Potential of algae-derived alginate oligosaccharides and β-glucan to counter inflammation in adult zebrafish intestine

Abstract

Alginate oligosaccharides (AOS) are natural bioactive compounds with anti-inflammatory properties. We performed a feeding trial employing a zebrafish (Danio rerio) model of soybean-induced intestinal inflammation. Five groups of fish were fed different diets: a control (CT) diet, a soybean meal (SBM) diet, a soybean meal+β-glucan (BG) diet and 2 soybean meal+AOS diets (alginate products differing in the content of low molecular weight fractions - AL, with 31% < 3kDa and AH, with 3% < 3kDa). We analyzed the intestinal transcriptomic and plasma metabolomic profiles of the study groups. In addition, we assessed the expression of inflammatory marker genes and histological alterations in the intestine. Dietary algal β-(1, 3)-glucan and AOS were able to bring the expression of certain inflammatory genes altered by dietary SBM to a level similar to that in the control group. Intestinal transcriptomic analysis indicated that dietary SBM changed the expression of genes linked to inflammation, endoplasmic reticulum, reproduction and cell motility. The AL diet suppressed the expression of genes related to complement activation, inflammatory and humoral response, which can likely have an inflammation alleviation effect. On the other hand, the AH diet reduced the expression of genes, causing an enrichment of negative regulation of immune system process. The BG diet suppressed several immune genes linked to the endopeptidase activity and proteolysis. The plasma metabolomic profile further revealed that dietary SBM can alter inflammation-linked metabolites such as itaconic acid, taurochenodeoxycholic acid and enriched the arginine biosynthesis pathway. The diet AL helped in elevating one of the short chain fatty acids, namely 2-hydroxybutyric acid while the BG diet increased the abundance of a vitamin, pantothenic acid. Histological evaluation revealed the advantage of the AL diet: it increased the goblet cell number and length of villi of the intestinal mucosa. Overall, our results indicate that dietary AOS with an appropriate amount of < 3kDa can stall the inflammatory responses in zebrafish.

Keywords: RNA seq; gut; macroalgae; metabolomics; microalgae; prebiotics; β-glucans.

Figure 1

Relative expression of immune genes in the intestine of zebrafish fed different diets. (A) interleukin-1b (il1b); (B) matrix metalloproteinase-9 (mmp9); (C) myeloid-specific peroxidase (mpx); (D) chemokine (C-X-C motif) ligand 8a (cxcl8a); (E) superoxide dismutase 1 (sod1); (F) catalase (cat); (G) mucin2.1 (muc2.1); (H) mucin5ac (muc5ac); (I) glutathione peroxidase (gpx1a); (J) interleukin-10 (il10). CT- control diet; SBM- soybean diet; BG- algal β-glucan; AL- AOS with 31% < 3kDa; AH- AOS with 3% < 3kDa. Asterisk * p < 0.05, ** p < 0.01 and *** p < 0.001, • p < 0.1. Each treatment group consisted of five biological replicates.

Figure 2

Transcriptome-based differences in the intestine of zebrafish from the soybean group compared to the control group. Volcano plot (A), PCA plot (B) and heatmap (C) of the differentially expressed genes in the soybean (SBM) group compared to the control (CT) group. Chord diagram showing the link between the enriched GO terms in the soybean group and the associated genes that were upregulated (D) and downregulated (E) in the soybean (SBM) group compared to the control (CT) group. The enriched GO terms are colour-coded and on the right side of the circles one finds the differentially expressed genes contributing to the enriched GO terms that are shown on the left of the circles. The gradient colour bar intensity varies with the Log2 fold change (adjusted p-value < 0.05 and |Log2 fold change| ≥ 1). There are five biological replicates in each study group.

Figure 3

Transcriptome-based differences in the intestine of zebrafish from the algal β-glucan group compared to the soybean group. Volcano plot (A), PCA plot (B) and heatmap (C) of the differentially expressed genes in the algal β-glucan (BG) group compared to the soybean (SBM) group. Transcripts with an adjusted p-value below 0.05 and |Log₂ fold change| ≥ 1 were considered as significantly differentially expressed. Dot plot (D) showing enriched GO terms in the BG group based on the genes that were differentially downregulated compared to the SBM group. The gradient colour bar intensity of GO terms varies with the p value of each GO term. There are five biological replicates in each study group.

Figure 4

Transcriptome-based differences in the intestine of zebrafish fed AOS with 31% <3kDa and AOS with 3% <3kDa compared to the soybean group. Volcano plot (A), PCA plot (B) and heatmap (C) of the differentially expressed genes in the AL (AOS with 31% <3kDa) group compared to the soybean (SBM) group. Dot plot (D) showing the enriched GO terms in the AL group based on the differentially downregulated genes compared to the SBM group. The gradient colour bar intensity of GO terms varies with the p value of each GO term. Volcano plot (E), PCA plot (F), heatmap (G) and differentially expressed genes (H) obtained by comparing the AH (AOS with 3% < 3kDa) group with the soybean (SBM) group. Transcripts with an adjusted p value below 0.05 and |Log₂ fold change| ≥ 1 were considered as significantly differentially expressed. There are five biological replicates in each study group.

Figure 5

Venn diagrams showing the total number of genes that were altered by the experimental diets. The genes that were differentially upregulated in the SBM vs CT comparison (A) but downregulated in the BG, AL and AH vs SBM comparisons. The genes that were differentially downregulated in the SBM vs CT comparison (B) but upregulated in the BG, AL and AH vs SBM comparisons. Genes that were altered in the SBM group had mRNA levels in the AL (C, D) and AH (E, F) groups, respectively, similar to that in the control group.

Figure 6

Diet-induced changes in plasma metabolites of zebrafish. Volcano plot (A), heatmap (B) and enriched KEGG pathways (C) by the differentially altered metabolites in the soybean (SBM) group compared to the control (CT) group (|Log₂ fold-change| ≥ 0.6, p value < 0.05). Volcano plot (D) showing the differentially abundant metabolites in the AL group (AOS diet with 31% < 3kDa) compared to the soybean (SBM) group. There are five biological replicates in each study group.

Figure 7

Diet-induced histomorphological changes in the intestine of zebrafish. Representative histological images (A) showing the changes in tissue architecture of the intestine of zebrafish stained with AB-PAS. Scale bar = 100 μm. The measured parameters include (B) Goblet cells per villus (C) Villi length (D) Goblet cell size (E) Eosinophils per villus (F) Lamina propria width. Horizontal bars indicate mean values. * indicates p < 0.05 and p < 0.1 (n = 6-9 per group). CT- control diet; SBM- soybean diet; BG- algal β-glucan diet; AL- AOS diet with 31% < 3kDa; AH- AOS diet with 3% < 3kDa.