Comprehensive Characterization of the Oxidative Stress Profiles in Neonatal Necrotizing Enterocolitis

Abstract

Objective: This study aims to portray the characteristics of oxidative stress (OS) in cases of Necrotizing enterocolitis (NEC), identify the hub genes and associated mechanisms involved, and explore potential drugs for NEC. Methods: We performed a comprehensive analysis integrating bulk-RNA sequencing and single-cell RNA sequencing datasets, coupled with various techniques including differential analysis, gene set enrichment analysis, and immune infiltration analysis. We aimed to systematically elucidate the variations in functions related to OS among distinct cell populations within both NEC and non-NEC tissues. Additionally, we depicted the longitudinal changes in immune cells, with a particular focus on macrophages, throughout the progression of NEC. NEC mice model was established and RT-qPCR was performed to validate the expression of the hub genes. Results: In total, 465 OS related genes were found, and 53 of them were significantly differentially expressed. These genes were mainly involved in several signaling pathways, such as TNF signaling pathway, IL-17 signaling pathway, FOXO signaling pathway, inflammatory bowel disease. The top 10 hub genes were MMP2, IL1A, MMP3, HGF, HP, IL10, PPARGC1A, TLR4, MMP9 and HMOX1. Ten kinds of drug were discovered as the potential treatment for NEC. Four specific macrophages subtypes and relative function were identified in NEC. RT-qPCR and immunofluorescence staining confirmed the expression of the hub genes in NEC model. Conclusions: This investigation yielded innovative insights into the immune environment and therapeutic methodologies directed at oxidative stress in the pathogenesis of NEC.

Keywords: immune infiltration; macrophages; neonatal necrotizing enterocolitis; oxidative stress; single cell RNA-sequencing..

Figure 1

(A) The volcano map of differentially expressed genes (DEGs) in GSE46619. (B) The heatmap and cluster analysis of the gene expression in NEC and CON group. (C) The Venn plot of DEGs and OS related genes showed an overlap of 53 OS related DEGs.

Figure 2

(A) PPI network diagram. Orange red indicates upregulated genes, and blue indicates downregulated genes. (B-C) GO and KEGG enrichment analyses of the modular genes. The size of the circle represents the number of genes involved, and the abscissa represents the frequency of the genes involved in the term total genes. PPI, protein-protein interaction; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3

(A) The Venn diagram shows that seven algorithms have screened out nine overlapping hub genes. (B) Hub genes and their co-expression genes were analyzed via GeneMANIA. (C-D) GO and KEGG enrichment analyses of the hub genes. The outermost circle is term on the right, and the inner circle on the left represents the significant p-value of the corresponding pathway of the gene. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4

(A) Scatter plot after quality control and filtering of cells. (B) The left panel indicates that the mitochondria were the same with sequencing depth; the right panel suggests that the numbers of the detected genes were significantly related to the sequencing depth. (C) UMAP visualization of single cell RNA-sequencing data in NEC and CON, which identified 17 cell clusters. (D) The heatmap displays the top 5 genes of the identified markers genes of each cluster. (E-F) UMAP visualization of cell populations distribution between NEC and CON group.

Figure 5

(A-B) UMAP visualization of cell subpopulations in macrophages in NEC and CON group. (C) Four different macrophage subtypes and their specific marker gene expression levels, with brightness indicating log-normalized average expression, and circle size representing the percent expressed. (D) The number cells of macrophage subtypes in NEC and CON group. (E) Bar plots showing the -log10 (p-value) from enrichment analysis of representative GO biological functions among different macrophage subtypes.

Figure 6

(A-B) Representative images of intestinal tissue sections in NEC and CON group stained by hematoxylin and eosin (n=5). Scale bar=20μm. (C) The pathological grade of the intestine tissue in NEC and CON group (n=5). *, p < 0.05; **, p < 0.01; ***, p < 0.001. (D-M) Validation of the ten hub genes' mRNA expression in intestinal tissue of NEC and CON group (n=3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. (N) Representative immunofluorescent staining images of MMP9 expression in intestinal tissue of NEC and CON group (n=5).