Animals

Eight-week-old C57BL/6J mice (20–30 g, SPF Biotechnology, Beijing, China) were obtained from the animal center of Army Medical University. The mice were kept in a Specific Pathogen-Free environment and fed a normal mouse diet. The mice can eat and drink freely, under a normal day and night cycle.

Animal models

C57BL/6 mice were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection), and placed on heating pads to maintain their body temperature. The right femoral arteries of the mice were cannulated with polyethylene tubing for arterial blood sampling. After ensuring the appropriate depth of anesthesia, the mice were intubated via tracheotomy and connected to a rodent ventilator (RWD Life Science, Shenzhen, China) with tidal volumes of 10 µl/g and a respiratory rate of 100 per minute. After thoracotomy, the hilum of the left lung of the I/R group was occluded with an atraumatic microclamp for 1 h to induce unilateral ischemia and then released for 1 h for reperfusion. The L-BAIBA treatment scheme is shown in Supplemental Fig. 1A. To determine whether L-BAIBA preconditioning has a protective effect on I/R injury, the mice were first preconditioned by the gavage of L-BAIBA (150 mg/kg/day, 150 mg L-BAIBA dissolved in 8 ml dH2O) for 10 consecutive days. Then, the left lung was injured by I/R, as described above. For post-treatment, L-BAIBA (150 mg/kg, 150 mg L-BAIBA dissolved in 20 ml saline) was given by intraperitoneal injection immediately after I/R. Only the left lung was used in these experiments.

After the lung I/R injury, blood samples were immediately obtained for arterial blood gas analysis (Gem primer 3000, Instrumentation Laboratory) and enzyme-linked immunosorbent assay (ELISA). To obtain BALF, the mouse neck skin was cut, and the thyroid gland was separated from the trachea by blunt dissection. Then a surgical suture was placed behind the separated trachea. A small incision in the upper end of the trachea was made, into which was quickly inserted a catheter (0.7 mm diameter) connected to a small animal indwelling needle (22G). The catheter and the trachea were tied together with the surgical suture. A 1 ml syringe was loaded with normal saline and gently injected into the trachea; 0.5 ml of the saline was drained by gentle suction and repeated 3 times; 0.5 ml of BALF from each mouse was collected for subsequent experiments.

Histological study

After performing the lung I/R injury, the mice were euthanized, and the lung tissues were rinsed with 30 ml pre-cooled PBS that were injected into the right ventricle to remove residual blood. The lung samples were stored in 4% paraformaldehyde for 24 h, then embedded in paraffin, sectioned, and placed on glass slides. After dewaxing and rehydrating with xylene and ethanol of different concentrations, the sections were processed by the following steps: stain with hematoxylin solution for 7 min, rinse with running water for 5 min, soak in 1% acid alcohol for 1 min, and counter-stain with eosin Y solution for 2 min. Finally, the plates were dehydrated and sealed with neutral resin. Smith pathological score was used to quantify the histological injury of the lungs, which included four indexes: pulmonary edema, alveolar and interstitial inflammation, alveolar and interstitial hemorrhage, and hyaline membrane formation (Smith et al. 1997).

Wet/dry lung weight ratio

After the mice were sacrificed, the lungs were taken and weighed (wet weight), then placed in an oven at 80 °C for 24 h, and reweighed to determine the dry weight for the calculation of the wet/dry weight ratio (Luo et al. 2022).

ELISA

To obtain the sera, the blood samples were collected from the right femoral artery by catheterization and allowed to coagulate at room temperature. Then the collected whole blood samples were quickly centrifuged at 1000 g for 5 min and the supernatants were collected. Care was taken to avoid hemolysis during the entire process. The BALF samples were centrifuged at 1000xg for 5 min and the supernatants were collected. The concentrations of IL-1β, IL-6, and TNF-α were measured by ELISA (Elabscience), according to the manufacturer’s instructions.

Measuring the activities of MDA, MPO, SOD, and the ratio of GSH and GSSG

Malondialdehyde (MDA), glutathione (GSH), and glutathione disulfide (GSSG) activities were measured using kits purchased from Beyotime Biotechnology. Myeloperoxidase (MPO) and superoxide dismutase (SOD) activity assay kits were purchased from Jiancheng Biological Engineering Institute. Briefly, the obtained lung tissues were homogenized in the sample preparation solution and then the assays were performed, according to the manufacturer’s instructions.

TUNEL staining

Paraffin sections of the lungs were dewaxed. Then, the sections were placed in an antigen repair solution and boiled in a water bath for 30 min. Thereafter, the boiled sections were sealed for 1 h after natural cooling. Then, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining solution, prepared according to the manufacturer, was added to the specimens and incubated at 37℃ for 30 min. Finally, the sections were dried and sealed with DAPI-containing immunostaining tablets. The images under a laser confocal microscope were obtained.

DHE staining

Superoxide production in the lung was measured using the fluorescent dye dihydroethidium (DHE, Beyotime). For lung tissues, frozen sections of the lung were stained with DHE (10− 5 M) for 30 min in 37 °C. For cell samples, the cells were cultured on the cell slide. After the cell treatments, the cell slides were stained with DHE (10-5 M) for 30 min in 37 °C. Then, the cells were fixed with 4% paraformaldehyde for 15 min. After the tissues or slides were washed by PBS three times, the images were taken by a confocal microscope at an excitation wavelength of 490 nm and an emission wavelength of 590 nm. All sections were processed under the same conditions. The DHE fluorescence intensity was quantified by ImageJ (NIH, Bethesda).

Western blotting

The total proteins were isolated from the lung tissues or cells. 20 mg of lung were lysed in 100 µl RIPA buffer, containing protease and phosphatase inhibitors cocktails (Solarbio), with Benchmark Beadblast tissue homogenizer (Benchmark Scientific). The protein concentration was measured with bovine serum albumin as a reference standard, according to BCA Protein Assay Kit (Beyotime Biotechnology). Nuclear and Cytoplasmic Protein Extraction Kit was purchased from Beyotime Biotechnology, and used according to the manufacturer’s instruction.

SDS-PAGE was performed using 8%, 10%, and 15% concentrations of SDS. The proteins separated by SDS-PAGE were transferred onto nitrocellulose filter membranes (Millipore Sigma). The nitrocellulose membranes were cropped according to molecular weights of the proteins and incubated with the appropriate antibodies. Image J (NIH, Bethesda, MD) was used to analyze the density of each band.

Antibodies used in this study were: anti-β-actin (1:500, Santa Cruz, SC-130,657), anti-H3 (1:3000, Cell Signaling Technologies, #4658), anti-GPX4 (1:500, Proteintech, 67763-1-Ig), anti-SLC7A11 (1:1000, Invitrogen, PA1-16893), anti-Nrf-2 (1:1000, Cell Signaling Technologies, #12721; 1:500, Proteintech, 80593-1-RR), anti-P53 (1:1000, Proteintech, 60283-2-Ig), anti-BAP1 (1:500, Affinity, AF7925), anti-PARP1 (1:1000, Proteintech, 22999-1-AP), anti-ATF3 (1:500, Santa Cruz, sc-81,189), anti-AMPK (1:1000, Cell Signaling Technologies, #2532), and anti-Phospho-AMPK (1:1000, Cell Signaling Technologies, #2535).

A/R (anoxia/reoxygenation)-induced injury in A549 or BEAS-2B cells

A549 cells (Procell Life Science & Technology, Wuhan, China), which are human pulmonary alveolar cells, obtained from lungs with carcinoma, were cultured in DMEM high glucose medium (Gibco), containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Beyotime). BEAS-2B cells, which are human bronchial epithelial cells (American Type Culture Collection), were cultured in bronchial epithelial cell basal medium, supplemented with BEGM (Lonza/Clonetics Corporation), containing 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were cultured at 37℃ in 95% air and 5% CO2 atmosphere. After changing the incubation medium to serum-free medium, the cells were placed in an anoxic chamber with 5% CO2 and 95% N2 at 37℃ for 2 h, followed by reoxygenation in 95% air and 5% CO2 atmosphere for another 2 h. Exogenous L-BAIBA was added to the cells (100 µM-100 nM, final concentrations) 48 h before they were placed inside the anoxic chamber.

Plasmid transfection and anti-oxidant response element (ARE) luciferase activity assays

DH5-competent E. coli was used for plasmid screening and amplification. A549 cells were seeded in 6-well plates and transfected with Lipofectamine 3000 at a cell confluency of approximately 50%, according to the standard experimental procedures provided in the manufacturer’s instructions. For the luciferase activity assay, the double-stranded oligonucleotide tandem repeats sequence spanning the Nrf-2 binding site 5-tgactcagca-3 was inserted into the restriction site of the pGL2 promoter plasmid to construct the ARE luciferase vector. Then, the cell lysate was mixed with the luciferase substrate solution, and the luciferase activity was measured with a luminometer (Xiao et al. 2018).

CCK8 and LDH release assay

A549 or BEAS-2B cells were seeded into 96-well culture plates (Corning, Lowell, MA). The L-BAIBA preconditioning group was treated with different concentrations (10− 7 to 10− 4 M) of L-BAIBA (Sigma) for 48 h. In addition, Nrf-2, AMPK, ERK, JNK, and PI3K inhibitors (Brusatol, Compound C, PD98059, SP600125, and LY294002, respectively) were added into the cell medium 30 min before preconditioning with L-BAIBA. RSL-3 and Erastin, two ferroptosis inducers, were added into the cell medium 24 h before hypoxia. Compound C, PD98059, SP600125, and LY294002 were purchased from Sigma-Aldrich. Brusatol, RSL-3 and Erastin were purchased from MCE.

A549 and BEAS-2B cells were allowed to grow to sub-confluence (70–80%). After the cells were subjected to A/R, CCK8 reagent (Bimake) and complete medium were added to the 96-well plates at a ratio of 1:9, then incubated at 37 °C for 1 h. Thereafter, the absorbance measured at 450 nm. LDH was measured by LDH cytotoxicity assay kit (Beyotime Biotechnology). 120 µl cell culture supernatants were collected and placed in a new 96-well plate. Then, 60 µl LDH detection working solution were added into each well, and incubated in the dark at 37℃ for 30 min. Finally, the absorbance was measured at 490 nm.

Fe2+ staining

The cells were seeded in glass-bottomed Petri dishes (Thermo Scientific). After the cells were treated, 1 µl Fe2+ indicator storage solution (1 mM, dissolved with DMSO, Maokang Biotechnology) was added to 1 ml cell culture medium (final concentration 1 µM) and incubated for 30 min at 37℃ with 95% air and 5% CO2. After loading, the cells were washed with PBS. Fluorescent images were captured at 40 X total magnification using Olympus IX83 microscope (Olympus, Lake Success, NY).

Immunofluorescence staining

The cells, cultured on glass slides, were fixed with 4% paraformaldehyde for 15 min after the treatments. Then, the glass slides with the cells were incubated with immunostaining blocking solution for 1 h at room temperature to prevent nonspecific antibody binding. Then, the cells in the glass slides were incubated with anti-Nrf-2 antibody (1:100) at 4℃ overnight. After washing with PBS 3 times (5 min each time), the cells in the glass slides were incubated with the secondary antibody, Cy3-labeled goat anti-rabbit IgG (1:200), at 37℃ for 30 min. After washing with PBS 3 times (5 min each time), the sections were stained with DAPI before being imaged under a confocal microscope.

Statistical analysis

All data are represented as mean ± SD. The distribution of the experimental data was determined by Kolmogorov-Smirnov test. Data with non-normal distribution were analyzed by Kruskal-Wallis test. For the data with normal distribution and homogeneity of variance, one-way analysis of variance (ANOVA) was used to analyze the differences among multiple groups, and least significant difference (LSD) was used for two-group comparison. The Tamhane test was performed when assumptions of homogeneity of variance were questionable. SPSS 26 (IBM Corporation, Armonk, NY) was used for the statistical analyses. For survival analysis, the Log-rank (Mantel-Cox) test was used to determine the significance of differences in cumulative survival. GraphPad prism 8.0.2 software was used for the statistical analysis. A value of P < 0.05 was considered significant.