Curcumin is a tumeric-derived, water-insoluble polyphenol with potential beneficial health effects for humans. It has been shown to have preventive as well as therapeutic effects in chemically induced murine models of colitis. To investigate whether curcumin exerts a similar effect on the spontaneous colitis in interleukin (IL)-10 gene-deficient mice, we gavaged these mice daily for 2 weeks with 200 mg/kg per day curcumin emulsified in carboxymethyl cellulose, a food additive generally used as a viscosity modifier. Mice fed the curcumin/carboxymethyl cellulose mixture and those receiving carboxymethyl cellulose alone demonstrated similar reductions in histological injury score and colon weight/length ratio compared to water-fed controls. However, significant reductions in pro-inflammatory cytokine release in intestinal explant cultures were only seen in mice treated with the curcumin mixture. Our data demonstrate that in IL-10 gene-deficient mice, both oral curcumin and carboxymethyl cellulose, appear to have modifying effects on colitis. However, curcumin has additional anti-inflammatory effects mediated through a reduced production of potent pro-inflammatory mucosal cytokines.
Curcumin, the agent responsible for the characteristic yellow–orange color and the strong spicy taste of curries, is the principal polyphenol derived from the root of the turmeric plant (Curcuma longa) . In addition to its uses in the food industry as a spice and food-coloring agent, curcumin has traditionally been used as a herbal remedy for inflammatory disorders, such as arthritis, colitis, and hepatitis . Past research has revealed that curcumin possesses pharmacological effects, including anti-tumor, anti-inflammatory, and anti-infectious activities [3–7]. The exact mechanism by which curcumin exerts its medicinal properties remains uncertain, but chemically induced rodent models of colitis have shown that curcumin suppresses NF-κB and p38 mitogen-activated protein-kinase (MAPK) activation as well as cyclo-oxygenase (COX-2) and pro-inflammatory gene expression [8–11]. A possible mechanism by which curcumin blocks NF-κB activation through the inhibition of inhibitory-factor I-κB kinase activity has been recently described .
Curcumin has been found to have both preventive and therapeutic effects in studies involving trinitrobenzene sulfonic acid (TNBS)-induced, dinitrobenzene sulfonic acid (DNBS)-induced, and dextran sodium sulfate (DSS)-induced murine colitis models [9, 11, 13, 14]. Various methods have been used to administer the curcumin to test animals, including the incorporation of curcumin into daily feed, daily intraperitoneal injection of a curcumin solution, and oral gavaging of curcumin emulsified in a carboxymethyl cellulose solution. Therapeutic concentrations range from 0.1 to 3 g/kg in rats and 0.3–8 g/kg in mice; the human dosage used in preclinical trials is 25–30 mg/kg.
The interleukin (IL)-10 gene-deficient mouse is a well-characterized model for inflammatory bowel disease (IBD) research. Due to the lack of this major anti-inflammatory cytokine, these mice spontaneously develop colitis with similarities to human Crohn’s disease.
The aim of this study was to investigate the possible preventive and therapeutic effects of curcumin in the genetically predisposed IL-10 gene-deficient mouse model of colitis.
Materials and Methods
Homozygous IL-10 gene-deficient mice, generated on a 129/SvEv background, were originally obtained from the DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California and thereafter maintained in a colony at the University of Alberta. Mice were used at age 12–13 weeks at the start of the study, and the results were analyzed at 15 weeks of age, which coincides with the onset of inflammation in untreated IL-10 gene-deficient mice in our colony. Mice were divided into three different treatment groups: those that received curcumin emulsified in 2% carboxymethyl cellulose (CMC), mice that received a 2% CMC solution, and water controls.
Curcumin Solution and Dosage
Curcumin (C21H20O6) is insoluble in water and was therefore emulsified in a 2% CMC solution at a concentration of 60 mg/ml . Technical grade curcumin (purity >75%) was purchased from Cayman Chemical Company (Ann Arbor, MI), and CMC powder was obtained from Sigma (Oakville, ON, Canada). The curcumin/CMC solution (200 mg/kg per day), 2% CMC, or water was administered daily by oral gavage (100 μl/day) for 14 days.
Assessment of Colitis
Colitis development was assessed daily for all control and experimental mice and included measurements of body weight, evaluations of stool consistency, and monitoring for the presence of blood in stools using Hemoccult testing paper (Fisher Scientific, Whitby, ON, Canada). Stool consistency was graded using a four point scale: 0, normal; 1, soft; 2, very soft but formed; 3, liquid. Intensity of the Hemoccult testing paper change was scored according to the following scale: 0, negative; 1, green/faintly blue; 2, moderately blue; 3, dark blue/blood visible in stool. Both scales are adapted from the stool consistency and guaiac paper test-scoring schemes of Deguchi et al. . At the time of cervical dislocation, weight and length of each colon were measured to establish colon weight-to-length ratios, which are used as an indicator of colitis severity .
Histologic Injury Score
On day 14 post-initiation of treatment, cervical dislocation was performed on control and experimental mice. Each colon was excised, and a longitudinally cut piece was embedded in paraffin in toto, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) for light microscopic examination. The slides were reviewed in a blinded fashion and assigned an inflammation histological score according to the sum of four scoring criteria: mucosal ulceration, epithelial hyperplasia, lamina propria mononuclear infiltration, and lamina propria neutrophilic infiltration (scoring scheme adapted from Saverymuttu et al. ).
In Vitro Intestinal Cytokines and Myeloperoxidase Activity
The colon, cecum, and ileum were excised and cut longitudinally; approximately half of each tissue sample was cultured in 1 ml of complete RPMI containing 5% FCS (Life Technologies, Burlington, ON, Canada) in the manner described previously . Following a 24-h incubation period at 37°C in 5% CO2, the supernatants of the cultures were collected, and the release of the pro-inflammatory cytokines interferon gamma (IFNγ) and IL-17 was quantified using the standard cytokine-specific enzyme-linked immunosorbent assay (ELISA). The culture supernatants were also used to determine myeloperoxidase (MPO) activity using an MPO kit (Cell Sciences, Canton, MA), which utilizes the basic ELISA principle to detect MPO activity.
Data are expressed as means ± standard error of the mean (SEM). Differences between mean values were evaluated using analysis of variance or paired t tests, where appropriate (SigmaStat program; Jandel, San Rafael, CA).
Administration of Curcumin/CMC and CMC-Alone Reduces Hemoccult and Stool Consistency Scores
Average Hemoccult and stool consistency scores were recorded for the mice in each treatment group on a daily basis over the 14-day gavage period. Both the curcumin/CMC and CMC-alone treatment groups showed reductions in blood in the stool and reduced stool consistency scores relative to the control water-fed mice (Fig. 1). Curcumin/CMC and CMC feeding alone reached significance by the second week of treatment.
Effects of Curcumin/CMC and CMC-Alone on Histology Injury Scores and Colonic Weight-to-Length Ratios
Control mice treated with water had high histopathologic injury scores and high colon weight/length ratios; these are similar to those reported previously for untreated IL-10 gene-deficient mice (Fig. 2) . The CMC-treated and curcumin/CMC-treated mice had reduced injury scores and weight-to-length ratios; however, statistical significance was only reached in the case of mice fed CMC-alone (Fig. 2).
Effects of Curcumin/CMC and CMC-Alone on Pro-Inflammatory Cytokine Release
Both IFN-γ and IL-17 are associated with an inflammatory response and are key cytokines implicated in the Th1-inflammatory pathway in IBD [21, 22]. At age 15 weeks, IL-10 gene-deficient mice demonstrate high levels of spontaneous IFN-γ and IL-17 release in the colon and cecum, which was verified with the data of our control water-fed mice (Fig. 3a) . The CMC-treatment alone had little effect on IFN-γ and IL-17 release. The administration of curcumin solution, however, significantly reduced the levels of these two pro-inflammatory cytokines in both the colon and cecum, suggesting different mechanisms of action for the reduction of IBD symptoms for CMC and curcumin (Fig. 3a, b).
Effects of Curcumin/CMC and CMC-Alone on MPO Activity
As it is a marker for the infiltration of inflammatory cells (primarily neutrophils), MPO activity was examined. Both the curcumin/CMC-treatment and CMC-alone treatment significantly reduced MPO activity in the colon (Fig. 4). A similar reduction of MPO activity in the cecum was observed in mice treated with both curcumin/CMC and CMC-alone (data not shown).
The effects of curcumin on chemically induced colitis models have been reasonably well detailed in the literature. However, it is not clear whether treatment with curcumin has similar effects in genetically deficient mouse models of colitis. We have therefore examined curcumin’s effects on the development of spontaneous colitis in the IL-10 gene-deficient mouse model.
As seen from the data collected, curcumin was effective in reducing the levels of inflammatory cytokines (IFN-γ and IL-17) and MPO in both the colons and cecums of these mice (Figs. 3, 4). This result is in agreement with the findings of chemically induced colitis studies, in which levels of pro-inflammatory cytokines were down-regulated, thus reducing the severity of the inflammation. Curcumin exerts its anti-inflammatory effects through various mechanisms, including inhibition of COX-2 [8, 9] and of the NF-κB pathway, the latter by inhibiting activation of the inhibitory-factor I-κB [12, 13]. However, curcumin’s reduction of histologic injury and visible symptoms, such as thickening of the large bowel (colon weight/length ratio) and rectal bleeding, seems to be limited in the case of the IL-10 gene-deficient model of IBD. Curcumin is able to reduce the release of pro-inflammatory cytokines; yet, the reduction of these cytokines alone may not be sufficient to counteract disease development. To restore the homeostatic balance in the intestinal milieu, the production of anti-inflammatory cytokines, such as IL-10, may be necessary. Alternatively, the lack of beneficial effects in the case of histologic injury and colon weight/length ratio may be attributed to curcumin’s non-universal effects and, therefore, the effects may be less useful in genetically induced IBD models of certain background strains of mice. Billerey-Larmonier et al.  have recently determined that the protective effects of curcumin in chemically induced murine colitis models are strain dependent. In their study, mice of different background strains (BALB/c and SJL/J) were administered curcumin post-chemical-induction of colitis, and the effects of curcumin were then assessed through various means. Although the colitis of the BALB/c mice responded favorably to curcumin administration, that of the SJL/J mice did not. A similar discrepancy in the efficacy of curcumin may exist for the genetically induced model of colitis, which may not mimic that of the most favorably affected chemically induced model. Furthermore, in chemically induced models, pathologic injury occurs concomitant to the measurable increase in inflammatory cytokine release. In the IL-10 gene-deficient mouse model, however, an increase in pro-inflammatory cytokine release precedes pathologic injury by several weeks, suggesting that additional parameters, such as direct bacterial–cell interactions, changes in cell surface marker expression, and cytokine-independent intracellular pathways, are activated for the injury to occur. Our study demonstrates that curcumin affects pro-inflammatory cytokine release but may not be effective in preventing other bacteria–cell interactions that ultimately lead to intestinal injury. Interestingly, CMC-treated mice had significantly lower histology scores and lower colon weight/length ratios, indicating that CMC-treatment alone may play a role in reducing colonic injury. Carboxymethyl cellulose appears to be beneficial despite high levels of pro-inflammatory cytokines, suggesting a mechanism of action independent of an imbalance in cytokine production. Possible mechanisms could include an increase in cellulose-derived nutrients to promote mucosal healing or immune function as well as variations in the intestinal flora. It has recently been shown that alterations to the viscosity of the mucosal lining affect bacterial mobility and spatial organization within the large intestine. Different bacterial shapes are better able to move and penetrate the mucosal lining at varying viscosities, while, at high viscosity levels, the movement of bacteria is prevented . Thus, CMC may have exerted its positive effects on the intestinal lumen by increasing the viscosity of the mucous layer, thereby altering patterns of pathogenic bacterial migration through the mucous layer, where they would have otherwise led to inflammation and damage to the intestine. Although the curcumin solution was prepared using the CMC solution, the gel-like matrix of the CMC solution may have been disrupted by the presence of the curcumin; thus, it is possible that the viscous barrier that the CMC solution would have formed would have been more easily penetrated by bacteria. This proposed mechanism would explain the differing results between curcumin/CMC and CMC-alone that were seen in the histology scores and the colon weight/length ratios. We are currently undertaking further examination of CMC’s mediation of intestinal inflammation.
While this manuscript was in review, a paper was published in which the effect of curcumin feeding in IL-10 gene-deficient mice was reported . In this study, IL-10 gene-deficient mice were provided curcumin in the feed at concentrations of 0.1, 0.5, and 1%, which would translate into a daily dose of 160, 800, and 1600 mg/kg per day given an average daily consumption of 4.5 g mouse chow. Although this study uses the same mouse model as our study, its experimental design differs slightly in the method of curcumin application (mixed in feed vs. gavaged in CMC solution) and the intestinal microbial condition of the mice leading to IBD (association with intestinal flora 2 days post start of curcumin feeding vs. intestinal flora from birth). Despite these differences, their low dose (160 mg/kg per day) results corresponds to the results we derived at a similar dosage (200 mg/kg per day), demonstrating a beneficial effect of curcumin on mucosal damage. While the investigator recorded reduced colonic IFN-γ RNA expression, cytokine secretion in colonic explant cultures was, contrary to our findings, not reduced. Surprisingly, the two higher dosages of curcumin feeding reversed the beneficial effect of the low-dose feeding; this result calls for caution when establishing a potential therapeutic dosage for curcumin.
In relation to the aims of our study, curcumin’s effects on chemically induced mouse colitis models seem to be replicated in terms of reduced pro-inflammatory cytokine production in this genetically-predisposed colitis model. However, curcumin alone is not able to reverse histologic intestinal injury in the IL-10 gene-deficient mouse model in a manner similar to the one described in chemically induced mouse colitis models, indicating that curcumin’s limitation as a treatment for colitis is dependent on the underlying cause of IBD development. This study contributes to the growing body of evidence for the potential therapeutic effects of curcumin, in both in vitro and in vivo studies, that has sparked numerous clinical trials on the efficacy of curcumin treatment in humans.
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This work was supported by a grant from the Crohn’s and Colitis Foundation of Canada.
Dr. Sydora and Dr. Fedorak share senior authorship.
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Ung, V.Y.L., Foshaug, R.R., MacFarlane, S.M. et al. Oral Administration of Curcumin Emulsified in Carboxymethyl Cellulose Has a Potent Anti-inflammatory Effect in the IL-10 Gene-Deficient Mouse Model of IBD. Dig Dis Sci 55, 1272–1277 (2010). https://doi.org/10.1007/s10620-009-0843-z
- Carboxymethyl cellulose
- Inflammatory bowel disease