Dihydrosanguinarine enhances tryptophan metabolism and intestinal immune function via AhR pathway activation in broilers

Abstract

Background: Tryptophan is essential for nutrition, immunity and neural activity, but cannot be synthesized endogenously. Certain natural products influence host health by modulating the gut microbiota to promote the production of tryptophan metabolites. Sanguinarine (SAN) enhances broiler immunity, however, its low bioavailability and underlying mechanisms remain unclear. This study aimed to decode the mechanisms by which sanguinarine enhances intestinal immune function in broilers.

Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to identify the main metabolites of sanguinarine in the intestine. Subsequently, equal concentrations of sanguinarine and its metabolites were separately added to the diets. The effects of sanguinarine and its metabolites on the intestinal immune function of broiler chickens were evaluated using 16S rRNA gene amplicon sequencing and tryptophan metabolomics approaches.

Results: We determined that dihydrosanguinarine (DHSA) is the main metabolite of sanguinarine in the intestine. Both compounds increased average daily gain and reduced feed efficiency, thereby improving growth performance. They also enhanced ileal villus height and the villus-to-crypt (V/C) ratio while decreasing crypt depth and upregulating the mRNA expression of tight junction proteins ZO-1, occludin and claudin-1. Furthermore, both compounds promoted the proliferation of intestinal Lactobacillus species, a tryptophan-metabolizing bacterium, stimulated short-chain fatty acid production, and lowered intestinal pH. They regulated tryptophan metabolism by increasing the diversity and content of indole tryptophan metabolites, activating the aryl hydrocarbon receptor (AhR) pathway, and elevating the mRNA levels of CYP1A1, CYP1B1, SLC3A1, IDO2 and TPH1. Inflammatory cytokines IL-1β and IL-6 were inhibited, while anti-inflammatory cytokines IL-10 and IL-22, serum SIgA concentration, and intestinal MUC2 expression were increased. Notably, DHSA exhibited a more pronounced effect on enhancing immune function compared to SAN.

Conclusions: SAN is converted to DHSA in vivo, which increases its bioavailability. DHSA regulates tryptophan metabolism by activating the AhR pathway and modulating immune-related factors through changes in the gut microbiota. Notably, DHSA significantly increases the abundance of Lactobacillus, a key tryptophan-metabolizing bacterium, thereby enhancing intestinal immune function and improving broiler growth performance.

Keywords: Broiler; Gut microbiota; Intestinal immunity; Sanguinarine; Tryptophan metabolism.

Fig. 1

Structural identificati of sanguinarine and its metabolites in the intestine. A Schematic diagram of the detection process of sanguinarine metabolites in intestinal contents. B MS² chromatogram of M₀ and fragmentation pathway. C Chromatogram of sanguinarine peak emergence time in ileum tissue. D Chromatogram of sanguinarine peak emergence time in cecum tissue. E MS² chromatogram of M₁ and fragmentation pathway. F Chromatogram of peak emergence time of dihydrosanguinarine in ileum tissue. G Chromatogram of peak emergence time of dihydrosanguinarine in cecum tissue

Fig. 2

Sanguinarine and dihydrosanguinarine enhanced ileal immune function in broilers. A Experimental grouping and treatment. B Optical microscopy was used to monitor HE-stained sections. C The villus height, crypt depth and villus height / crypt depth (V/C) of the ileum were analyzed using HE staining. D The mRNA expression levels of ZO-1, occludin and claudin-1 were examined using qRT-PCR. E The levels of IL-1β, IL-6, IL-10, IL-22 and SIgA in the ileum were analyzed by ELISA kits. F The relative expression levels of IL-1β, IL-6, IL-10, IL-22 and MUC2 in the ileum were analyzed by qRT-PCR. CON Control group (basal diet), SAN Sanguinarine group (basal diet containing 0.225 mg/kg sanguinarine), DHSA Dihydrosanguinarine group (basal diet containing 0.225 mg/kg dihydrosanguinarine). Values are expressed as mean ± SEM (n = 5). *P < 0.05; **P < 0.01

Fig. 3

Sanguinarine and dihydrosanguinarine regulated tryptophan metabolism in the intestine. A PCA. B Venn diagram comparing tryptophan species in the ileum (left) and cecum (right) of broilers between experimental groups and CON group. C Heatmap of tryptophan metabolite species. D The contents of β-Indole-3-acetic acid, Indole-3-lactic acid, Indole-3-β-acrylic acid, Indolylpropionic acid, serotonin and quinolinic acid in intestinal of broilers. CON Control group (basal diet), SAN Sanguinarine group (basal diet containing 0.225 mg/kg sanguinarine), DHSA Dihydrosanguinarine group (basal diet containing 0.225 mg/kg dihydrosanguinarine). Values are expressed as mean ± SEM (n = 3). *P < 0.05; **P < 0.01

Fig. 4

Sanguinarine and dihydrosanguinarine could activate AhR pathway in the intestine. Stereographic and closeup images of A sanguinarine and B dihydrosanguinarine docked into TPH1 protein and their hydrogen bonding interactions. Stereographic and closeup images of C sanguinarine and D dihydrosanguinarine docked into CYP1A2 protein and their hydrogen bonding interactions. E The relative expression levels of CYP1A1, CYP1A2, CYP1B1, IDO2, TPH1 and SLC3A1 in the ileum were analyzed by qRT-PCR. F To evaluate the correlation between the types and contents of tryptophan and its metabolites and AhR pathway gene expression in ileum of broilers by spearman correlation analysis. G The relative expression levels of CYP1A1, CYP1A2, CYP1B1, IDO2, TPH1 and SLC3A1 in the cecum were analyzed by qRT-PCR. H To evaluate the correlation between the types and contents of tryptophan and its metabolites and AhR pathway gene expression in cecum of broilers by spearman correlation analysis. CON Control group (basal diet), SAN Sanguinarine group (basal diet containing 0.225 mg/kg sanguinarine), DHSA Dihydrosanguinarine group (basal diet containing 0.225 mg/kg dihydrosanguinarine). Values are expressed as mean ± SEM (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

Sanguinarine and dihydrosanguinarine regulated the intestinal flora structure and abundance. A Evaluation of α diversity of the intestinal microbiota in broilers. B PCoA plot of the ileum (left) and cecum (right) microbiota of broilers. C Venn diagram analysis of ileum flora in broilers. D Composition of the top 15 abundant microorganisms in ileal of broilers based on genus level. E The difference of microflora abundance in ileum content samples between experimental groups and CON group. F Venn diagram analysis of cecum flora in broilers. G Composition of the top 15 abundant microorganisms in cecal of broilers based on genus level. H The difference of microflora abundance in cecum content samples between experimental groups and CON group. I Correlation analysis between the top 15 abundant microorganisms in the ileum of broilers and the contents of tryptophan and its metabolites based on genus level. J Correlation analysis between the top 15 abundant microorganisms in the cecum of broilers and the contents of tryptophan and its metabolites based on genus level. CON Control group (basal diet), SAN Sanguinarine group (basal diet containing 0.225 mg/kg sanguinarine), DHSA Dihydrosanguinarine group (basal diet containing 0.225 mg/kg dihydrosanguinarine). Values are expressed as mean ± SEM (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 6

Sanguinarine and dihydrosanguinarine could enhance the immune function of cecum. A The relative expression levels of ZO-1, occludin, claudin-1, MUC2, IL-1β, IL-6, IL-10 and IL-22 in the cecum were analyzed by qRT-PCR. B Correlation analysis between the top 15 abundant microorganisms in the ileum of broilers and the contents of ileum immune factor based on genus level. C Correlation analysis between the top 15 abundant microorganisms in the cecum of broilers and the contents of cecum immune factor based on genus level. CON Control group (basal diet), SAN Sanguinarine group (basal diet containing 0.225 mg/kg sanguinarine), DHSA Dihydrosanguinarine group (basal diet containing 0.225 mg/kg dihydrosanguinarine). Values are expressed as mean ± SEM (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001