Anti-inflammatory effects of freeze-dried black raspberry powder in ulcerative colitis

Abstract

Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa that can dramatically increase the risk of colon cancers. In the present study, we evaluated the effects of a dietary intervention of freeze-dried black raspberries (BRB), a natural food product with antioxidant and anti-inflammatory bioactivities, on disease severity in an experimental mouse model of UC using 3% dextran sodium sulfate (DSS). C57BL/6J mice were fed either a control diet or a diet containing BRB (5 or 10%) for 7-14 days and then the extent of colonic injury was assessed. Dietary BRB markedly reduced DSS-induced acute injury to the colonic epithelium. This protection included better maintenance of body mass and reductions in colonic shortening and ulceration. BRB treatment, however, did not affect the levels of either plasma nitric oxide or colon malondialdehyde, biomarkers of oxidative stress that are otherwise increased by DSS-induced colonic injury. BRB treatment for up to 7 days suppressed tissue levels of several key pro-inflammatory cytokines, including tumor necrosis factor α and interleukin 1β. Further examination of the inflammatory response by western blot analysis revealed that 7 day BRB treatment reduced the levels of phospho-IκBα within the colonic tissue. Colonic cyclooxygenase 2 levels were also dramatically suppressed by BRB treatment, with a concomitant decrease in the plasma prostaglandin E₂ (276 versus 34 ng/ml). These findings demonstrate a potent anti-inflammatory effect of BRB during DSS-induced colonic injury, supporting its possible therapeutic or preventive role in the pathogenesis of UC and related neoplastic events.

Fig. 1.

The effects of BRB on DSS-induced body weight changes. Mice were administered 3% DSS in drinking water for 7 days and then switched to plain drinking water for an additional 7 days and fed either a control diet or a diet containing 5 or 10% BRB powder (n = 10 per group) for the entire 14 day period. (A) Body weights were measured daily and reported as a percentage of body weight at the start of the experiment (day 0). (B) Area under the curve (days 0–14) was determined for each animal using the trapezoidal rule (GraphPad Prism, version 5). The effects of DSS and BRB were evaluated using one-way analysis of variance with Bonferroni's post-test to evaluate group mean difference. An α-level of P < 0.05 was set for statistical significance. Means not sharing a common superscript are significantly different from each other.

Fig. 2.

The effects of BRB on DSS-induced colonic disease. Mice were administered 3% DSS in the drinking water and concomitantly fed either a control diet or a diet containing BRB powder (5 or 10%) until sacrifice at 7 days [n = 10 per group; means ± standard errors of the mean (SEMs)]. (A) Colon lengths were determined immediately upon sacrifice and (B) percentage of colonic ulceration was quantified in hematoxylin- and eosin (H&E)-stained sections as described in Materials and Methods. Representative H&E staining of colonic ulcerations from mice given DSS plus control diet (C), 5% BRB diet (D) and 10% BRB diet (E) (×100); greater magnification of each panel is indicated by the boxes (×400); arrows indicate intact crypts retained within damaged mucosa. Data are means ± SEMs, n = 10 mice per group. Significance was determined by one-way analysis of variance with Bonferroni's post-test. Groups not sharing a common superscript are significantly different (P < 0.05).

Fig. 3.

The effects of BRB on DSS-induced activation of biomarkers of oxidative stress and inflammatory cell infiltrate in the colon. Mice were administered 3% DSS-incorporated drinking water and concomitantly fed either a control diet or a diet containing 10% BRB powder for 7 days. A control group of mice was administered plain drinking water along with either diet. Data are means ± standard errors of the mean, n = 4 mice per group. (A) Plasma nitrite levels were determined by nitric oxide production using a colorimetric assay kit. (B) MDA concentrations were determined in colon tissue by high-performance liquid chromatography-fluorescence as described under Materials and Methods. Two-way analysis of variance with Bonferroni's post-test was used to evaluate the effects of DSS, BRB and their interaction on nitrite and MDA. Nitrite and MDA were affected, P < 0.05, by DSS only. ‘*’ Indicates effects due to DSS. (C) fluorescence-activated cell sorting analysis was performed on cells isolated from the colonic lamina propria, as described in Materials and Methods. Cell populations are reported as fold changes after 7 days of DSS administration as compared with untreated mice for each diet.

Fig. 4.

The effects of BRB on DSS-induced pro-inflammatory cytokine production. Mice were administered 3% DSS-incorporated drinking water and concomitantly fed either a control diet or a diet containing 10% BRB powder for 7 days. A control group of mice was administered plain drinking water along with either diet. RNA was isolated from fresh-frozen colon samples and used for QRT-PCR analysis, as described under Materials and Methods. The relative messenger RNA expression levels of (A) TNF α and (B) IL-1β are shown. Data are means ± standard errors of the mean, n = 5 per group. Two-way analysis of variance with Bonferroni's post-test was used to evaluate the effects due to DSS, BRB and their interaction. TNFα was affected by DSS and BRB (P < 0.05). ‘*’ Indicates differences due to DSS and ‘$’ indicates differences due to BRB. A significant increase in IL-1β was observed in the control diet group after DSS administration. No significant increase in IL-1β was observed in mice given BRB. A trend (P = 0.075) was found for BRB to reduce IL-1β in comparison with control diet-fed mice after DSS administration.

Fig. 5.

The effects of BRB on DSS-induced changes in IκBα phosphorylation. Mice were administered 3% DSS in the drinking water concomitantly with either control or 10% BRB-incorporated powder diet. After 7 days, colons were harvested and protein lysates prepared for (A) immunoblotting analysis of P-IκBα and total IκBα protein expression. Numbers over lanes represent individual mice within treatment groups. (B) Band intensities were quantified as described in Materials and Methods. (C) Representative immunohistochemical staining for P-IκBα in a colonic section from a DSS-exposed mouse given control diet (×10). (D) Magnification of panel (C) (box), arrows indicate positively stained cells (×20). (E) Representative immunohistochemical staining for P-IκBα in a colonic section from a DSS-exposed mouse given BRB-incorporated diet (×10). (F) Magnification of panel (E) (box), arrows indicate positively stained cells (×20). (G) Representative immunohistochemical staining for NF-κB p65 in a colonic section from a DSS-exposed mouse given control diet (×10). (H) Magnification of panel (G) (box), arrows indicate positively stained cells (×20). (I) Representative immunohistochemical staining for NF-κB p65 in a colonic section from a DSS-exposed mouse given BRB-incorporated diet (×10). (J) Magnification of panel (I) (box), arrows indicate positively stained cells (×20). Images of entire slides containing immunohistochemically stained colon tissue sections we captured using ImageScope software (Aperio, Vista, CA). The magnifications listed for all representative images of stained sections are those designated by the software. Data are means ± standard errors of the mean, n = 5 mice per group. *P < 0.05, comparing control diet group and BRB diet group (unpaired t-test).

Fig. 6.

The effects of BRB on DSS-induced changes in COX-2 and PGE₂ levels. Mice were administered 3% DSS in the drinking water along with either control or 10% BRB-incorporated powder diet. After 7 days, colons were harvested and protein lysates were prepared for (A) immunoblotting analysis of protein expression. Numbers over lanes represent individual mice within treatment groups and (B) band intensities were quantified as described in Materials and Methods. (C) Plasma was also prepared from mice and PGE₂ levels were determined by enzyme-linked immunosorbent assay. Data are means ± standard errors of the mean, n = 5 mice per group. *P < 0.05, comparing control diet group and BRB diet group (unpaired t-test).