Fish Sidestream-Derived Protein Hydrolysates Suppress DSS-Induced Colitis by Modulating Intestinal Inflammation in Mice

Abstract

Inflammatory bowel disease is characterized by extensive intestinal inflammation, and therapies against the disease target suppression of the inflammatory cascade. Nutrition has been closely linked to the development and suppression of inflammatory bowel disease, which to a large extent is attributed to the complex immunomodulatory properties of nutrients. Diets containing fish have been suggested to promote health and suppress inflammatory diseases. Even though most of the health-promoting properties of fish-derived nutrients are attributed to fish oil, the potential health-promoting properties of fish protein have not been investigated. Fish sidestreams contain large amounts of proteins, currently unexploited, with potential anti-inflammatory properties, and may possess additional benefits through bioactive peptides and free amino acids. In this project, we utilized fish protein hydrolysates, based on mackerel and salmon heads and backbones, as well as flounder skin collagen. Mice fed with a diet supplemented with different fish sidestream-derived protein hydrolysates (5% w/w) were exposed to the model of DSS-induced colitis. The results show that dietary supplements containing protein hydrolysates from salmon heads suppressed chemically-induced colitis development as determined by colon length and pro-inflammatory cytokine production. To evaluate colitis severity, we measured the expression of different pro-inflammatory cytokines and chemokines and found that the same supplement suppressed the pro-inflammatory cytokines IL-6 and TNFα and the chemokines Cxcl1 and Ccl3. We also assessed the levels of the anti-inflammatory cytokines IL-10 and Tgfb and found that selected protein hydrolysates induced their expression. Our findings demonstrate that protein hydrolysates derived from fish sidestreams possess anti-inflammatory properties in the model of DSS-induced colitis, providing a novel underexplored source of health-promoting dietary supplements.

Keywords: IL-10; IL-6; chemokines; colitis; cytokines; fish protein hydrolysates; inflammation.

Figure 1

The effect of dietary supplements on colon length and spleen weight of mice with DSS-induced colitis. (A) Graphical illustration of the experimental design. (B) Representative intestines from each diet group. (C) Length measurements of the total intestines. (D) Hematoxylin and eosin tissue staining of colon tissue sections. White asterisks indicate tissue damage and loss of crypts; white arrow indicates infiltration of inflammatory cells. (E) Blinded histological scoring performed on H&E-stained colonic tissue. (F) Representative spleens from each diet group. (G) Weight measurements of the total spleens normalized to total body weight. Graphs represent median ± SD and an unpaired t-test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 1

The effect of dietary supplements on colon length and spleen weight of mice with DSS-induced colitis. (A) Graphical illustration of the experimental design. (B) Representative intestines from each diet group. (C) Length measurements of the total intestines. (D) Hematoxylin and eosin tissue staining of colon tissue sections. White asterisks indicate tissue damage and loss of crypts; white arrow indicates infiltration of inflammatory cells. (E) Blinded histological scoring performed on H&E-stained colonic tissue. (F) Representative spleens from each diet group. (G) Weight measurements of the total spleens normalized to total body weight. Graphs represent median ± SD and an unpaired t-test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Body weight change of mice exhibiting DSS-induced colitis. (A–F). Total body weight was monitored daily and expressed as percentage of initial weight prior to experimental start in each diet group. Graphs represent median ± SD and 2-way ANOVA statistical analysis was performed. * p < 0.05, ** p < 0.01.

Figure 3

DSS-induced colitis disease progression. (A–F) Disease severity in each diet group was monitored daily and scored as follows: Score = 0: normal stools. Score = 1: soft stools with positive hemoccult. Score = 2: very soft stools with traces of blood. Score = 3: watery stools with visible rectal bleeding. Graphs represent median ± SD and 2-way ANOVA statistical analysis was performed. * p < 0.05, ** p < 0.01.

Figure 4

The effect of dietary supplements on mRNA levels of pro-inflammatory cytokines in colon tissue of DSS-treated mice. The expression profile of (A) Tnfa, (B) Il-6 and (C) Il-1β was measured using real-time PCR in colon tissue. Graphs represent median ± SD and an unpaired t-test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

The effect of dietary supplements on protein levels of (A) intestinal IL-6, (B) serum IL-6 and (C) serum Cxcl1 quantified using ELISA. Graphs represent median ± SD and an unpaired t-test was performed. * p < 0.05, ** p < 0.01.

Figure 6

Evaluation of the expression of selected chemokines in DSS-induced colitis in mice. mRNA expression of (A) Cxcl1, (B) Cxcl2, (C) Ccl2 and (D) Ccl3 was quantified using real-time PCR in colon tissue of the selected diet groups. Graphs represent median ± SD and an unpaired t-test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Monitoring the effect of dietary supplements on the expression of anti-inflammatory mediators in colon tissue of DSS treated mice. (A) Il-10 and (B) Tgfb mRNA expression levels were measured using real-time PCR in the selected diet groups. Graphs represent median ± SD and an unpaired t-test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.