Pathological analysis of sinomenine’s effect on the intestinal mucosal fold. (A) HE staining of intestinal mucosa in adult fish fed with FM, SBM, and SN diets. Scale bar: 100 μm. (B) HE staining of intestinal mucosa in 27 dpf larva fed with FM, SBM, and SN diets. FM, fish meal diet; SBM, soybean meal diet; SN, sinomenine supplementary SBM diet. Scale bar: 20 μm. ** represented p < 0.01, and *** represented p < 0.001.

Lymphocyte-related intestinal protein expression reflected by immunofluorescent signals. CD4⁺(A) or Foxp3⁺(B) immunofluorescence signals (green) in adult fish fed with FM, SBM, and SN diets. The quantification of the ratio, which was calculated by dividing positive cell number with intestinal villi’s length, was shown beside the typical images (at the right side). Typical signals are indicated by arrows. The blue signals represented DAPI-stained cell nucleus. FM, fish meal diet; SBM, soybean meal diet; SN, sinomenine supplementary SBM diet. Scale bar: 100 μm. ** represented p < 0.01, and *** represented p < 0.001.

qPCR validation. (A) qPCR validation of transcriptomic data; (B) qPCR analysis of enteritis-related pro-/anti-inflammatory factors, which could reflect α7nAchR-mediated anti-inflammatory effect in fish. "ns" represented p > 0.05, * represented p < 0.05, ** represented p < 0.01, and *** represented p < 0.001.

Enriched GO terms and KEGG pathways for DEGs from the comparison between the SN and SBM groups in the zebrafish SBMIE adult model. (A) The GO terms of biological process; (B) the KEGG pathways. SBM, soybean meal diet; SN, sinomenine supplementary SBM diet.

Imaging analysis of immune cells in the hindgut of the zebrafish SBMIE larva model. (A) Hindgut innate immune cells, including neutrophils (lyz-DsRed labeled cells) and macrophages (mpeg-EGFP labeled cells); (B) the histogram of lyz or mpeg signals in the hindgut of larva. (C) Hindgut adaptive immune cells, including immature lymphocytes (rag2-DsRed-labeled red cells) and mature T lymphocytes (lck-EGFP-labeled green cells). (D) The histogram of rag2⁺ or lck ⁺ signals in the hindgut of larva. FM, fish meal diet; SBM, soybean meal diet; SN, sinomenine (35 ppm) supplementary SBM diet. The significant differences were indicated by p value, and *** represented p < 0.001. The scale bar in whole hindgut pictures was 100 μm, while the scale bar in the enlarged view was 20 μm.

Analysis of intestinal microbiota OTU and taxa composition influenced by sinomenine inclusion in zebrafish SBMIE adult model. (A) Venn diagram of OTU in the FM, SBM, and SN groups; (B) hindgut bacteria composition at phylum level; (C) hindgut bacteria composition at the genus level. FM, fish meal diet; SBM, soybean meal diet; SN, sinomenine supplementary SBM diet.

The hypothetical mechanism of SN’s α7nAchR-mediated anti-inflammatory effect on the intestinal immune barrier during foodborne enteritis in the fish hindgut (A) at the immune level; (B) at the microbiota and metabolic level. IEL, intestinal epithelial layer; LP, lamina propria layer; Mø, macrophage; Th, T helper; Treg, regulatory T cell.