Nintedanib Alleviates Experimental Colitis by Inhibiting CEBPB/PCK1 and CEBPB/EFNA1 Pathways

Abstract

The super-enhancer, a cluster of enhancers with strong transcriptional activity, has become one of the most interesting topics in recent years. This study aimed to investigate pathogenic super-enhancer-driven genes in IBD and screen therapeutic drugs based on the results. In this study, through the analysis of differentially expressed genes in colitis patients from the GEO database and the analysis of the super-enhancer-associated database, we found that the super-enhancer pathogenic genes PCK1 and EFNA1 were simultaneously regulated by transcription factor CEBPB through two super-enhancers (sc-CHR20-57528535 and sc-CHR1-155093980). Silencing CEBPB could significantly inhibit the expression of PCK1 and EFNA1 and enhance the expression of epithelial barrier proteins claudin-1, occludin, and ZO-1. In LPS-induced Caco-2 cells, drugs commonly used in clinical colitis including tofacitinib, oxalazine, mesalazine, and sulfasalazine inhibited mRNA levels of CEBPB, PCK1, and EFNA1. In the drug screening, we found that nintedanib significantly inhibited the mRNA and protein levels of CEBPB, PCK1, and EFNA1. In vivo experiments, nintedanib significantly alleviated DSS-induced colitis in mice by inhibiting CEBPB/PCK1 and CEBPB/EFNA1 signaling pathways. At the genus level, nintedanib improved the composition of the gut microbiota in mice with DSS-induced experimental colitis. In conclusion, we found that PCK1 and EFNA1 are highly expressed in colitis and they are regulated by CEBPB through two super-enhancers, and we further demonstrate their role in vivo and in vitro. Nintedanib may be a potential treatment for IBD. Super-enhancers may be a new way to explore the pathogenesis of colitis.

Keywords: CEBPB/EFNA1; CEBPB/PCK1; inflammatory bowel disease; nintedanib; super-enhancer.

FIGURE 1

Identification of potential pathogenic super-enhancer–driver genes in IBD. (A) Volcano diagram of differentially expressed genes between IBD patients and normal people in datasets GSE107499, GSE75214, and GSE59071 in the GEO database. (B) Venn diagram formed by cross-linking of super-enhancer–associated genes and differentially expressed genes in dataset in inflammatory bowel disease. (C) Interaction network of TF, super-enhancer, and related genes in inflammatory bowel disease. (D) The KEGG pathway analysis. (E) TFs binding PCK1 and EFNA1 super-enhancers.

FIGURE 2

Silencing transcription factor CEBPB significantly inhibited the expression of PCK1\EFNA1 and increased the expression of intestinal epithelial barrier protein. The protein expression of super-enhancer–associated gene PCK1\EFNA1 (A) and intestinal barrier proteins ZO-1, occludin, and claudin-1 (B) after transfection of siCEBPB. Data are presented as the mean ± SD (n = 3). ^* p < 0.01 and ^** p < 0.05 significantly different from the control group. (C) Inhibitory effect of IBD marketed drugs (olsalazine, mesalazine, tofacitinib, and sulfasalazine) on CEBPB, PCK1, and CEBPB mRNA. Data are presented as the mean ± SD (n = 3). ^#### p < 0.001 significantly different from the control group; ^* p < 0.1, ^*** p < 0.001 and ^**** p < 0.0001, significantly different from LPS groups.

FIGURE 3

Screening of tyrosine kinase inhibitors and the effect of nintedanib on barrier protein in Caco-2 cells. (A) Screening of tyrosine kinase inhibitors in LPS-induced Caco-2 cells. (B) Nintedanib significantly inhibited the protein expression levels of CEBPB, PCK1, and EFNA1. (C) Nintedanib improves the injury of intestinal epithelial barrier protein in vitro. Data are presented as the mean ± SD (n = 3). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 and ^#### p < 0.0001 significantly different from the control group; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001, significantly different from LPS groups.

FIGURE 4

Effect of nintedanib on DSS-induced experimental colitis in mice. (A) 5% DSS-induced experimental colitis in mice. Effects of nintedanib on weight change (B), the DAI score (C), and colon length change (D) in experimental colitis mice. (E) Histological sections of colonic tissue stained with hematoxylin and eosin are shown using a microscope. Scale bars represent 50 μm. (F) Representative images of Alcian blue-stained inner mucus layer of colonic sections. Scale bars represent 50 μm. (G) The effect of nintedanib on the expression of intestinal epithelial barrier proteins ZO-1, occludin, and claudin-1. Data are presented as the mean ± SD (n = 5). ^## p < 0.01, ^### p < 0.001, significantly different from the control group; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, significantly different from DSS groups.

FIGURE 5

Inhibition of CEBPB/PCK1 and CEBPB/EFNA1 pathways by nintedanib in vivo. (A) Effect of nintedanib on the level of CEBPB, PCK1, and EFNA1 mRNA in the colon tissue. (B) Effect of nintedanib on the expression of CEBPB, PCK1, and EFNA1 proteins in the colon tissue. The IHC staining analysis of CEBPB in mouse (C) and human (D) colon tissues. Scale bars represent 50 μm. Data are presented as the mean ± SD (n = 5). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 and ^#### p < 0.0001, significantly different from the control group; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 and ^**** p < 0.0001, significantly different from DSS groups.

FIGURE 6

Nintedanib regulates the gut microbiota composition of DSS-induced experimental colitis. (A) The Venn diagram showing the unique and shared OTUs from control, DSS, and nintedanib groups. (B) The difference of OTUs among the control group, the DSS group, and the nintedanib group was determined by linear discriminant analysis combined with effect size (lefse). (C) PCOA of gut microbiota communities based on the OTU level. (D) The clustering analysis of the evolution of the gut microbiotas between control, DSS, and nintedanib groups. Based on the distance matrix, the gut microbiota tree was constructed using the UPGMA (unweighted pair group method with arithmetic mean) clustering method for analysis. (E) Histograms of relative abundance of grouped species at the genus level. (F) Gut microbiota differentially expressed at genus level between control and DSS groups. (G) Gut microbiota differentially expressed at genus level between DSS and nintedanib groups.