β-glucan and its preparation
The β-glucan used in this study was β-glucan peptide, a high molecular weight polysaccharide extracted from the fungus Trametes versicolor (Invivogen, category code: tlrl-bgp). The β-glucan consists of a highly ramified glucan portion, including a β-(1, 4) main chain and β-(1, 3) side chain, with β-(1, 6) side chains covalently linked to a polypeptide portion rich in aspartic, glutamic and other amino acids. The main structure of β-glucan is shown in Fig. 1B. β-glucan was diluted in sterile PBS to a concentration of 1 mg/ml.
NEC induction and drug treatment
All operations performed in our experiment were approved by the Animal Ethics Committee at Chongqing Medical University. Following protocol previously described (36), 10-day-old C57BL/6 mice were separated from their mothers and were fed by gavage with hyperosmolar formula (Similac Advance (Abbott Nutrition, USA)/Esbilac puppy milk replacer (PetAg, USA) = 1.7) every 4 h, subjected to hypoxia and hypothermia (100% N2 for 90 s subsequently with 4 ℃ for 10 minutes, 3 times per day) for 3 days to induce NEC. In our study, newborn 3-day-old mice were gavage with either 1mg/ml β-glucan or PBS at 0.03 ml/g for consecutive 7 days before NEC induction. In addition, age-matched and untreated mice were left with their mothers as control group. Body weight and survival condition were recorded daily throughout the establishment of NEC. On postnatal day 13, mice were sacrificed by cervical decapitation and intestine were harvested for further analysis.
Gut histology
The intestines were completely removed and a 1-cm portion of the distal ileum was fixed in 4% paraformaldehyde solution overnight. Then, the samples were dehydrated, embedded in paraffin and cut into 4µm sections. Subsequently, 4-µm tissue sections were stained with hematoxylin and eosin, and the histological injury was assessed by an established scoring criteria (37) in a double-blinded manner as follow: 0: no damage; 1: epithelial cell lifting or separation; 2: necrosis to the mid-villous level; 3: necrosis of the entire villus; 4: transmural necrosis. Animals with a histologic tissue injury score ≥ 2 were considered positive for NEC.
Immunohistochemistry
Paraffin sections from three groups were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol, followed by antigen retrieval with citric acid antigen repair buffer (PH6.0) in a microwave oven. The slides were placed in a wet box and incubated at 4°C overnight with the following primary antibodies diluted in PBS (PH7.4) respectively against primary antibodies. After slightly shaken dry, the slides were incubated with anti-rabbit antibody (HRP labeled) at room temperature for 50min. Finally, slides were stained with DAB, and nuclei were stained using hematoxylin. After incubating with the appropriate secondary antibody for 60 min, slides were stained with DAB, and nuclei were stained using hematoxylin for about 1min. Images were collected under microscope after dehydration and sealing.
Real-time PCR
Total RNA was extracted from the intestine tissue using TRIzol (Life Technologies CA, USA). The purity of RNA was quantified using a nanodrop spectrophotometer (Thermo Fisher Scientific, CA, USA) and eligible RNA samples (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0) were used. cDNAs were synthesized using a Prime Script RT Reagent (Takara, Japan) and were used for qRT-PCR assay using TB Green Premix Ex Taq II (Tli RNase H Plus) Kit (Takara, Japan). β-actin was used as an internal control, and the relative expression of mRNA (TLR4, IL-1β, IL-6, IL-10 and TNF-α) in intestine tissue was determined using ΔΔCT method. The detailed information of RT-PCR primer sequences is shown in Table 1.
Western blotting
Intestine tissue soaked in RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitor (Beyotime, China) was homogenized using an electric homogenizer and centrifuged to obtain the supernatant. Protein concentrations were measured by Pierce BCA Protein Assay Kit (Beyotime, China). The protein supernatant was mixed with sodium dodecyl sulfate sample buffer (Beyotime, China) at a ratio of 4:1 and denatured in 100℃ for 10 minutes. Protein samples were separated in 10% polyacrylamide gels and transferred to 0.45 µm PVDF membranes, and measured using anti-TLR4 (Servicebio, China), anti-NF-κB P65 (Servicebio, China), anti-ZO-1, anti-Occludin, Claudin-1 (Proteintech, China) and β-actin (ZENBIO Biotechnology, China) at 4℃ overinight. Signals were detected using chemiluminescence (ECL Western Blotting Substrate, Bio-Rad). The relative intensity of target bands was quantified by Image J analysis system (Bio-Rad).
Fecal Sample Collection and Microbiota Analysis
The ileum and colon feces of mice were collected in 1.5 ml sterile tubes and immediately placed in liquid nitrogen and transferred to -80℃ refrigerator for fecal sample microbiota analysis. Total genomic DNA was extracted from fecal samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s instructions. The quality and concentration of DNA were measured using 1% agarose gel electrophoresis and a NanoDrop® ND-2000 spectrophotometer (Thermo Scientific Inc., USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene were amplified with primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R(5'-GGACTACHVGGGTWTCTAAT-3') by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). PCR amplification cycling conditions were as follows: initial denaturation at 95 ℃ (3 min), 27 cycles of denaturing at 95 ℃ (30 s), annealing at 55 ℃ (30 s) and extension at 72 ℃ (45 s), and single extension at 72 ℃ (10 min), and end at 4 ℃. All samples were amplified in triplicate. The PCR product was extracted from 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to manufacturer’s instructions and quantified using Quantus™ Fluorometer (Promega, USA). Purified amplicons were sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego,USA) and the data were deposited into database. The original data were analyzed. Briefly, reads containing bases with a quality score < 20 were truncated, and sequences longer than 10 bp were combined together with their overlapped sequence. Reads that exceed the maximum mismatch ratio of 0.2 were discarded. Using UPARSE 7.1, the optimal sequences were clustered into operational taxonomic units (OTUs) which 97% sequence were in the similarity level. The most abundant sequence was selected as a representative sequence from each OUT. To minimize the effects of sequencing depth on alpha and beta diversity measure, the number of 16S rRNA gene sequences from each sample were rarefied to 27555. Bioinformatic analysis of the gut microbiota was conducted using the Majorbio Cloud platform (https://cloud.majorbio.com). Using Mothur software (http://www.mothur.org/wiki/Calculators), Alpha diversity, Chao1 richness and Shannon index were figured out. Differences of Alpha diversity among groups were analyzed using Wilcoxon rank sum test. The similarity among the microbial communities in different samples was measured by principal coordinate analysis (PCoA) based on Bray-curtis dissimilarity and the PERMANOVA test was applied to assess whether the variation could be explained by the treatment accompanied with its statistical significance. The linear discriminant analysis (LDA) effect size (LEfSe) (LDA score > 2, P < 0.05)was used to identify the significantly different taxa (phylum to genera) of bacteria among the different groups.
Molecular docking
Molecular docking was used to identify the binding mode between the β-glucan and the TLR4 using AutoDock4.2. The structure of TLR4 (PDB ID: 3FXI) was downloaded from Research Collaboratory for Structural Bioinformatics Protein Data Bank (38). (RCSB PDB, RRID:SCR_012820) (http://www.rcsb.org/pdb/). The 3D structure of β-glucan peptide was drawn by RDKit. The protein Amber14SB charge and the protonation state were allocated respectively using UCSF Chimera software and H++ (39, 40), and the structure was optimized using the classical MMFF94 force field. The optimized molecules were employed for AM1-BCC local charge calculation with UCSF Chimera software. The geometric center of the binding site that was predicted by SiteMap was applied as the docking center. The docking centre of the TLR4 was identified as center_x: -7.88, center_y: −13.00, and center_z: 45.55. The docking calculation was limited to the rectangular box with the center of each protein docking and the side length was 22.5 Å, and the Spacing step was set to 0.375 Å. The maximum number of search conformations was set to 10000. Amino acids in the docking center as well as ligands were regarded as flexible objects, and the outside amino acids were regarded as rigid objects, allowing amino acid side chains, such as aspartic acid and tryptophan, to flip over. Semi-flexible docking method was carried out to docking and genetic algorithm was used for conformational sampling and scoring.
Statistical analysis
Data analysis was performed by the GraphPad Prism (version 9.3.0). Normally distributed data were expressed as the mean ± SD and significance was identified by One-way ANOVA. Median and interquartile range (IQR) were used to describe nonnormally distributed data, and differences were determined by the Kruskal-Wallis test. P < 0.05 was considered statistically significant.