Sanguinarine Improves Intestinal Health in Grass Carp Fed High-Fat Diets: Involvement of Antioxidant, Physical and Immune Barrier, and Intestinal Microbiota

Abstract

An eight-week trial was conducted to investigate the effects of sanguinarine supplementation (600 μg and 1200 μg/kg) in high-fat (crude fat: 10%) diets (HF) on the intestinal physiological function of Ctenopharyngodon idellus (initial weight 50.21 ± 0.68 g), based on a basic diet (5% crude fat, CON), which were named HFLS and HFHS, respectively. The results showed that the HF diet significantly impaired the intestinal immune and physical barrier function, and disrupted the balance of the intestinal microbiota in grass carp. Compared to the HF diet, sanguinarine supplementation significantly improved the levels of serum C4, C3, AKP, IgA, and IgM, and enhanced the intestinal antioxidant capacity (gr, CuZnsod, gpx4, cat, gsto, and nrf2 expression were significantly up-regulated). Sanguinarine significantly down-regulated the expression of claudin-15 and up-regulated the expression of claudin-b, claudin-c, occludin, and zo-1 by inhibiting MLCK signaling molecules. Additionally, sanguinarine significantly down-regulated the expression of il-6, il-1β, and tnf-α and up-regulated the expression of il-10, tgf-β2, and tgf-β1 by inhibiting NF-κB signaling molecules, thereby alleviating intestinal inflammation caused by HF diets. Furthermore, compared to the HF diet, the abundance of Fusobacterium and Cetobacterium in the HFHS diet increased significantly, while the abundance of Firmicutes and Streptococcus showed the opposite trend. In conclusion, the HF diet had a negative impact on grass carp, while sanguinarine supplementation enhanced intestinal antioxidant ability, alleviated intestinal barrier damage, and ameliorated the homeostasis of the intestinal microbiota.

Keywords: Chinese herbal extract; Ctenopharyngodon idellus; high-fat diet; intestinal barrier; oxidative stress.

Figure 1

Effects of adding sanguinarine to a high-fat diet on intestinal antioxidant enzyme activities of grass carp. Reactive oxygen species (ROS, U/mg); Malondialdehyde (MDA, nmol/kg); Superoxide dismutase (SOD, U/mg); Glutathione peroxidase (GPX, U/mg); Catalase (CAT, U/mg); Glutathione (GSH, μmol/mg). Bars represent the mean ± SE. Significant differences are expressed by different superscripts (p < 0.05).

Figure 2

Effects of adding sanguinarine to a high-fat diet on intestinal antioxidant enzyme-related gene expression of grass carp. (A) cat; (B) CuZnsod; (C) gpx1; (D) gpx4; (E) gsto; (F) gr; (G) nrf2; (H) keap1. Bars represent the mean ± SE. Significant differences are expressed by different superscripts (p < 0.05).

Figure 3

Effects of adding sanguinarine to a high-fat diet on intestinal morphology of grass carp. (A) control (CON) group; (B) high-fat (HF) group; (C) HF diet with 1200 μg/kg sanguinarine (HFHS) group; (D) intestinal villi length and goblet cell quantity. Bars represent the mean ± SE. *: p < 0.05, ns: p > 0.05.

Figure 4

Effects of adding sanguinarine to a high-fat diet on intestinal physical barrier-related gene expression of grass carp. Bars represent the mean ± SE. Significant differences are expressed by different superscripts (p < 0.05).

Figure 5

Effects of adding sanguinarine to a high-fat diet on MLCK gene expression and protein expression of grass carp. (A) relative gene expression level of mlck; (B) relative protein level of MLCK; (C) Blot of MLCK and β-actin. Bars represent the mean ± SE. Significant differences are expressed by different superscripts (p < 0.05).

Figure 6

Influences of dietary mulberry leaf flavonoids on antioxidant relative genes expression in the livers of rice field eels fed a high-carbohydrate diet. (A) pro-inflammatory factor related expression; (B) anti-inflammatory factor related expression; (C) the expression of regulatory factors. Different letters show significant difference (p < 0.05).

Figure 7

Effects of adding sanguinarine to a high-fat diet on NF-κB gene expression and protein expression of grass carp. (A) relative gene expression level of nf-κb; (B) relative protein level of NF-κB p65; (C) Blot of NF-κB p65 and β-actin. Bars represent the mean ± SE. Significant differences are expressed by different superscripts (p < 0.05).

Figure 8

Effects of adding sanguinarine to a high-fat diet on α-, β-diversity, and evolutionary tree in intestinal microbiota of grass carp. (A) α-diversity; (B) β-diversity; (C) evolutionary tree. Bars represent the mean ± SE. * p < 0.05.

Figure 9

Effects of adding sanguinarine to high-fat diets on the microbial composition of grass carp. (A) phylogenetic tree plot; (B) classification hierarchy tree diagram (The largest circle represents phylum level, and the decreasing circle represents class, order, family, genus and species in descending order); (C) species abundance at the phylum level (Top 10); (D) showing significant variations of the relative abundance of intestinal microbiota at the phylum level; (E) species abundance at the genus level (Top 10); (F) showing significant variations of the relative abundance of intestinal microbiota at the genus level. Bars represent the mean ± SE. * p < 0.05.

Figure 10

Effects of adding sanguinarine to high-fat diets on the microbial composition of grass carp. Bars represent the mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001.