Effect of peptide LLTRAGL on TNBS-induced migration of zebrafish to intestinal macrophages. (A) Typical fluorescence images of Tg (zlyz: EGFP) macrophage migration in transgenic zebrafish juveniles. (B) Box chart showing the number of macrophages migrating from zebrafish larvae to the intestine. (A) the image on the right is an enlarged image of the white box on the left. Compared with the blank group, ## p ≤ 0.01; compared with the TNBS group, * p ≤ 0.05 and ** p ≤ 0.01.

LLTRAGL improves TNBS-induced intestinal peristalsis damage. (A) Typical fluorescence images of intestinal efflux rate (IEE) of wild zebrafish juveniles. The blue box shows that after staining with calcein for 1.5 h, the drug was administered for 16 h. (B) Box chart showing the intestinal efflux rate of zebrafish juveniles. (C) Violin chart showing the number of intestinal peristalses in zebrafish juveniles. Compared with the blank group, ## p ≤ 0.01; compared with the TNBS group, * p ≤ 0.05 and ** p ≤ 0.01.

Effects of the peptide on TNBS-induced intestinal tissue pathology and ultrastructure of zebrafish. (A) H&E staining of intestinal tissues. Black arrow: sparse arrangement of cells; Blue arrow: Mucosal layer necrosis, cell lysis, enhanced cytoplasmic basophilia, disappearance of intestinal folds. Scale bar is 100 µm. (B) Alcian blue (AB) staining of intestinal tissues. Scale bar is 100 µm. (C) Electron microscopy of intestinal ultrastructure. Red arrow: intestinal microvilli status; Mv represents microvilli; Green arrow: goblet cell state; GC stands for goblet cell. Scale bar is 10.0 µm.

The differentially expressed genes among the control group, TNBS group and peptide treatment group in RNA-Seq. (A) Statistical histogram of differentially expressed genes in TNBS vs. Control and LLTRAGL vs. TNBS. (B) TNBS vs. Control differential gene grouping cluster diagram. (C) LLTRAGL vs. TNBS differential gene grouping cluster diagram. (D) Venn diagram of common and unique differentially expressed genes between the TNBS vs. Control and LLTRAGL vs. TNBS comparative groups. The red color in the figure represents genes encoding relatively high expression proteins, while the blue color represents genes encoding relatively low expression proteins.

GO enrichment analysis of DEGs and GSEA of all tested genes. (A) Circle plots of the distribution of DEGs in different GO categories. The circle chart shows the distribution of DEGs in the molecular function (MF), biological process (BP), and cellular composition (CC) categories. (B) Grouping clustering diagram of inflammatory response in the GSEA analysis. (C) Analysis Results of the Inflammatory Response Gene Set, mainly including the distribution map of enrichment score (ES), gene distribution map of gene set, and distribution map of the measurement and control bar sorting matrix.

KEGG pathway enrichment analysis of DEGs. (A) KEGG-enriched top 10 chord diagram of TNBS vs. Control. (B) KEGG-enriched top 10 chord diagram of LLTRAGL-vs-TNBS. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Effect of LLTRAG on the inflammation-related gene expression in zebrafish, determined by RT-PCR. Compared with the blank group, # p ≤ 0.05 and ## p ≤ 0.01; compared with the TNBS group, * p ≤ 0.05 and ** p ≤ 0.01.

Effect of LLTRAG on gene expression in TNBS-induced colitis in zebrafish. (A) NOD-like receptor signal pathway-related genes. (B) Inflammatory factors-related genes. (C) necroptosis signal pathway-related gene. Compared with the blank group, # p ≤ 0.05 and ## p ≤ 0.01; compared with the TNBS group, * p ≤ 0.05 and ** p ≤ 0.01.