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. 2022 Oct 13:9:957391.
doi: 10.3389/fnut.2022.957391. eCollection 2022.

Collagen peptide promotes DSS-induced colitis by disturbing gut microbiota and regulation of macrophage polarization

Xiaoxia Li  1 , Luwen Cui  1 , Guilin Feng  1 , Shengnan Yu  1 , Guanglong Shao  1 , Ningning He  1 , Shangyong Li  1
Affiliations

Collagen peptide promotes DSS-induced colitis by disturbing gut microbiota and regulation of macrophage polarization

Xiaoxia Li et al. Front Nutr. .

Abstract

Ulcerative colitis (UC) is an inflammatory bowel disease caused by mucosal immune system disorder, which has increased steadily all over the world. Previous studies have shown that collagen peptide (CP) has various beneficial biological activities, it is not clear whether the effect of CP on UC is positive or negative. In this study, 2.5% dextran sulfate sodium (DSS) was used to establish acute colitis in mice. Our results suggested that CP supplementation (200, 400 mg/kg/day) promoted the progression of colitis, increased the expression of inflammatory factors and the infiltration of colonic lamina propria macrophages. Gut microbiota analysis showed the composition changed significantly and inflammation promoted bacteria was after CP treatment. Meanwhile, the effect of CP on macrophage polarization was further determined in Raw264.7 cell line. The results showed that CP treatment could increase the polarization of M1 macrophages and promote the expression of inflammatory factors. In conclusion, our results showed that CP treatment could disrupt the gut microbiota of host, promote macrophage activation and aggravate DSS-induced colitis. This may suggest that patients with intestinal inflammation should not take marine derived CP.

Keywords: DSS-induced colitis; collagen peptide; gut microbiota; intestinal barrier; macrophage polarization.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
CP aggravated UC progression in DSS-induced colitis mice. (A) Molecular weight of CP. (B) Designed experiment flow. Body weight change (C) and DAI index (D) for 7 days DSS induction. (E) Survival rate of four experiment groups. (F) Colon pictures and colon length of four experiment groups. (G) Representative H&E staining. Compare with DSS group, *P < 0.05, **P < 0.01, ***P < 0.001.
FIGURE 2
FIGURE 2
CP maintained the colonic epithelial barrier. (A) Western blot results of ZO-1, Occludin, Claudin-1 (n = 5). Quantitative analysis of western blot image for ZO-1 (B), Occludin (C), Claudin-1 (D). (E) Real-time PCR results of MUC2 (n = 5). (F) Western blot and its quantitative analysis of MUC2 (n = 5). (G) Representative images of Alcian blue staining. Compare with DSS group, **P < 0.01, ***P < 0.001.
FIGURE 3
FIGURE 3
CP changed composition of gut microbiota. (A) Common OTUs for three groups. (B) PCoA analysis (n = 4). (C) Composition of microbiota at phylum level. Relative abundance of Bacteroidota (D), Firmicutes (E), Proteobacteria (F) and Verrucomicrobiota (G). Shannon index (H), Chao 1 index (I), Ace index (J) and PD_while_tree index (K) for three groups. Compare with DSS group, *P < 0.05, **P < 0.01, ***P < 0.001.
FIGURE 4
FIGURE 4
Change of gut microbiota by CP treatment. Heatmap of microbiome species abundance at the family (A) and genus level (B). Color intensity was used to represent the abundance of bacteria. (C) Linear discriminant analysis (LDA) effect size (LEfSe) and (D) cladogram of species annotated by OTU were used to identify deferentially abundant taxa. Family (f), Genera (g), Class (c), Phylum (p), Order (o), Species (s) with P < 0.05 and LDA score (log 10) > 2 were considered significant.
FIGURE 5
FIGURE 5
CP enhanced the inflammatory response in DSS-induced colitis. The expression of level of TNF-α (A), IL-1β (B), IL-6 (C) and IL-10 (D) in serum measured by ELISA (n = 5). Representative immunohistochemistry staining images for CD206 (E) and iNOS (F). Panels (G,H) are quantitative analysis of panels (E,F) by IntDen/Area using Image J software. Western blot results of p-P65, P65 (I) and the quantitative analysis of western blot image for P65 (J) and p-P65 (K) in colonic tissue samples. Compare with DSS group, *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant.
FIGURE 6
FIGURE 6
CP aggravate inflammation in macrophages using Raw264.7 cells. The mRNA expression changes of IL-6, IL-1β and TNF-α after 24 h treatment with 0.1, 0.5 or 5 mg/ml CP in murine Raw264.7 cells (A) or LPS-stimulated Raw264.7 cells (B). The ELISA assay for expression changes of IL-6, IL-1β and TNF-α after 24 h treatment with 0.1, 0.5 or 5 mg/ml CP in murine Raw264.7 cells (C) or LPS-stimulated Raw264.7 cells (D). *P < 0.05, **P < 0.01, ***P < 0.001.
FIGURE 7
FIGURE 7
CP aggravate inflammation in macrophages through NF-κB signaling. Representative flow cytometry analysis of CD80 levels in different treatment groups of Raw264.7 cells (A). The proportion of CD80-positive cells of three independent experiments (B). The phagocytosis changes of Raw264.7 cells with or without CP treatment indicated by relative MFI (mean fluorescence intensity) from FACS (C). Western blot results of p-P65, P65 (D) and the quantitative analysis of western blot image for p-P65, P65 (E) in Raw264.7 cells. *P < 0.05, **P < 0.01, ***P < 0.001.
FIGURE 8
FIGURE 8
The proposed model for CP on colitis. CP disturbs gut microbiota and impairs colonic epithelial barrier, which then triggers the activation of macrophage and aggravated colitis progression.
See this image and copyright information in PMC

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