Reagents and Antibodies

Lipopolysaccharide (LPS), cobalt chloride (CoCl2), and deferoxamine (DFO) were procured from Sigma‒Aldrich (St. Louis, MO, USA). NAC and ferrostatin-1 (Fer-1) were obtained from Selleckchem (Houston, TX, USA). Antibodies against GPX4 (ab125066), Tfrc (ab269513), PTGS2 (ab62331), FTH1 (ab183781), FTL (ab109373), SLC7A11 (ab175186), SESN2 (ab178518) and 4-HNE (ab46545) were procured from Abcam (Cambridge, UK). E-cadherin antibody (#14,472) was sourced from Cell Signaling Technology (Danvers, MA, USA), while ZO-1 (21,773–1-AP) and Claudin-1 (13,050–1-AP) antibodies were acquired from Proteintech (Wuhan, China). Furthermore, β-actin antibody (AC026) was obtained from ABclonal Technology (Wuhan, China). HRP-conjugated secondary antibodies (GAM007 and GAR007) were procured from MultiSciences (Hangzhou, China).

Human Tissue Samples

Specimens from infants with NEC (n = 11) and from “noninflammatory” control infants (n = 13), including those with intestinal perforation (n = 1), volvulus (n = 1), congenital small bowel atresia (n = 2), intestinal obstruction (n = 2) and intestinal malrotation (n = 7), were obtained from the Department of General Surgery at Children’s Hospital of Soochow University. The use of these tissue samples was sanctioned by the Medical Ethics Committee of the Children’s Hospital of Soochow University (CS180), with all guardians providing written informed consent. This study was carried out in adherence to the principles of the Declaration of Helsinki. The baseline characteristics of the infants with NEC and control infants are detailed in Table 1.

Table 1 Characteristics of NEC and Control InfantsAnimal Experiments

The animal experiments were reviewed and approved by the Ethics Committee of Soochow University (SUDA20230719A01). C57BL/6 mouse pups (five to seven days old) were purchased from JOINN Laboratories (Suzhou, China), and experimental NEC was induced as per established protocols [26]. Briefly, the mice were fed a specific formula (a combination of 10 g of Similac Plus [Abbott, Saint-Laurent, Canada] and 50 ml of Esbilac [PetAg, Hampshire, USA]), 40 μl/g through gavage every 6 h, followed by exposure to hypoxia (95% N2 + 5% O2 for 10 min after half an hour of feeding) and intragastric administration of LPS (4 μg/g added to the second feeding of formula on the second and third days of NEC modeling). The pups were randomly assigned to four experimental groups: the control group (Group 1), which included pups that were breastfed by their mothers; the NAC group (Group 2), which included pups that stayed with their mothers and were administered NAC (150 μg/g of body weight) by intraperitoneal injection every day; the NEC group (Group 3), which included pups that were separated from their mothers and used to model NEC by the method described above; and the NAC + NEC group (Group 4), which included pups that were separated from their mothers and subjected to the same conditions as Groups 2 and 3. The surviving pups in all groups were euthanized after 96 h to collect ileal tissues and serum samples.

Cell Culture and Treatments

FuHeng Biotechnology Co., Ltd., provided the IEC-6 cell line (RRID: CVCL_0343), which was cultured in high-glucose DMEM (HyClone, UT, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and maintained in an incubator at 37 °C with 5% CO2. The cells were exposed to LPS (10 μg/mL) and CoCl2 (400 μM) for 24 h to establish the in vitro NEC model. Unless specified otherwise, the cells were subjected to 2 μM Fer-1, 200 μM DFO or 3 mM NAC for 2 h before LPS stimulation.

Cell Transfection

Control small interfering RNA (siRNA) and SESN2 siRNAs were obtained from GenePharma (Shanghai, China). The sequences can be found in Table 2. SESN2 cDNA was cloned and inserted into a pcDNA3.1 expression vector expressing a Flag tag for transient transfection to establish SESN2-overexpressing cells. The empty vector and SESN2 plasmid were purchased from Youbio Biological Technology (Hunan, China). Transient transfection was conducted utilizing Lipofectamine™ 3000 (Invitrogen, USA), and the transfected cells were used in the subsequent experiments.

Table 2 SESN2 mRNA-Specific siRNAs and Negative Control (NC) siRNACell Viability

IEC-6 cells were seeded in 96-well plates at a density of 8,000 cells per well (BD Falcon, Corning Inc., Corning, NY) and cultured to form a monolayer for approximately 48 h. The medium was then changed to serum-free medium. After treatment, cell viability was determined utilizing a cell counting kit-8 (CCK-8, NCM Biotech, Suzhou, China).

Hematoxylin and Eosin (H&E) Staining

The collected ileum samples were fixed in 4% paraformaldehyde, embedded in paraffin blocks, sectioned at 5 μm, and stained with H&E. Based on the published scoring system, intestinal injury severity scores were assessed in a blinded manner by histological evaluation.

Immunohistochemistry (IHC)

Intestinal tissue was fixed in 4% paraformaldehyde, embedded in paraffin, dewaxed, and subjected to antigen retrieval. To inhibit endogenous peroxidase activity, 3% H2O2 and 5% BSA were used successively. Subsequently, primary antibody incubation was performed. Visualization was performed using an immunochromogenic kit (Gene Tech, Shanghai, China). A secondary antibody was applied, followed by the addition of DAB color development solution. After a few minutes, the slices were washed and stained with hematoxylin. After washing, the slices were dehydrated using graded ethanol solutions, followed by clearing with xylene and sealing with neutral resin. Finally, images of these slices were taken under a light microscope.

Immunofluorescence (IF)

Intestinal tissues that had undergone antigen repair and fixed IEC-6 cells were blocked with 5% BSA before being incubated with the indicated primary antibodies. After washing with PBS, the specimens were incubated in the dark with a secondary antibody labeled with fluorescent dye. Nuclei were stained with DAPI for 5 min. Fluorescence images were acquired using a Zeiss confocal microscope (Zeiss Microsystems, Germany).

Enzyme-linked Immunosorbent Assay (ELISA)

ELISA kits (RK00020, ABclonal, Wuhan, China; P16599, CUSABIO, Wuhan, China) were used to assess the amounts of IL-6 and TNFα in the cell culture supernatant.

Quantitative Reverse Transcription-polymerase Chain Reaction (qRT‒PCR)

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was utilized to extract the total RNA, and a cDNA 1st Strand Synthesis kit (Novoprotein, China) was used to reverse transcribe the RNA into cDNA. SYBR qPCR SuperMix (Novoprotein, China) was used for qRT‒PCR. Utilizing the 2−△△Ct technique, mRNA quantities were assessed and adjusted against β‐actin levels for normalization. The sequences for both the forward (F) and reverse (R) primers are provided in Table 3.

Table 3 Primers Used in This StudyWestern Blot Analysis

A protease inhibitor cocktail (ab271306, Abcam, Cambridge, UK) was added to RIPA buffer (Beyotime, Shanghai, China) for the extraction of total protein. Protein separation was achieved through SDS‒PAGE. The membranes were incubated with primary antibodies after 1 h in a blocking solution at room temperature. Subsequently, the membranes were incubated with an HRP-conjugated secondary antibody. Identification of the protein bands was performed using a Syngen GeneGnome XRQ system (Syngene, UK) and enhanced chemiluminescence (ECL) detection reagents (Yeasen Biotechnology, Shanghai, China).

Wound Healing Assay

The cells were cultivated at a density of 5 × 105 cells per well in 6-well plates (BD Falcon, Corning Inc., Corning, NY). Once reaching confluence, the cells were separated into groups and treated with different agents in serum-free medium following the application of a scratch through the layer of cells. The documentation of wound closure was carried out after 0 h and 24 h of incubation. The healed area of each wound was calculated using the following formula: acellular area at 24 h/acellular area at 0 h × 100%.

RNA Sequencing (RNA-seq) ANALYSIS

RNA-seq was performed and the results were analyzed by LC Bio Technology (Hangzhou, China) on the Illumina NovaSeq™ 6000 system. For the differentially expressed genes (DEGs), both Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted.

Transmission Electron Microscopy Imaging

After fixation with electron microscopy fixative, IEC-6 cells were collected and centrifuged. Subsequently, the samples were postfixed with 1% osmium tetroxide, dehydrated with ethanol and acetone and embedded in epoxy resin. Polymerization was carried out at 60 ℃ for 48 h, followed by the cutting of ultrathin Sects. (60 nm thick) and staining with 2% uranyl acetate and lead citrate. The cellular ultrastructural morphology was observed by TEM, and images were taken.

Measurement of the Reduced Glutathione (GSH)/Oxidized Glutathione Disulfide (GSSG) Ratio

The GSH/GSSG ratio was evaluated using a GSH and GSSG assay kit (Beyotime, Shanghai, China) according to the manufacturer’s protocols. The concentrations of total glutathione and GSSG were measured successively at 412 nm by using an enzyme labeling instrument. The GSH/GSSG ratio was calculated using the following formula: (total glutathione—GSSG × 2)/GSSG.

MDA Measurement

The relative MDA concentration was measured with a lipid peroxidation MDA assay kit (Beyotime, Shanghai, China). Tissues or cells were homogenized or lysed before centrifugation to obtain the supernatant. Then, the supernatant was added to the MDA working solution and incubated at 100 ℃ for 15 min, after which the absorbance was measured at 532 nm.

Lactate Dehydrogenase (LDH) Release Assay

IEC-6 cells grown in 96-well plates were treated and cultured for a period of time. Then, LDH release was determined utilizing a cytotoxicity LDH assay kit (Dojindo, Kumamoto, Japan). After the addition of lysis buffer or working solution to some wells, LDH levels in the medium were measured. Subsequently, the absorbance was measured at 490 nm.

Assessment of Lipid Peroxidation by Flow Cytometry or Fluorescence Imaging

IEC-6 cells were incubated with Liperfluo (Dojindo, Kumamoto, Japan) at 37 °C for 0.5 h. Following incubation, the cells were rinsed, harvested, and resuspended in HHBS. A flow cytometer (Beckman, Germany) was used to quantify the cell fluorescence, which was analyzed using FlowJo software.

The cells were exposed to BODIPY™ 581/591 C11 (Thermo Fisher Scientific, MA, USA), followed by washing with PBS and staining of the nuclei. Fluorescence images were acquired by a Zeiss confocal microscope.

Assessment of Intracellular Fe2+ by Fluorescence Imaging

IEC-6 cells were washed with HHBS and then treated with FerroOrange (Dojindo, Kumamoto, Japan) at 37 °C for 0.5 h. Cell fluorescence was visualized by a Zeiss confocal microscope.

Molecular Docking

The stereo structures of the protein and compound were obtained from the UniProt and PubChem databases and then processed through the ADT tool. Subsequently, the parameters of the docking box were calculated by PyMol 2.2.0, which led to the docking and scoring executed by AutoDock Vina 1.2.3. Following the completion of docking, the results were examined and interpreted with PyMOL and Discovery Studio 2021.

Statistical Analysis

Data analyses were carried out using GraphPad Prism 8.3.0. All of the data were obtained from at least three independent experiments. One-way ANOVA was used to compare normally distributed data between groups. The data in this study were expressed as the mean ± standard deviation. Using the nonparametric Mann‒Whitney U test, nonnormally distributed quantitative data were compared between different groups. These data were presented as the medians and interquartile ranges. The Kaplan‒Meier method was used to assess the survival data. A p value less than 0.05 was considered to indicate statistical significance.