Animals

The Animal Department of the Research and Development Center of Shengjing Hospital, China Medical University provided adult Sprague–Dawley (SD) rats, with a female to male mating ratio of 3 to 1, all neonatal SD rats were spontaneously delivered by females. All animal experiments were conducted in accordance with the national animal protection regulations of China and the guidelines of the Animal Protection and Use Committee of China Medical University (Approval No.: 2018PS178K). And all animal experiments should be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments or the National Research Council's Guide for the Care and Use of Laboratory Animals. Furthermore, reporting (not performance) of animal testing experiments should comply with the ARRIVE guidelines.

Animal model and tissue harvest

Within 12 h after birth, newborn SD rats were randomly divided into two groups: a control group (FiO2 = 21%) and a hyperoxia group (FiO2 = 85%), each consisting of 8 rats. To avoid differences between the groups caused by oxygen poisoning, the female rats were exchanged every 24 h. On the 3rd, 7th, 10th, and 14th day after birth, rats were randomly selected from both groups and euthanized for intestinal tissue harvesting.

Cell lines and cell cultivation

The NCM460 cells were cultured in RPMI 1640 incomplete medium (cat. no. Kgm31800-500 KeyGENBioTECH, Jiangsu, China), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin double antibiotic solution. The cells in the logarithmic growth phase were digested and passaged. In the control group, the cells were cultured in a ordinary incubator (FiO2 21%, 37 ℃, 5% CO2). In the hyperoxia group, after the cells were cultured in the ordinary incubator for 24 h, then were cultured in a hyperoxia incubator (FiO285%, 37 ℃, 5%CO2) for 24 h, 48 h, and 72 h, respectively. In the cells of the hyperoxia experimental group with antagonists and inhibitors, the culture medium added the drugs was changed every 24 h, and the cells were collected after the specified culture time was reached, and the specific concentrations were as follows, tBHQ (cat. no. S4990, Selleck; USA; 20 μM); ML385 (cat. no. S8790, Selleck; USA; 5 μM); Fer-1 (cat. no. A4371, APExBIO; USA; 1 μM); Celecoxib (cat. no. S1261, Selleck; USA; 50 μM); TG4-155 (cat. no. HY-18971, MCE; USA; 2 μM); CJ-42794 (cat. no. HY-10797, MCE; USA; 10 μM).

Immunohistochemical (IHC) stainingSmall intestinal tissue samples

A paraffin section of the intestinal tissue was taken, and dewaxing, antigen repair, blocking, and then were incubated with the primary antibodies: rabbit anti-recombinant divalent metal transporter 1(DMT1) (cat.no.20507-1-AP,Proteintech,Wuhan,China); rabbit anti-transferrin receptor(TFRC)(cat.no.A5865, Abclonal, Wuhan, China); rabbit anti-GPX4(cat.no.A13309,Abclonal,Wuhan,China); rabbit anti-FTH1(cat.no.A1144,Abclonal,Wuhan,China); rabbit anti-recombinant solute carrier family 7, member 11 (SLC7A11)(cat.no.A2413,Abclonal,Wuhan,China) were carried out at 4 ℃ overnight. Next the sections were in turn incubated with biotin-labeled goat anti-rabbit IgG and horseradish enzyme-labeled streptavidin working solution (cat. no. SP9001, Zhongshan Golden Bridge Biotech, Beijing, China) for 30 min. Images were taken using a light Microscope and a Nikon image acquisition system (Eclipse NI, Nikon, Tokyo, Japan). The expressions of proteins were analyzed using Image J 1.48 (National Institutes of Health) software.

Intestinal epithelial cells

NCM460cells were fixed on cover glass with 4% paraformaldehyde. Endogenous peroxidase of the cells was blocked with 3% H2O2 and 10% goat serum. Then as described above, the cells were in turn incubated with the primary antibodies (rabbit anti-DMT1, TFRC, GPX4, FTH1, SLC7A11, respectively), biotin-labeled goat anti-rabbit IgG and horseradish enzyme-labeled streptavidin working solution. Finally, DAB and hematoxylin were used for staining. Image acquisition and analysis were carried out as described above.

Measurement of lipid peroxidation levels

The Lipid Peroxidation MDA Assay Kit (cat.no.BC0025,Solarbio,Beijing,China), the GSH Assay Kit (cat.no.BC1170,Solarbio,Beijing,China), and the Total SOD Assay Kit (cat.no.BC0175,Solarbio,Beijing,China) were used to analyse levels of malondialdehyde (MDA), GSH, and superoxide dismutase (SOD), respectively, following the kit instructions for all procedures.

Detection of ROS level

NCM460 cells in the logarithmic growth phase were routinely digested and plated in 6-well plates. After the cells were attached, the cells in the control group were placed in an ordinary incubator and continued to be cultured for 24 h. The cells of the hyperoxia group were placed in the hyperoxia incubator and continued to be cultured for 24 h, 48 h and 72 h respectively, and then taken out, and the cells of the hyperoxia 72 h needed to be replaced once when the hyperoxia was 48 h. The cells were incubated with DCFH-DA probe in the dark at 37 ℃ and were observed by the fluorescence microscope, and the green fluorescence produced is the reactive oxygen species in the cell, and the brighter the green fluorescence, the higher the content of reactive oxygen species contained in the cell. The multimode microplate reader was adjusted to an excitation wavelength of 488 nm and a reception wavelength of 525 nm to measure the absorbance of each well in a 6-well plate. The higher the measured absorbance (OD) value, the more reactive oxygen species are produced.

MMP assay

The cell culture is the same as the Detection of ROS level. The 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benz imidazole carbon iodide (JC-1) fluorescent probe (cat. no. C2006, Beyotime, Shanghai, China) was used to detect MMP (mtΔΨ). The cells were incubate in a cell culture incubator at 37 ℃ for 20 min in the dark, wash off the probes and set aside on ice. Observation and photography were taken under a fluorescence microscope. The absorbance (OD) values of each well were measured with a multimode microplate reader. JC-1 monomer is green, JC-1 polymer is red, their maximum excitation wavelength is 490 nm, 525 nm, and the maximum emission wavelength is 530 nm and 590 nm, respectively.

Cell mortality rate

Propidium iodide (PI) penetrates the membranes of dead cells, and can be inserted into double-stranded DNA, so the nuclei of dead cells are stained, but not living cells. And the cell mortality was measured by flow cytometry. Take out the treated cells, digest the cells with EDTA-free trypsin, pay attention to the supernatant of the original culture medium in the medium dish and store it in the centrifuge tube, terminate the digestion of the cells with the culture medium at the end of digestion, move the cell suspension to the centrifuge tube, resuspend the cells with pre-cooled PBS after centrifugation, repeat 2 times, transfer the cells to the flow cytometry tube, add PI solution, protect from light at room temperature for 15 min, insert the flow cytometer into ice, and go up to the flow cytometer for detection within 1 h.

Western blot analysis

Protein was extracted from cells or intestinal tissue and quantified by BCA kit (cat.no.P0013C, Beyotime, Shanghai, China). The samples were transferred to 10% SDS-PAGE gel, then transferred to a polyvinylidene fluoride (PVDF) membranes and sealed using 5% skim milk. The membranes were incubated with primary antibodies: rabbit anti-DMT1; TFRC; GPX4; FTH1; SLC7A11; Nrf2 (cat.no. ab31163, Abcam, Cambridge, USA); COX-2 (cat.no.ab179800, Abcam, Cambridge, USA);TNF alpha (cat. no. 17590-1-AP, Proteintech, Wuhan, China); prostaglandin E receptor 4(EP4)(cat. no. 24895-1-AP, Proteintech, Wuhan, China); prostaglandin E receptor(EP2)(cat.no.ab167171, Abcam, Cambridge, USA) and mouse anti-IL-4 (cat.no.66142-1-Ig, Proteintech, Wuhan, China); IL-6 (cat.no.66146-1-Ig, Proteintech, Wuhan, China); β-actin (cat.no.66009-1-Ig, Proteintech, Wuhan, China) overnight at 4 ℃. Then the membranes were incubated with goat anti-rabbit or mouse IgG (cat.no.SA00001-2 or SA00001-1, Proteintech, Wuhan, China) and with an enhanced chemiluminescent substrate. Images were captured using Amersham Imager 680 (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Band density values were calculated using ImageJ 6.0 (National Institutes of Health) and normalized to β-actin.

Statistical analysis

Experimental data are presented as the mean ± SD. SPSS25.0 software (IBM Corp, Armonk, NY, USA) was used for statistical analysis, and GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA) was used to prepare charts. The unpaired t-test was used for comparison between groups. Two-factor analysis of variance was used for comparisons between multiple groups, followed by Bonferroni post-hoc tests.