Dietary Red Raspberry Reduces Colorectal Inflammation and Carcinogenic Risk in Mice with Dextran Sulfate Sodium-Induced Colitis

Abstract

Background: Ulcerative colitis causes recurring intestinal mucosal injury and sustained inflammation, increasing the likelihood of colorectal cancer (CRC) development. Dietary red raspberry (RB) is a rich source of phytonutrients known to have anti-inflammatory activity; however, the role of RB on CRC prevention in chronic colitis has not been examined.

Objective: This study examined the effects of dietary RB supplementation on inflammation, epithelium repair, and oncogenic signaling in dextran sulfate sodium (DSS)-induced chronic colitis in mice.

Methods: Six-week-old male C57BL/6J mice were fed a control or RB (5% of dry feed weight; n = 12/group) diet for 10 wk. Starting from the fourth week, mice were administered 2 repeated cycles of 1% DSS (7-d DSS treatment plus 14-d recovery) and were monitored daily for disease activity index (DAI) score. Colonic tissues were collected at the end of the study for histochemical, immunohistochemical, and biochemical analysis of inflammation, differentiation and proliferation markers.

Results: RB supplementation reduced the DAI score and histologic damage (by 38.9%; P ≤ 0.01), expression of inflammatory mediators (by 20-70%; P ≤ 0.01), infiltration of CD4 T cells (by 50%; P ≤ 0.05), and α4β7 integrin and related adhesion molecules (by 33.3%; P ≤ 0.01). Furthermore, RB supplementation facilitated epithelium repair, as evidenced by enhanced goblet cell density, expression of transcription factors including Kruppel-like factor 4 (Klf4) and Hairy and enhancer of split 1 (Hes1), terminal differentiation markers, mucin 2 (Muc2), and intestinal alkaline phosphatase (by 20-200%; P ≤ 0.01). Conversely, proliferating cell nuclear antigen (by 70%; P ≤ 0.01), β-catenin, and signal transducer and activator of transcription 3 (STAT3) signaling (by 19-33%; P ≤ 0.05) were reduced by RB supplementation. In addition, RB supplementation enhanced p53 stability (by 53%) and reduced oncogenic gene expression (by 50-60%).

Conclusion: RB supplementation reduced DAI score and the risk of CRC development during recurring colitis in mice, suggesting that RB is a possible dietary supplement for patients with ulcerative colitis and related gut inflammatory diseases.

Keywords: colitis; colorectal cancer; inflammation; intestine; proliferation; red raspberry.

FIGURE 1

Disease activity index scores (A) and histopathologic scores (B) of male mice fed a CON diet or a 5% RB diet subjected to two 7-d cycles of 1% DSS treatment. Values are means ± SEMs,n = 12. **Different from CON,P ≤ 0.01. Panel B shows representative images of hematoxylin and eosin-stained colonic tissue. (Representative images are at 200× magnification.) CON, control; DSS, dextran sulfate sodium; RB, red raspberry.

FIGURE 2

Relative protein content and mRNA expression of IL-6 and COX-2 (A), mRNA expression ofIl17 andIfng (B) and CD4-positive cells (C), mRNA expression of adhesion molecules (D, E), and α4β7 integrin filtration score (F) in male mice fed a CON diet or a 5% RB diet subjected to two 7-d cycles of 1% dextran sulfate sodium treatment. Values are means ± SEMs,n = 12. *,**Different from CON: *P ≤ 0.05, **P ≤ 0.01. C and F are representative images of CD4 and α4β7 integrin immunohistochemical stained colonic tissue, respectively. (Representative images are at 200× magnification.) CON, control; COX-2, cyclooxygenase 2;Cxcl1, chemokine (C-X-C motif) ligand 1;Icam1, intercellular adhesion molecule 1;Ifng, interferon γ;Madcam1, mucosal vascular addressin cell adhesion molecule 1; RB, red raspberry;Vcam1, vascular cell adhesion molecule 1.

FIGURE 3

Goblet cell density (A); mRNA expression ofMuc2, Klf4, andAlpi (B); and mRNA expression and protein content of HES1 and PCNA (C) in male mice fed a CON diet or a 5% RB diet subjected to two 7-d cycles of 1% dextran sulfate sodium treatment. Values are means ± SEMs,n = 12. *,**Different from CON: *P ≤ 0.05, **P ≤ 0.01. Panel A shows representative images of Alcian blue–stained colonic tissue. (Representative images are at 200× magnification.)Alpi, intestinal alkaline phosphatase; CON, control; HES1, Hairy and enhancer of split 1;Klf4, Kruppel-like factor 4;Muc2, mucin 2; PCNA, proliferating cell nuclear antigen; RB, red raspberry.

FIGURE 4

Protein contents of β-catenin and p-β-catenin (Ser 552) (A), mucin 1 and E-cadherin (B), mRNA expression ofMuc1 (C), and protein contents of total STAT3, p-STAT3 (Tyr 705), and Ac-STAT3 (Lys 685) (D) in male mice fed a CON diet or a 5% RB diet subjected to two 7-d cycles of 1% dextran sulfate sodium treatment. Values are means ± SEMs,n = 12.^#,*,**Different from CON:^#P ≤ 0.10, *P ≤ 0.05, **P ≤ 0.01. Ac-STAT3, acetylated STAT3; CON, control;Muc1, mucin 1; p-β-catenin, phosphorylated-β-catenin; p-STAT3, phosphorylated STAT3; RB, red raspberry; STAT3, signal transducer and activator of transcription 3.

FIGURE 5

Protein contents of total p53 and p-p53 and p19^ARF (A), mRNA expression ofp21 (B), and antiapoptotic and oncogenic genes (C, D) in male mice fed a CON diet or a 5% RB diet subjected to two 7-d cycles of 1% dextran sulfate sodium treatment. Values are means ± SEMs,n = 12.^#,*,**Different from CON:^#P ≤ 0.10, *P ≤ 0.05, **P ≤ 0.01.Bcl2, B cell lymphoma 2;Ccdn1, proliferative cyclin D1; CON, control;Mcl1, myeloid cell leukemia 1;Myc, myelocytomatosis oncogene; p-p53, phosphorylated p53; RB, red raspberry.