatant were determined using kits from Beyotime (S0131S, S0053, Shanghai, China), following the manufacturer’s instructions. MDA and GSH levels were measured individually using a microplate reader at 450 and 405 nm, respectively.

Flow cytometry

The apoptotic rate of A549 cells was measured using an Annexin V-FITC/PI detection kit (CA1020, Solarbio, Beijing, China). Briefly, A549 cells from different groups were harvested after trypsin digestion and fixed with 70% ethanol. Then, cells were stained with Annexin V-FITC and PI in the presence of 50 mg/mL RNase A for 1 h at room temperature in darkness, which were subsequently assessed with flow cytometry.

Transmission electron microscopy

A549 cells from various groups underwent fixation with 1% osmic acid at room temperature in the dark for 2 h, dehydration using acetone, polymerization at 60 °C for 48 h, and slicing into 60–80 nm ultrathin sections. These sections were then stained with a 2% saturated uranyl acetate alcohol solution and a 2.6% lead citrate solution and observed for any changes in mitochondrial morphology under a transmission electron microscope (×50 000 magnification, Hitachi, Tokyo, Japan).

Immunofluorescence staining

The A549 cells from different groups were subjected to three washes with PBS, then fixation with 4% paraformaldehyde for 30 min. Subsequently, the cells were treated with 1% Triton X-100 for 20 min. Following three washes with PBS, the cells were blocked with 5% BSA for 1 h at 37 °C. Subsequently, they were incubated overnight at 4 °C with the primary antibody against GNPAT (14931-1-AP, Proteintech). Afterward, a fluorescent secondary antibody (1:300) was added and incubated for 2 h at 37 °C. Finally, the cells were stained with DAPI (1:4000) for 5 min before observation under a fluorescence microscope (×500 magnification).

Quantitative real-time PCR

Total RNA was extracted from lung tissues or cells using Trizol reagent from Invitrogen, USA. The extracted RNA was then reverse-transcribed into cDNA using the PrimeScript™ RT reagent kit by Takara. The PCR amplification was carried out using the SYBR Green PCR kit by Takara on the ABI PRISM Step-One Real-time PCR System, manufactured by Applied Biosystems, Carlsbad, CA, USA. The relative expression levels were determined by the 2−ΔΔCT method. Table 1 contains the PCR primer sequences used in the experiment.

Table 1 Primer pairs used for quantitative RT-PCR analysisWestern blot analysis

Total protein was isolated from lung tissues or cells using RIPS lysis buffer (Beyotime, Shanghai, China), and corresponding protein concentration was determined with BCA protein assay (Beyotime). After being mixed with loading buffer and boiled in boiling water, 30 µg proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). After being blocked with 5% non-fat milk for 2 h, the membranes were incubated with primary antibodies against GPX4 (ab219592), FAR-1 (ab202298), AGPS (ab236621), GNPAT (ab75060) and β-actin (ab8227) from Abcam overnight at 4 °C. Afterwards, the membranes were incubated with horseradish peroxidase-linked secondary antibody (1:5000) for 2 h at room temperature. Protein bands were visualized with enhanced chemiluminescence reagent (Thermo Fisher Scientific).

Immunoprecipitation assay

To perform the immunoprecipitation assay, we first lysed the cells using a lysis buffer. Next, the cell lysates were incubated with a cross-linked resin and 10 µg of GNPAT antibody (ab75060, Abcam) overnight at 4 °C. We eluted the protein complexes bound to the antibody the following day using an elution buffer. These eluted complexes were then subjected to a western blot assay using an acetyl-lysine antibody (ab190479, Abcam).

Statistical analysis

GraphPad Prism 8 software (San Diego, CA, USA) was utilized for data analysis. The results were presented as mean ± standard deviation (SD). Statistical comparisons were conducted using two-sided, unpaired Student’s t-test or one-way analysis of variance (ANOVA), followed by Dunnett’s test or Tukey’s post-hoc test as appropriate. A p-value less than 0.05 was considered statistically significant.