Animals
10 male and 16 female transgenic C57BL/6 strain T027452RETNLB mice, each between the ages of 4–6 weeks, were purchased from Nanjing Jicui Yaokang Co., Ltd. 10 male and 16 female C57BL/6J mice, each between the ages of 4–8 weeks, were acquired from Zhuhai Baishitong (animal batch number: SCXK (Guangdong) 2004-0011, female: male = 1:2). The female and male mating cages were crossed, and the genes of their progeny were identified after one week of adaptive rearing. The homozygotes of 7-day-old suckling mice were included in the experiment if all the offspring were homozygotes. Animal experiments were carried out in accordance with institutional guidelines and protocols approved by the Animal Ethics Committee of Shenzhen Baoan Women's and Children's Hospital, Jinan University (protocol # LLSC 2018-12-10). All the study methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
Identification of mouse genotypes
DNA extraction from the mouse tail was performed using a tissue genome DNA extraction kit. Mouse tails were counted and cut to perform DNA extraction according to manufacturer’s instructions.
PCR was used to carry out amplification and identification of genotype. The PCR amplification system used in this study was as follows: a total 20uL volume was made of 2uL DNA, 2uL of 10Taq Buffer (mg2 + plus), 0.5uL of dNTP Mix (10 mmol/L), 0.5 uL of Primer Mix (10 umol/L), 0.5 uL of TaqDNA Polymerase (5 u/L) and 14.5uL of H20. PCR amplification conditions were as follows: pre-denaturation at 95% for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 30 seconds for a total of 20 cycles. The samples underwent re-denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 3 minutes followed by storage at 25°C. The PCR primer sequence used is shown in Table 1.
Table 1
List of Primers Used in This Study
| Primer name | Forward primer | Reverse primer |
| RELMβ-KO | 5'-CTGGAAACGGAGAGATATGGG-3' | 5'-ACCAGGGTGGATGCTTTAGTCTT-3' |
| RELMβ | 5'-GCCTGAAAACACTGCTCATTGGTG-3' | 5'-GCTGGATGGAGGGAAT-3' |
Gel imaging results (Shanghai Tianneng Technology Co., Ltd.) were observed using PCR nucleic acid agarose gel electrophoresis. The electrophoresis bands of each genotype were as follows: RETNLB KO heterozygote (RETNLB+/-):301 bp, wild type (RETNLB+/+):265 bp, while the negative genotype (RETNLB-/-) and the RETNLB homozygous KO had no bands.
NEC model preparation and specimen collection
Wild-type (WT) C57BL/6 mouse pups and RETNLB KO mouse pups were divided into four groups and NEC experimental models were subsequently induced at postnatal days (PND) 5–7. Group A consisted of WT mice, Group B consisted of KO mice, Group C consisted of WT mice with NEC, and Group D consisted of KO mice with NEC. Each group contained 9 mice. Groups A and B were fed normally, while groups C and D were fed artificially, and underwent hypoxia and cold stimulation with LPS administration (for more information, see Supplementary table S1-S3).
C57BL/6 mouse pups in each group were euthanized by cervical dislocation after three days of NEC experimental modeling to collect intestinal tissue. Classic NEC signs were observed in the small intestine and included intestinal dilatation, intestinal wall emergence and necrosis. Photos were taken post-incision and removal of the digestive system.
The end of the small intestine was cut by 5 cm. Pathological staining was performed after 1 cm of intestinal tissue had been fixed with 4% paraformaldehyde (PFA) and kept at room temperature. Additionally, 2 cm of the remaining intestinal tissue was cut into pieces no larger than 0.5 cm. Trizol was added to the tissue, which was subsequently frozen and stored at -80°C. qRT-PCR was subsequently performed on the tissues.
Hematoxylin-eosin staining (HE staining)
Intestinal tissue was placed in a dehydration basket and dehydrated by gradient alcohol. The samples were embedded, sliced (4 mm) and subsequently dewaxed. The samples were dehydrated, stained with hematoxylin for 8 min, washed with running water and dyed with eosin for 3 min. Afterwards, the samples were washed with running water, dehydrated, sealed with transparent neutral gum, and examined under light microscope to collect imaging data. The cytoplasm is red in the images, while the nucleus is blue.
qRT-PCR
A homogenizer was used to grind 50 mg of fresh intestinal tissue into powder post-shattering in liquid nitrogen in a mortar. Total RNA was extracted from the tissue in accordance with the manufacturer’s instructions (Trizol).
ThermoFisher reverse transcriptase instructions were used to determine reverse transcription operation specifications and convert whole cell RNA into cDNA. Afterward, qRT-PCR detection was performed. Cycle threshold (Ct) values were used to determine RNA expression levels, and the comparative (2-ΔΔCt) method (fold change) was used to calculate RNA levels.
Statistical analysis
The differences among the four groups were assessed using one-way ANOVA testing (GraphPad Prism). A relative expression software tool (REST) was used to compare the expression of TLR2 and TLR4 in each of the four groups. Results are shown as Mean ± SE, while significance is determined by p < 0.05.