Clinical study

Lung tissue specimens from 23 cases of ARDS patients and 27 healthy volunteers (non-ARDS) in Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine were collected and stored in liquid nitrogen for use. All tissue samples were verified by pathological examination. Patients signed informed consent. This study was approved by Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Basic information of all patients and volunteers was collected and shown in Table 1.

Table 1 General characteristics of ARDS patients and Non-ARDS volunteersCell culture

Human lung microvascular endothelial cell line-5a (HULEC-5a) was purchased from American Type Culture Collection (ATCC) and cultured with Dulbecco’s modified eagle medium (DMEM; Gibco, New York, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin and streptomycin (FBS; Gibco). The cells were cultured in an incubator with 37 ℃ and 5% CO2.

Cell transfection and treatment

Negative control pcDNA 3.1 vector, pcDNA 3.1-TFRC overexpression vector, short hairpin (sh) negative control (sh-NC) plasmid, and sh-NAT10 plasmid were synthesized by Genomeditech Biotechnology Co., LTD (Shanghai, China). HULEC-5a cells (5 × 105 cells/well) were inoculated in 6-well plates (Corning, NY, USA). After the cell confluence reached 80%, transfection was performed for 48 h using Lipofectamine 3000 (Thermo Fisher Scientific; Waltham, MA, USA).

Besides, HULEC-5a cells were stimulated with lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO,USA; 1 μg/mL) for 24 h (Wang et al. 2021), tauroursodeoxycholic acid (TUDCA; 500 mM; Yeason Biotechnology Co., LTD, Shanghai, China) for 12 h, necrosulfonamide (NSA; 20 μmol/L; Abcam, Cambridge, MA, USA) for 2 h, and ferrostatin-1 (Fer-1; 5 μM; Sigma) for 36 h.

Western blot

Cells were lysed using the RIPA lysis buffer (Thermo Fisher) on ice to extract proteins. After detecting protein concentration using the BCA method (Sigma), 50 μg of the proteins were loaded on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for separation. Then, the proteins were transferred to the PVDF membrane (Sigma). The membrane was blocked in western blocking buffer (Thermo Fisher) for 1.5 h at room temperature, and incubated with the primary antibodies overnight at 4 °C for immunoblotting. The primary antibodies used were rabbit antibodies specific for GPX4 (1/1000; ab125066; Abcam), solute carrier family 7 member 11 (SLC7A11; 1 µg/mL; 711,589; Thermo Fisher), achaete-scute complexlike 4 (ACSL4; 1/1000; PA5-27,137; Thermo Fisher), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1/1000; A-11008; Thermo Fisher). Then, the membrane was washed thrice by Tris-buffered saline Tween (TBST; Yeason), and incubated with the secondary antibody (1/10000; 31,460; Thermo Fisher) for 1 h at room temperature. Finally, the membranes were washed with TBST and a SuperPico ECL Chemiluminescence kit (Vazyme Biotechnology Co., LTD, Nanjing, China) was used to expose the protein bands.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from cells and tissues was extracted using Trizol regent (Yeason). The quality of isolated RNA was measured with a Nanodrop 2000 spectrophotometer (Thermo Fisher) and the RNA concentration was adjusted to 500 ng/μL. To obtain cDNA, the reverse transcription were carried out using the PrimeScript RT reagent Kit (Takara, Tokyo, Japan), and the qPCR amplification experiment was performed using the TB Green® Premix Ex Taq II FAST qPCR (Takara) with the provided reaction conditions. All primers used in this study were synthesized by Genescript Biotechnology Co., LTD, (Nanjing, China) and listed as follows: N-acetyltransferase (NAT)10, forward, 5′-ATAGCAGCCACAAACATTCGC-3′ and reverse, 5′-ACACACATGCCGAAGGTATTG-3′; transferrin receptor (TFRC), forward, 5′-ACCATTGTCATATACCCGGTTCA-3′ and reverse, 5′-CAATAGCCCAAGTAGCCAATCAT-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′. The gene expression was calculated by the 2−ΔΔCT method and GAPDH was used as the internal control.

Cell counting kit-8 (CCK-8) assay

The cells were first seeded into a 96-well plate at the density of 1 × 103 cells/well. Then, the cells were maintained in the incubator for 24 h, and 10 μL of CCK-8 solution (Vazyme) was added to each well to incubate with cells for 2 h. A microplate reader (Thermo Fisher) was used to assess the absorbance at 450 nm. Three replicate wells were set up.

Determination of cellular ferrous iron (Fe2+), malondialdehyde (MDA), and GSH

Fe2+ level, MDA content, and GSH concentration in cells or tissues were detected by commercial Ferrous Ion Content Assay (Solarbio, Beijing, China), MDA Assay (Jiancheng Biotechnology Co. Ltd., Nanjing, China), and GSH Assay kits (Jiancheng) according to the provided instructions.

Flow cytometric analysis of lipid ROS production

ROS production detection was performed as previously described (Jiang et al. 2022). Cells were incubated with the Molecular Probes BODIPY 581/591C11 (Invitrogen, Carlsbad, CA, USA) working solution (5 μmol/L) at 37 °C for 30 min without light. Next, the cells were washed with phosphate buffer solution three times and measured using flow cytometric analysis.

ac4C-RNA immunoprecipitation (RIP)

The ac4C-RIP assay was performed according to the published literature (Chen et al. 2023). Briefly, RNA fragments were incubated with an anti-ac4C antibody (Abcam, ab252215) or IgG (Abcam, ab172730), and ac4C-modified RNA was then eluted for ac4C enrichment analysis by qPCR.

RIP assay

RIP assay was used to explore the interaction between TFRC and NAT10 in HULEC-5a cells using the Imprint RIP Kit (Merck Millipore, Billerica, MA, USA). In brief, cells were lysed in RIP buffer for 30 min at 4 °C. Then, the cell supernatant was incubated with magnetic beads (Merck Millipore) bound with IgG and NAT10 antibodies (Abcam) for 4 h at 4 °C. After incubation, the beads were washed and eluted. Then, proteinase K was added to remove protein at 55 °C for 30 min. The RNA was isolated and qPCR was performed to detect TFRC expression.

Dual-luciferase reporter assay

The cDNA containing full-length 3'-untranslated regions (UTR) of FTRC was cloned into the pGL3 luciferase reporter vector (Promega, Madison, WI,USA) to obtain pGL3-FTRC-wild type (WT). Besides, pGL3-FTRC-mutant type (MUT) was obtained by the introduction of mutations into pGL3-FTRC-WT using the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). Next, sh-NC/sh-NAT10 and pGL3-FTRC-WT/pGL3-FTRC-MUT were co-transfected into HULEC-5a cells using Lipofectamine 3000 for 48 h. Afterwards, luciferase activity was measured using Dual-Luciferase® Reporter Assay System Kit (Promega) and normalized to the activity of Renilla luciferase. The ratio of firefly/Renilla luciferase activity was used as the relative luciferase activity.

RNA stability assessment

RNA stability assessment was performed to verify the stability of TFRC after NAT10 silenced in HULEC-5a cells. HULEC-5a cells were treated with actinomycin (Act) D (0.5 µg/mL, Yeason), then existing TFRC expression at different time points (1, 4, 8, and 12 h) was analyzed by qPCR.

Animal study

A total of 24 male Wistar (6–8 weeks old) rats were purchased from Charles River (Beijing, China) and housed in cages with 24 ℃, a 12 h alternating light/dark cycle and free access to water and food. After one-week adaptive feeding, the rats were randomly divided into four groups (n = 6 per group): sham, cecum ligation and puncture (CLP), CLP + sh-NC, and CLP + sh-NAT10 groups.

The sepsis rat model was established according to a previous study (Li et al. 2022b). Briefly, rats were anesthetized with 2% sodium pentobarbital (0.3 mL/100 g; Sigma) by abdominal cavity injection after fasting for 12 h. Next, median abdomen hair of the rats was scraped and disinfected using iodine and medical alcohol. Incision was made (about 2 cm) along the linea alba to locate the cecum and ligation with 5–0 suture at about 1/3 of the cecum. The cecum was punctured with a needle, ligated twice. A small amount of intestinal contents were squeezed out of the puncture hole to ensure that the cecum is unobstructed. Next, the treated cecum was reintroduced into the abdominal cavity and the inner layer was sutured with 5–0 thread, and the outer layer, with 3–0 thread. Finally, the rats were placed on a heating pad and the rectal temperature was maintained at 37 ± 0.5 °C. When the rats woke up, they were fed and drank freely. The sham group rats received the same operation except cecal ligation and puncture. For NAT10 knockdown, lentivirus containing sh-NAT10 and sh-NC (0.2 mL, 1 × 109 pfu/mL) were injected into the caudal vein 4 days before modeling, respectively. Finally, all rats were sacrificed by administration of an overdose of anesthesia of sodium pentobarbital. The lung tissues from each rat were collected and stored at − 80 °C for subsequent experiments.

Hematoxylin & eosin (H&E) staining

The lung tissues were fixed with 4% paraformaldehyde (Sigma). Next, the samples were embedded with paraffin. After that, the tissue sections were cut into 5 μm and stained with hematoxylin and eosin (Yeason) for 5 min. Finally, the sections were observed by a light microscope.

Statistical analysis

The SPSS 21.0 software was used to analyze data. Data are expressed as mean ± standard deviation (SD). Student’s t-test was used for comparison between the two groups. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis was used for comparison among groups. R language and HemI 1.0 software (http://hemi.biocuckoo.org/index.php) were used to generate a heatmap showing the expression of ferroptosis-related genes in sh-NC and sh-NAT10 groups. Potential diagnostic value of NAT10 in SPI was presented by receiver operating characteristics (ROC) curve analysis. Statistical analyses were performed using GraphPad Prism software (v8.0.1, GraphPad Software Inc., San Diego, CA, USA). p < 0.05 indicates that the difference is statistically significant.