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doi:10.1111/jgh.12485
GASTROENTEROLOGY
Effects of dietary supplementation of glucosamine sulfate on intestinal inflammation in a mouse model of experimental colitis Youn-Kyung Bak,* Johanna W. Lampe† and Mi-Kyung Sung* *Department of Food and Nutrition, Sookmyung Women’s University, Seoul, Korea; and †Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Key words colitis, colorectal cancer, glucosamine, inflammation, tight junction protein. Accepted for publication 5 November 2013. Correspondence Professor Mi-Kyung Sung, Department of Food and Nutrition, Sookmyung Women’s University, Cheongpa-ro 47-gil 100 Yongsan-gu, Seoul 140-742, Korea. Email: [email protected]
Abstract Background and Aim: Epidemiological evidences suggested an inverse association between the use of glucosamine supplements and colorectal cancer (CRC) risk. In this study, the efficacy of glucosamine to attenuate dextran sodium sulfate (DSS)-induced colitis, a precancerous condition for CRC, was evaluated. Methods: C57BL/6 mice were separated into three groups receiving glucosamine sulfate at concentrations of 0, 0.05, and 0.10% (w/w) of AIN-93G diet, respectively for 4 weeks. Colitis was induced by supplying two cycles (5 days per cycle) of 2% DSS in the animals’ drinking water. Results: Glucosamine supplementation at the level of 0.10% of the diet (w/w) reduced colitis-associated symptoms as measured by disease activity index (DAI). Tumor necrosis factor-α (TNF-α), interleukin-1β, and nuclear factor-kappa B mRNA expression in the colonic mucosa was significantly lower in animals fed 0.10% glucosamine compared with those of the control group. Expression of the tight junction proteins ZO-1 and occludin was significantly higher in the 0.10% glucosamine-supplemented group compared with the other groups. Also, colonic protein expression of lipocalin 2, and serum concentrations of interleukin-8 and amyloid P component (SAP) were significantly reduced in the 0.10% glucosamine-supplemented group compared with the control group. Conclusion: These results suggest that glucosamine attenuates the colitis disease activity by suppressing NF-κB activation and related inflammatory responses.
Introduction Inflammatory bowel diseases (IBD) are autoimmune diseases characterized by serious inflammation of the gastrointestinal tract due to dysregulated mucosal immune responses.1 Crohn’s disease and ulcerative colitis (UC) are the major types of IBD, and their incidence is increasing in many parts of the world, particularly in industrialized countries.2,3 UC is characterized by chronic inflammation in the colon and rectum causing epithelial apoptosis and ulceration, which is mediated by pro-inflammatory cytokines, including interleukin-8 (IL8), interleukin-1 (IL-1), and tumor necrosis factor-α (TNF-α).4–7 Mitogen-activated protein kinase (MAPK) phosphorylation followed by nuclear factor-kappa B (NF-κB) activation has been suggested to mediate cytokine production in IBD.8 Therapies for UC rely heavily on the use of immunosuppressive drugs such as 5-aminosalicylic acid, corticosteroids, and other immunosuppressants.9 However, the side effects of these types of drugs, which include nausea, vomiting, headaches, rash, fever, agranulocytosis, pancreatitis, nephritis, hepatitis, and male infertility, may severely disrupt patients’ quality of life.10 In addition,
patients taking sulfasalazine should also take folic acid supplements, as the sulfa portion of the drug interferes with folic acid absorption.11 Also, biological agents including anti-TNF-α agents improved IBD therapy, however a large proportion of the patients are either nonresponsive or develop side effects.12 In a recent cross-sectional study, 21% of IBD patients reported that they used complementary and alternative medicine (CAM).13 Another study reported that 44.1% of IBD patients use oral CAM, although the frequency of CAM use reported by the IBD patients was similar to that reported by the healthy control subjects in the study.14 These results indicate that there is a significant need to provide scientific evidence on the usage of CAM in these patients. Glucosamine is an essential component of glycosaminoglycans, and the resulting aggrecans are major constituents of joint cartilage.15–18 Glucosamine has been used to reduce the clinical symptoms of osteoarthritis by enhancing the synthesis of aggrecans.19 Recently, epidemiological evidence based on the VITamins And Lifestyle cohort study suggested that the use of glucosamine and chondroitin is associated with reduced total mortality and the decreased risk of death from cancer.14 Also, prolonged (> 3 years) and regular (> 4 times/week) use of
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glucosamine + chondroitin tended to be associated with reduced risk of colorectal cancer (CRC) (hazard ratio [HR] 0.66; 95% confidence interval [CI] 0.30–1.01), and this association was significant among overweight and obese subjects (HR 0.28; 95% CI 0.10–0.76).20 The same group of researchers found that C-reactive protein (CRP) was inversely associated with regular use of glucosamine.21 CRP is an acute-phase protein and its circulating concentration rise in response to inflammatory process. Although CRP has been used as a marker of colitis disease activity in other studies,22,23 mechanistic explanations for the negative relationship seen between the use of glucosamine supplementation and the risk of CRC have not been provided. Based on the evidence indicating that individuals with colitis are predisposed to develop CRC, this study investigated the efficacy of glucosamine supplementation to alleviate dextran sodium sulfate (DSS)-induced colitis and elucidated its mechanisms of action.
Materials and methods Animals and experimental scheme. Seven-week-old male C57BL/6J mice were purchased from Central Laboratory (Seoul, Korea). The animals were housed in plastic cages, three to four mice per cage, at a constant temperature (25°C) and humidity (50 ± 10%) and maintained in air-conditioned quarters with 12-h light/dark cycles. Body weight and food intake were measured once a week. Animals were separated into three groups: group 1 received AIN-93G control diet (CD), group 2 received the CD supplemented with glucosamine sulfate at a concentration of 0.05% w/w (LGS), and group 3 received the CD supplemented with glucosamine sulfate at a concentration of 0.1% w/w (HGS). Glucosamine sulfate was substituted in place of sucrose. The mice received experimental diets throughout experimental period. Two weeks after starting experimental diets, colitis was induced by supplying two cycles (5 days per cycle) of 2% DSS (MW 50 000, MP Biochemicals, lllkirch, France) in distilled water as the animals’ drinking water for 5 days. The two cycles were separated by 7 days (Fig. 1). The care, maintenance, and treatment of the animals followed protocols approved by the Institutional Animal Care and Use Committee (SM-IACUC-2012-0917-012).
Preparation of the blood and tissue samples. The animals were sacrificed at the end of week 4. Blood was collected from the inferior vena cava into ethylenediaminetetraacetic acidfree tubes and centrifuged at 1550 × g for 20 min. The colon and liver were quickly removed, rinsed with cold saline, and weighed. The colonic mucosa was laid flat on a glass slide and was then scraped with glass slides, frozen instantly in liquid nitrogen, and stored at −80°C until assayed. Disease activity index (DAI) and histological observation. The DAI was determined by scoring changes in weight loss (score: 0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; and 4, > 20%), stool consistency (score: 0, formed; 1, mildly soft; 2, very soft; and 3, watery stool), and bleeding (score: 0, normal; 1, brown; 2, reddish and 3, bloody).24 All parameters were scored daily during DSS treatment (day 14 to day 31). Scores assigned for each parameter were used to calculate the average DAI. Colon tissue was flushed with saline and the length was measured. The colon was cut open longitudinally along the main axis and the lumen was flushed with saline. Excess saline was removed and the colon was rolled using a wooden stick. Each roll was fixed in 10% buffered formalin. Paraffin-embedded sections of the colon were prepared according to routine procedures, and stained with Alcian blue, which stains for mucin-producing goblet cells. The number of goblet cells stained with Alcian blue per villus was counted in three random regions without erosion. Measurement of serum IL-8 and serum amyloid P component (SAP). Serum IL-8 and SAP concentrations were determined using ELISA kits (TSZ Scientific LLC, Waltham, MA, USA and R&D Systems, Inc., Minneapolis, MN, USA). RNA isolation and real-time quantitative polymerase chain reaction (qPCR) analysis. The mRNA expression levels of TNF-α and IL-1β were determined in colon tissue samples using real-time qPCR analysis. Total RNA was extracted from scraped colonic mucosa using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The total RNA (1 μg) prepared from the colonic mucosa was reverse-transcribed using a cDNA Synthesis Kit
Figure 1 Experimental design. Male C57BL/6 mice were separated into three experimental groups (n = 10/group). Glucosamine sulfate was supplemented at concentrations of 0% (control), 0.05% (low glucosamine group, LGS) and 0.10% (high glucosamine group, HGS) of AIN-93G basal diet. Two weeks after feeding the experimental diet, colitis was induced by supplying two cycles (5 days per cycle) of 2% DSS in drinking water. The two cycles were separated by 7 days. Animals were killed at the end of week 4. DSS, dextran sodium sulfate.
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(PhileKorea Technology, Seoul, Korea) according to the manufacturer’s instructions. Real-time qPCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using a QuantiMix SYBR Kit (PhileKorea Technology). The primers for TNF-α, IL-1β, and β-actin were synthesized by Bioneer Corporation (Daejeon, Korea). Relative fold changes in gene expression were determined using the 2ΔΔCT (relative quantification) analysis protocol. Protein extraction and Western blot analysis. Colon tissue was homogenized in a PRO-PREP protein extraction solution (iNtRON Biotechnology Inc., Seongnam, Gyeonggi, Korea) for 20 min on ice, then centrifuged (16 600 × g, 10 min, 4°C). Protein content was determined against a standardized control, using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 50 μg of protein from each sample was separated by 4∼12% and 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Polyvinylidene difluoride membranes (Koma Biotech Inc., Seoul, Korea). The membranes were blocked with 2% skim milk (Amersham Biosciences, Arlington Heights, IL, USA) and incubated with specific antibodies for NF-κB (dilution 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), lipocalin 2 (Lnc2) (dilution 1:1000; Abcam Inc., Cambridge, MA, USA), ZO-1 (dilution 1:200; Abcam Inc.), occludin (dilution 1:200; Invitrogen, Carlsbad, CA, USA), and β-actin (dilution 1:4000; Sigma-Aldrich Corp., St. Louis, MO, USA). Peroxidaseconjugated anti-rabbit IgG was used as the secondary antibody for all proteins except occludin. For the determination of occludin protein expression, peroxidase-conjugated anti-mouse IgG was used as the secondary antibody (Sigma-Aldrich Corp., St. Louis, MO, USA) The membranes were washed with PBS/Tween 20 (PBST) containing 0.1% Tween 20 (Sigma-Aldrich Corp., St. Louis, MO, USA). Reactive bands were visualized using an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Arlington Heights, IL, USA). Following visualization, the antibodies were stripped from the membranes, and the stripping was confirmed by re-exposure to enhanced ECL and detection with an LAS-3000 Imager (Fujifilm, Tokyo, Japan). The membranes were subsequently blocked again, and reprobed with one of the other primary antibodies. The intensity of the bands was quantified using a Bio-Rad GS-800 Calibrated Densitometer equipped with Quantity One software (Bio-Rad Laboratories, Inc.). Statistical analysis. Statistical analysis was conducted using SAS software (Version 9.0, SAS Institute Inc., Cary, NC, USA). All data were expressed as mean ± standard deviation. Data were analyzed by one-way analysis of variance followed by Duncan’s multiple range test. Mann–Whitney and Kruskal–Wallis tests were used in between-group comparisons for DAI scores. Results were considered statistically significant at P < 0.05.
Results Final body weights, organ weights, and colon lengths of animals with induced colitis. There were no differences in body weights among the experimental groups
Glucosamine and colitis
Table 1 Final body weight, organ weights, and colon length of experimental animals Organ (g)
CD
LGS
HGS
Body weight Liver Absolute Relative* Spleen Absolute Relative Colon length
22.94 ± 2.03
22.17 ± 1.86
22.94 ± 2.06
1.08 ± 0.07 5.49 ± 0.34
1.06 ± 0.10 6.23 ± 0.56
1.02 ± 0.10 5.06 ± 0.48
0.11 ± 0.04 0.58 ± 0.20 4.90 ± 0.47b
0.13 ± 0.03 0.76 ± 0.16 5.11 ± 0.72b
0.12 ± 0.03 0.62 ± 0.13 5.73 ± 0.32a
Values are presented as a mean ± SD of 10 animals per group. Data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test. *Relative organ weight was calculated percentage of body weight. Values with different superscript letters are significantly different (P = 0.0043). CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.1% glucosamine sulfate diet.
(Table 1), and liver and spleen weights did not differ significantly. In contrast, the average length of the colon in the control group was significantly shorter compared with that of the mice in the HGS group (Table 1). There were no differences in small intestine length among the experimental groups. Evaluation of DAI in colitic mice. Two weeks after starting the experimental diets, the animals were exposed to the two 5-day cycles of 2% DSS in drinking water, separated by 7 days (Fig. 1). The DAI began to increase beginning on day 3 and reached a first peak on day 7 (Fig. 2). Both the LGS and HGS groups had lower DAI scores compared with the CD group, although the difference was not significant. Clinical symptoms were slowly improved at the end of the first DSS cycle. After the second administration of DSS, the DAI scores of all the groups became higher compared with those during the first cycle. The DAI scores in animals fed a diet supplemented with 0.1% glucosamine were significantly lower compared with those of the control group during the second DSS cycle (days 13–17). Histological changes in colon mucosa. Figure 3 shows mucin-producing goblet cells of colon tissue section stained with Alcian blue. Our results indicated that the number of goblet cells per crypt-villus axis was slightly higher, although the difference was not statistically significant (P = 0.43), in animals fed the glucosamine-supplemented diet. Serum concentrations of IL-8 and SAP. Serum concentrations of IL-8, a representative inflammatory cytokine shown to be related to disease severity of colitis,25 and SAP, a protein formed during acute inflammation, were measured. Compared with animals in the CD group, animals in the HGS group had 48% and 45% lower concentrations of serum IL-8 and SAP, respectively (P < 0.05) (Fig. 4). With the lower level of glucosamine sulfate supplementation, however, although dose-dependent decreases in IL-8 and SAP concentrations were observed compared with the CD group, these differences were not statistically significant.
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Figure 2 Effect of glucosamine sulfate supplementation on disease activity index (DAI). DAI was determined by scoring changes in weight loss, stool consistency and bleeding. Scores assigned for each parameter were used to calculate average DAI. Values with different letters at each time point are significantly different at P < 0.05 as determined by Mann– Whitney and Kruskal–Wallis tests. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.10% glucosamine sulfate diet. ; LGS, ; HGS, . CD,
Figure 3 Histological changes in the colon tissue of experimental animals. Tissues were stained with Alcian blue (×400). Goblet cells producing mucin stain blue. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.10% glucosamine sulfate diet.
Figure 4 Effects of glucosamine sulfate supplementation on serum concentrations of IL-8 and SAP. Values are presented as a mean ± SD of 10 animals per group. Values with different letters are significantly different (P = 0.012 for IL-8 and P = 0.024 for SAP) as determined by Duncan’s multiple range test. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.10% glucosamine sulfate diet.
mRNA and protein expressions in colon tissue. TNF-α and IL-1β are critical pro-inflammatory cytokines contributing to colon tissue damage in DSS-induced colitis. Glucosamine sulfate supplementation markedly reduced the relative mRNA expression of colonic tissue TNF-α and IL-1β in a dose-dependent manner (Fig. 5). NF-κB has been observed to be activated in the inflamed colonic mucosa in IBD, and steroid-induced healing of mucosa was accompanied by the disappearance of NF-κB.8 As shown in Figure 6, 0.10% glucosamine sulfate supplementation diminished NF-κB protein expression by 54% compared with the expression in the CD group; however, no dose–response relationship was observed. Lnc2, also known as neutrophil gelatinase-associated lipocalin (NGAL), is a protein from secondary granules of human neutrophils. In our study, the expression of Lnc2 protein was suppressed 960
by 38% in the HGS group compared with the CD group (Fig. 6). However, no significant effect was observed in the LGS group. ZO-1, a tight junction (TJ) protein, is part of the apical junctional complex. Our results revealed that ZO-1 protein expression was significantly higher (∼twofold) in the HGS group compared with the CD group (Fig. 6). Expression of occludin, another TJ protein, was also significantly higher in animals in the HGS group compared with those in the CD group (Fig. 6).
Discussion Glucosamine has been widely used as a complementary therapeutic supplement to suppress osteoarthritis. Results from a recent cohort study suggest that the use of glucosamine supplements is
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Glucosamine and colitis
Figure 5 Effect of glucosamine sulfate supplementation on colonic TNF-α and IL-1β mRNA expression. Values are presented as a mean ± SD of 10 animals per group. Values with different letters are significantly different (P = < 0.0001 for TNF-α and P = 0.0028 for IL-1β) as determined by Duncan’s multiple range test. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.10% glucosamine sulfate diet.
Figure 6 Effects of glucosamine sulfate supplementation on colonic NF-κB, lipocalin 2, ZO-1 and occludin protein expressions. Values are presented as a mean ± SD of 10 animals per group. Values with different letters are significantly different (P = 0.011 for NF-κB, P = 0.046 for lipocalin 2, P = 0.006 for ZO-1 and P = 0.044 for occludin) as determined by Duncan’s multiple range test. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.1% glucosamine sulfate diet.
inversely associated with CRC.20 Based on evidence showing antiinflammatory actions of glucosamine, as well as a close association between chronic inflammation and CRC, it is possible to hypothesize that glucosamine suppresses colonic inflammation, and thereby reduces CRC risk. Therefore, in this study, we used an animal model of colitis to examine the in vivo efficacy of glucosamine supplementation to alleviate colonic inflammation, and elucidated possible mechanisms of action of this effect. DSSinduced colitis is a well-established experimental model with many signs and symptoms that resemble those of human UC, including diarrhea, bloody feces, weight loss, mucosal ulceration, and shortening of the large intestine.26,27 Our study results showed that glucosamine supplementation at a concentration of 0.1% (w/w diet) improved the clinical symptoms and pathological features of colitis, while a lower concentration of glucosamine (0.05%) slightly, but not significantly, improved the DAI. Similarly, glucosamine supplementation reduced blood
markers of inflammation. In this study, we measured serum SAP, a major acute phase reactant in mice,28 as an indicator of inflammation. Our results showed that mean serum SAP concentration was 45% lower in the HGS group as compared with the CD group. Another major acute phase protein synthesized in response to inflammation is CRP. Although CRP is highly induced in humans, it never rises above 2–3 mg/L in mice.29 A recent study reported that CRP was inversely associated with the use of glucosamine supplements in a subgroup of the NHANES population.21 Two small human intervention studies have also evaluated the association between glucosamine supplementation and circulating CRP concentration.30,31 Glucosamine supplementation did not reduce plasma CRP in rheumatoid arthritis patients, but 3-month supplementation of glucosamine hydrochloride significantly decreased serum prostaglandin E2 concentrations. The findings from our study, coupled with other human studies, suggest a potential role of glucosamine in reducing systemic inflammation.
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Glucosamine supplementation was shown to reduce the inflammatory responses of joint cartilage by inhibiting the activation of NF-κB, which lies upstream of inflammatory mediators such as CRP, IL-1β, IL-8,32,33 and TNF-α. Studies have demonstrated that an imbalance of pro-inflammatory and anti-inflammatory cytokines is related to the pathogenic mechanisms of UC.34 Binding of pro-inflammatory cytokines to their respective receptors amplifies the immune response by enhancing the proliferation of T cells, promoting leukocyte infiltration, and facilitating cellcell signaling.35 A previous study in a DSS-induced colitis model indicated that IL-1, which consists of IL-1α and IL-1β, increases the production of IL-8, IL-6, and TNF-α by monocytes and macrophages,36 and IL-8 expression is upregulated by IL-1β and TNF-α stimulation in human epithelial cells via MAPK phosphorylation and NF-κB activation. In our study, glucosamine supplementation led to significant decreases in both circulating IL-8 concentrations and colonic expression of IL-1β and TNF-α, which may be mediated by the observed decrease in NF-κB expression. Therefore, it is reasonable to hypothesize that glucosamine may protect against inflammation-related intestinal tissue damage and related mucosal barrier disruption by suppressing the synthesis of major inflammatory mediators.8 Glucosamine is a precursor for glycosaminoglycans (GAGs) that are major components of joint cartilage. The antiinflammatory activity of glucosamine is thought to be derived from the direct interactions of GAGs with the protein structure of NF-κB due to the presence of charged groups such as sulfate and carboxyl groups in GAGs.37,38 Previous studies reported that certain chemokines, including pro-inflammatory cytokines, require interactions with GAGs for their in vivo function.39,40 These interactions are thought to play a role in the sequestration of chemokines and subsequent presentation to the receptor expressed on the leukocyte cell surface.39–41 We also observed that intestinal TJ protein expression was increased in the animals that received the glucosaminesupplemented diets. A widely accepted hypothesis regarding the mechanism of chronic relapsing intestinal inflammation in IBD is that gut epithelial barrier dysfunction permits luminal antigens to enter the subepithelial tissues, resulting in the recruitment and activation of leukocytes.42,43 Pro-inflammatory cytokines play a key role in the induction of barrier defects in IBD.44 TNF-α and IFN-γ released during mucosal inflammation can induce an increase in epithelial permeability through regulation of the TJ proteins.45,46 Three of the key proteins of the TJ are ZO-1, occludin, and the more recently identified family of claudins. Expression of these TJ proteins has been shown to be decreased in the intestinal inflammation of IBD.47–49 Intestinal membrane barrier disruption due to inflammation evokes the influx of neutrophils into the mucosa, followed by transepithelial migration of neutrophils, which is a first line of defense against inflammationassociated barrier disruption. IL-8 is known to play an essential role in directing the sequential process of neutrophil rolling, adhesion, and transmigration in the inflamed microvasculature. In addition, pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 increase the expression of adhesion molecules on endothelial cells and neutrophils. In this study, Lcn2 was used as a neutrophil infiltration marker in the DSS-induced colitis model. Our results showed that 0.1% glucosamine supplementation significantly decreased colonic protein expression of Lcn2 compared with the 962
CD group as control. Therefore, it is possible to assume that the production of inflammatory mediators induced less damage to the intestinal barrier suppressing neutrophil infiltration. In summary, this study demonstrated that glucosamine sulfate attenuates DSS-induced colitis symptoms, possibly by suppressing the NF-κB-mediated TNF-α and IL-1β production and neutrophil activation that contribute to intestinal barrier disruption in colitic mice. The effective dose of glucosamine to suppress the DAI was 0.1% of the total diet, which corresponds to approximately 0.5 g/ day based on daily calorie consumption. If one uses the body surface area normalization formula to translate the effective dose in animals to humans,50 a dose of glucosamine of 0.1% of the total diet corresponds to approximately 9 mg/d. This study provides insights on possible utilization of glucosamine sulfate in a practical manner to prevent colitis and CRC. Further efficacy studies in humans are required in the future.
Acknowledgment This research was supported by the SRC program (Center for Food & Nutritional Genomics: grant number 2012-0000642) and the Mid-Career Research Program (2012R1A2A2A01046228) of the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Science, and Technology.
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12 Silva LCR, Ortigosa LCM, Benard G. Anti-TNF-α agents in the treatment of immune-mediated inflammatory diseases: mechanisms of action and pitfalls. Immnunotherapy 2010; 2: 817–33. 13 Opheim R, Bernklev T, Fagermoen MS, Cvancarova M, Moum B. Use of complementary and alternative medicine in patients with inflammatory bowel disease: results of a cross-sectional study in Norway. Scand. J. Gastroenterol. 2012; 47: 1436–47. 14 Koning M, Ailabouni R, Gearry RB, Frampton CM, Barclay ML. Use and predictors of oral complementary and alternative medicine by patients with inflammatory bowel disease: a population-based, case-control study. Inflamm. Bowel Dis. 2013; 19: 767–78. 15 Pavelká K, Gatterová J, Olejarová M, Machacek S, Giacovelli G, Rovati LC. Glucosamine sulfate use and delay of progression of knee osteoarthritis: a 3-year, randomized, placebo-controlled, double-blind study. Arch. Intern. Med. 2002; 162: 2113–23. 16 Bruyere O, Pavelka K, Rovati LC et al. Glucosamine sulfate reduces osteoarthritis progression in postmenopausal women with knee osteoarthritis: evidence from two 3-year studies. Menopause 2004; 11: 138–43. 17 McAlindon TE, LaValley MP, Gulin JP, Felson DT. Glucosamine and chondroitin for treatment of osteoarthritis: a systematic quality assessment and meta-analysis. JAMA 2000; 283: 1469–75. 18 Reginster JY, Deroisy R, Rovati LC et al. Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial. Lancet 2001; 357: 251–6. 19 Reginster JY, Neuprez A, Lecart MP, Sarlet N, Bruyere O. Role of glucosamine in the treatment for osteoarthritis. Rheumatol. Int. 2012; 32: 2959–67. 20 Kantor ED, Lampe JW, Peters U, Shen DD, Vaughan TL, White E. Use of glucosamine and chondroitin supplements and risk of colorectal cancer. Cancer Causes Control 2013; 24: 1137–46. 21 Kantor ED, Lampe JW, Vaughan TL, Peters U, Rehm CD, White E. Association between use of specialty dietary supplements and C-reactive protein concentrations. Am. J. Epidemiol. 2012; 176: 1002–13. 22 Brandhorst G, Weigand S, Eberle C et al. CD4+ immune response as a potential biomarker of patient reported inflammatory bowel disease (IBD) activity. Clin. Chim. Acta 2013; 421C: 31–3. 23 de Villiers WJ, Varilek GW, de Beer FC, Guo JT, Kindy MS. Increased serum amyloid a levels reflect colitis severity and precede amyloid formation in IL-2 knockout mice. Cytokine 2000; 12: 1337–47. 24 Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab. Invest. 1993; 69: 238–49. 25 Xia B, Han H, Zhang KJ et al. Effects of low molecular weight heparin on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid-induced colitis. World J. Gastroenterol. 2004; 10: 729–32. 26 Holma R, Salmenperä P, Virtanen I, Vapaatalo H, Korpela R. Prophylactic potential of montelukast against mild colitis induced by dextran sulphate sodium in rats. J. Physiol. Pharmacol. 2007; 58: 455–67. 27 Kwon HS, Oh SM, Kim JK. Glabridin, a functional compound of liquorice, attenuates colonic inflammation in mice with dextran sulphate sodium-induced colitis. Clin. Exp. Immunol. 2008; 151: 165–73. 28 Pepys MB, Baltz M, Gomer K, Davies AJ, Doenhoff M. Serum amyloid P-component is an acute phase reactant in the mouse. Nature 1979; 278: 259–61. 29 Pepys MB. Isolation of serum amyloid P-component (protein SAP) in the mouse. Immunology 1979; 37: 637–41.
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30 Nakamura H, Masuko K, Yudoh K, Kato T, Kamada T, Kawahara T. Effects of glucosamine administration on patients with rheumatoid arthritis. Rheumatol. Int. 2007; 27: 213–8. 31 Nakamura H, Nishioka K. Effects of glucosamine/chondroitin supplement on osteoarthritis: involvement of PGE2 and YKL-40. J. Rheum. Joint Surg. 2002; 21: 175–84. 32 Largo R, Alvarez-Soria MA, Díez-Ortego I et al. Glucosamine inhibits IL-1beta-induced NFkappaB activation in human osteoarthritic chondrocytes. Osteoarthr. Cartilage 2003; 11: 290–8. 33 Chan PS, Caron JP, Orth MW. Short-term gene expression changes in cartilage explants stimulated with interleukin beta plus glucosamine and chondroitin sulfate. J. Rheumatol. 2006; 33: 1329–40. 34 Hicks A, Monkarsh SP, Hoffman AF, Goodnow R Jr. Leukotriene B4 receptor antagonists as therapeutics for inflammatory disease: preclinical and clinical development. Expert Opin. Investig. Drugs 2007; 16: 1909–20. 35 Mosmann TR, Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 1996; 17: 138–46. 36 Akira S, Hirano T, Taga T, Kishimoto T. Biology of multi-functional cytokines: IL 6 and related molecules (IL 1 and TNF). FASEB J. 1990; 4: 2860–7. 37 Campo GM, Avenoso A, Campo S et al. Glycosaminoglycans modulate inflammation and apoptosis in LPS-treated chondrocytes. J. Cell. Biochem. 2009; 106: 83–92. 38 Tannock LR, Kirk EA, King VL, LeBoeuf R, Wight TN, Chait A. Glucosamine supplementation accelerates early but not late atherosclerosis in LDL receptor-deficient mice. J. Nutr. 2006; 136: 2856–61. 39 Mulloy B, Rider CC. Cytokines and proteoglycans: an introductory overview. Biochem. Soc. Trans. 2006; 34: 409–13. 40 Proudfoot AE. The biological relevance of chemokine-proteoglycan interactions. Biochem. Soc. Trans. 2006; 34: 422–6. 41 Scott JE. Supramolecular organization of extracellular matrix glycosaminoglycans, in vitro and in the tissues. FASEB J. 1992; 6: 2639–45. 42 DeMeo MT, Mutlu EA, Keshavarzian A, Tobin MC. Intestinal permeation and gastrointestinal disease. J. Clin. Gastroenterol. 2002; 34: 385–96. 43 Podolsky DK. Inflammatory bowel disease. N. Engl. J. Med. 2002; 347: 417–29. 44 John LJ, Fromm M, Schulzke JD. Epithelial barrkers in intestinal inflammation. Antioxid. Redox Signal. 2011; 15: 1255–70. 45 Berkes J, Viswanathan V, Savkovic S, Hecht G. Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation. Gut 2003; 52: 439–51. 46 Nusrat A, Turner J, Madara JL. Molecular physiology and pathophysiology of tight junctions, IV: regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G851–G857. 47 Gassler N, Rohr C, Schneider A et al. Inflammatory bowel disease is associated with changes of enterocytic junctions. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 281: G216–G228. 48 Kucharzik T, Walsh SV, Chen J, Parkos CA, Nusrat A. Neutrophil transmigration in inflammatory bowel disease is associated with differential expression of epithelial intercellular junction proteins. Am. J. Pathol. 2001; 159: 2001–9. 49 Poritz LS, Garver KI, Green C, Fitzpatrick L, Ruggiero F, Koltun WA. Decreased expression of occludin in the intestine of patients with inflammatory bowel disease. FASEB J. 1997; 11: A310. 50 Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J. 2007; 22: 659–61.
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Effects of dietary supplementation of glucosamine sulfate on intestinal inflammation in a mouse model of experimental colitis Youn-Kyung Bak,* Johanna W. Lampe† and Mi-Kyung Sung* *Department of Food and Nutrition, Sookmyung Women’s University, Seoul, Korea; and †Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
Key words colitis, colorectal cancer, glucosamine, inflammation, tight junction protein. Accepted for publication 5 November 2013. Correspondence Professor Mi-Kyung Sung, Department of Food and Nutrition, Sookmyung Women’s University, Cheongpa-ro 47-gil 100 Yongsan-gu, Seoul 140-742, Korea. Email: [email protected]
Abstract Background and Aim: Epidemiological evidences suggested an inverse association between the use of glucosamine supplements and colorectal cancer (CRC) risk. In this study, the efficacy of glucosamine to attenuate dextran sodium sulfate (DSS)-induced colitis, a precancerous condition for CRC, was evaluated. Methods: C57BL/6 mice were separated into three groups receiving glucosamine sulfate at concentrations of 0, 0.05, and 0.10% (w/w) of AIN-93G diet, respectively for 4 weeks. Colitis was induced by supplying two cycles (5 days per cycle) of 2% DSS in the animals’ drinking water. Results: Glucosamine supplementation at the level of 0.10% of the diet (w/w) reduced colitis-associated symptoms as measured by disease activity index (DAI). Tumor necrosis factor-α (TNF-α), interleukin-1β, and nuclear factor-kappa B mRNA expression in the colonic mucosa was significantly lower in animals fed 0.10% glucosamine compared with those of the control group. Expression of the tight junction proteins ZO-1 and occludin was significantly higher in the 0.10% glucosamine-supplemented group compared with the other groups. Also, colonic protein expression of lipocalin 2, and serum concentrations of interleukin-8 and amyloid P component (SAP) were significantly reduced in the 0.10% glucosamine-supplemented group compared with the control group. Conclusion: These results suggest that glucosamine attenuates the colitis disease activity by suppressing NF-κB activation and related inflammatory responses.
Introduction Inflammatory bowel diseases (IBD) are autoimmune diseases characterized by serious inflammation of the gastrointestinal tract due to dysregulated mucosal immune responses.1 Crohn’s disease and ulcerative colitis (UC) are the major types of IBD, and their incidence is increasing in many parts of the world, particularly in industrialized countries.2,3 UC is characterized by chronic inflammation in the colon and rectum causing epithelial apoptosis and ulceration, which is mediated by pro-inflammatory cytokines, including interleukin-8 (IL8), interleukin-1 (IL-1), and tumor necrosis factor-α (TNF-α).4–7 Mitogen-activated protein kinase (MAPK) phosphorylation followed by nuclear factor-kappa B (NF-κB) activation has been suggested to mediate cytokine production in IBD.8 Therapies for UC rely heavily on the use of immunosuppressive drugs such as 5-aminosalicylic acid, corticosteroids, and other immunosuppressants.9 However, the side effects of these types of drugs, which include nausea, vomiting, headaches, rash, fever, agranulocytosis, pancreatitis, nephritis, hepatitis, and male infertility, may severely disrupt patients’ quality of life.10 In addition,
patients taking sulfasalazine should also take folic acid supplements, as the sulfa portion of the drug interferes with folic acid absorption.11 Also, biological agents including anti-TNF-α agents improved IBD therapy, however a large proportion of the patients are either nonresponsive or develop side effects.12 In a recent cross-sectional study, 21% of IBD patients reported that they used complementary and alternative medicine (CAM).13 Another study reported that 44.1% of IBD patients use oral CAM, although the frequency of CAM use reported by the IBD patients was similar to that reported by the healthy control subjects in the study.14 These results indicate that there is a significant need to provide scientific evidence on the usage of CAM in these patients. Glucosamine is an essential component of glycosaminoglycans, and the resulting aggrecans are major constituents of joint cartilage.15–18 Glucosamine has been used to reduce the clinical symptoms of osteoarthritis by enhancing the synthesis of aggrecans.19 Recently, epidemiological evidence based on the VITamins And Lifestyle cohort study suggested that the use of glucosamine and chondroitin is associated with reduced total mortality and the decreased risk of death from cancer.14 Also, prolonged (> 3 years) and regular (> 4 times/week) use of
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glucosamine + chondroitin tended to be associated with reduced risk of colorectal cancer (CRC) (hazard ratio [HR] 0.66; 95% confidence interval [CI] 0.30–1.01), and this association was significant among overweight and obese subjects (HR 0.28; 95% CI 0.10–0.76).20 The same group of researchers found that C-reactive protein (CRP) was inversely associated with regular use of glucosamine.21 CRP is an acute-phase protein and its circulating concentration rise in response to inflammatory process. Although CRP has been used as a marker of colitis disease activity in other studies,22,23 mechanistic explanations for the negative relationship seen between the use of glucosamine supplementation and the risk of CRC have not been provided. Based on the evidence indicating that individuals with colitis are predisposed to develop CRC, this study investigated the efficacy of glucosamine supplementation to alleviate dextran sodium sulfate (DSS)-induced colitis and elucidated its mechanisms of action.
Materials and methods Animals and experimental scheme. Seven-week-old male C57BL/6J mice were purchased from Central Laboratory (Seoul, Korea). The animals were housed in plastic cages, three to four mice per cage, at a constant temperature (25°C) and humidity (50 ± 10%) and maintained in air-conditioned quarters with 12-h light/dark cycles. Body weight and food intake were measured once a week. Animals were separated into three groups: group 1 received AIN-93G control diet (CD), group 2 received the CD supplemented with glucosamine sulfate at a concentration of 0.05% w/w (LGS), and group 3 received the CD supplemented with glucosamine sulfate at a concentration of 0.1% w/w (HGS). Glucosamine sulfate was substituted in place of sucrose. The mice received experimental diets throughout experimental period. Two weeks after starting experimental diets, colitis was induced by supplying two cycles (5 days per cycle) of 2% DSS (MW 50 000, MP Biochemicals, lllkirch, France) in distilled water as the animals’ drinking water for 5 days. The two cycles were separated by 7 days (Fig. 1). The care, maintenance, and treatment of the animals followed protocols approved by the Institutional Animal Care and Use Committee (SM-IACUC-2012-0917-012).
Preparation of the blood and tissue samples. The animals were sacrificed at the end of week 4. Blood was collected from the inferior vena cava into ethylenediaminetetraacetic acidfree tubes and centrifuged at 1550 × g for 20 min. The colon and liver were quickly removed, rinsed with cold saline, and weighed. The colonic mucosa was laid flat on a glass slide and was then scraped with glass slides, frozen instantly in liquid nitrogen, and stored at −80°C until assayed. Disease activity index (DAI) and histological observation. The DAI was determined by scoring changes in weight loss (score: 0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; and 4, > 20%), stool consistency (score: 0, formed; 1, mildly soft; 2, very soft; and 3, watery stool), and bleeding (score: 0, normal; 1, brown; 2, reddish and 3, bloody).24 All parameters were scored daily during DSS treatment (day 14 to day 31). Scores assigned for each parameter were used to calculate the average DAI. Colon tissue was flushed with saline and the length was measured. The colon was cut open longitudinally along the main axis and the lumen was flushed with saline. Excess saline was removed and the colon was rolled using a wooden stick. Each roll was fixed in 10% buffered formalin. Paraffin-embedded sections of the colon were prepared according to routine procedures, and stained with Alcian blue, which stains for mucin-producing goblet cells. The number of goblet cells stained with Alcian blue per villus was counted in three random regions without erosion. Measurement of serum IL-8 and serum amyloid P component (SAP). Serum IL-8 and SAP concentrations were determined using ELISA kits (TSZ Scientific LLC, Waltham, MA, USA and R&D Systems, Inc., Minneapolis, MN, USA). RNA isolation and real-time quantitative polymerase chain reaction (qPCR) analysis. The mRNA expression levels of TNF-α and IL-1β were determined in colon tissue samples using real-time qPCR analysis. Total RNA was extracted from scraped colonic mucosa using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The total RNA (1 μg) prepared from the colonic mucosa was reverse-transcribed using a cDNA Synthesis Kit
Figure 1 Experimental design. Male C57BL/6 mice were separated into three experimental groups (n = 10/group). Glucosamine sulfate was supplemented at concentrations of 0% (control), 0.05% (low glucosamine group, LGS) and 0.10% (high glucosamine group, HGS) of AIN-93G basal diet. Two weeks after feeding the experimental diet, colitis was induced by supplying two cycles (5 days per cycle) of 2% DSS in drinking water. The two cycles were separated by 7 days. Animals were killed at the end of week 4. DSS, dextran sodium sulfate.
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(PhileKorea Technology, Seoul, Korea) according to the manufacturer’s instructions. Real-time qPCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using a QuantiMix SYBR Kit (PhileKorea Technology). The primers for TNF-α, IL-1β, and β-actin were synthesized by Bioneer Corporation (Daejeon, Korea). Relative fold changes in gene expression were determined using the 2ΔΔCT (relative quantification) analysis protocol. Protein extraction and Western blot analysis. Colon tissue was homogenized in a PRO-PREP protein extraction solution (iNtRON Biotechnology Inc., Seongnam, Gyeonggi, Korea) for 20 min on ice, then centrifuged (16 600 × g, 10 min, 4°C). Protein content was determined against a standardized control, using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 50 μg of protein from each sample was separated by 4∼12% and 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Polyvinylidene difluoride membranes (Koma Biotech Inc., Seoul, Korea). The membranes were blocked with 2% skim milk (Amersham Biosciences, Arlington Heights, IL, USA) and incubated with specific antibodies for NF-κB (dilution 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), lipocalin 2 (Lnc2) (dilution 1:1000; Abcam Inc., Cambridge, MA, USA), ZO-1 (dilution 1:200; Abcam Inc.), occludin (dilution 1:200; Invitrogen, Carlsbad, CA, USA), and β-actin (dilution 1:4000; Sigma-Aldrich Corp., St. Louis, MO, USA). Peroxidaseconjugated anti-rabbit IgG was used as the secondary antibody for all proteins except occludin. For the determination of occludin protein expression, peroxidase-conjugated anti-mouse IgG was used as the secondary antibody (Sigma-Aldrich Corp., St. Louis, MO, USA) The membranes were washed with PBS/Tween 20 (PBST) containing 0.1% Tween 20 (Sigma-Aldrich Corp., St. Louis, MO, USA). Reactive bands were visualized using an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Arlington Heights, IL, USA). Following visualization, the antibodies were stripped from the membranes, and the stripping was confirmed by re-exposure to enhanced ECL and detection with an LAS-3000 Imager (Fujifilm, Tokyo, Japan). The membranes were subsequently blocked again, and reprobed with one of the other primary antibodies. The intensity of the bands was quantified using a Bio-Rad GS-800 Calibrated Densitometer equipped with Quantity One software (Bio-Rad Laboratories, Inc.). Statistical analysis. Statistical analysis was conducted using SAS software (Version 9.0, SAS Institute Inc., Cary, NC, USA). All data were expressed as mean ± standard deviation. Data were analyzed by one-way analysis of variance followed by Duncan’s multiple range test. Mann–Whitney and Kruskal–Wallis tests were used in between-group comparisons for DAI scores. Results were considered statistically significant at P < 0.05.
Results Final body weights, organ weights, and colon lengths of animals with induced colitis. There were no differences in body weights among the experimental groups
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Table 1 Final body weight, organ weights, and colon length of experimental animals Organ (g)
CD
LGS
HGS
Body weight Liver Absolute Relative* Spleen Absolute Relative Colon length
22.94 ± 2.03
22.17 ± 1.86
22.94 ± 2.06
1.08 ± 0.07 5.49 ± 0.34
1.06 ± 0.10 6.23 ± 0.56
1.02 ± 0.10 5.06 ± 0.48
0.11 ± 0.04 0.58 ± 0.20 4.90 ± 0.47b
0.13 ± 0.03 0.76 ± 0.16 5.11 ± 0.72b
0.12 ± 0.03 0.62 ± 0.13 5.73 ± 0.32a
Values are presented as a mean ± SD of 10 animals per group. Data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test. *Relative organ weight was calculated percentage of body weight. Values with different superscript letters are significantly different (P = 0.0043). CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.1% glucosamine sulfate diet.
(Table 1), and liver and spleen weights did not differ significantly. In contrast, the average length of the colon in the control group was significantly shorter compared with that of the mice in the HGS group (Table 1). There were no differences in small intestine length among the experimental groups. Evaluation of DAI in colitic mice. Two weeks after starting the experimental diets, the animals were exposed to the two 5-day cycles of 2% DSS in drinking water, separated by 7 days (Fig. 1). The DAI began to increase beginning on day 3 and reached a first peak on day 7 (Fig. 2). Both the LGS and HGS groups had lower DAI scores compared with the CD group, although the difference was not significant. Clinical symptoms were slowly improved at the end of the first DSS cycle. After the second administration of DSS, the DAI scores of all the groups became higher compared with those during the first cycle. The DAI scores in animals fed a diet supplemented with 0.1% glucosamine were significantly lower compared with those of the control group during the second DSS cycle (days 13–17). Histological changes in colon mucosa. Figure 3 shows mucin-producing goblet cells of colon tissue section stained with Alcian blue. Our results indicated that the number of goblet cells per crypt-villus axis was slightly higher, although the difference was not statistically significant (P = 0.43), in animals fed the glucosamine-supplemented diet. Serum concentrations of IL-8 and SAP. Serum concentrations of IL-8, a representative inflammatory cytokine shown to be related to disease severity of colitis,25 and SAP, a protein formed during acute inflammation, were measured. Compared with animals in the CD group, animals in the HGS group had 48% and 45% lower concentrations of serum IL-8 and SAP, respectively (P < 0.05) (Fig. 4). With the lower level of glucosamine sulfate supplementation, however, although dose-dependent decreases in IL-8 and SAP concentrations were observed compared with the CD group, these differences were not statistically significant.
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Figure 2 Effect of glucosamine sulfate supplementation on disease activity index (DAI). DAI was determined by scoring changes in weight loss, stool consistency and bleeding. Scores assigned for each parameter were used to calculate average DAI. Values with different letters at each time point are significantly different at P < 0.05 as determined by Mann– Whitney and Kruskal–Wallis tests. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.10% glucosamine sulfate diet. ; LGS, ; HGS, . CD,
Figure 3 Histological changes in the colon tissue of experimental animals. Tissues were stained with Alcian blue (×400). Goblet cells producing mucin stain blue. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.10% glucosamine sulfate diet.
Figure 4 Effects of glucosamine sulfate supplementation on serum concentrations of IL-8 and SAP. Values are presented as a mean ± SD of 10 animals per group. Values with different letters are significantly different (P = 0.012 for IL-8 and P = 0.024 for SAP) as determined by Duncan’s multiple range test. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.10% glucosamine sulfate diet.
mRNA and protein expressions in colon tissue. TNF-α and IL-1β are critical pro-inflammatory cytokines contributing to colon tissue damage in DSS-induced colitis. Glucosamine sulfate supplementation markedly reduced the relative mRNA expression of colonic tissue TNF-α and IL-1β in a dose-dependent manner (Fig. 5). NF-κB has been observed to be activated in the inflamed colonic mucosa in IBD, and steroid-induced healing of mucosa was accompanied by the disappearance of NF-κB.8 As shown in Figure 6, 0.10% glucosamine sulfate supplementation diminished NF-κB protein expression by 54% compared with the expression in the CD group; however, no dose–response relationship was observed. Lnc2, also known as neutrophil gelatinase-associated lipocalin (NGAL), is a protein from secondary granules of human neutrophils. In our study, the expression of Lnc2 protein was suppressed 960
by 38% in the HGS group compared with the CD group (Fig. 6). However, no significant effect was observed in the LGS group. ZO-1, a tight junction (TJ) protein, is part of the apical junctional complex. Our results revealed that ZO-1 protein expression was significantly higher (∼twofold) in the HGS group compared with the CD group (Fig. 6). Expression of occludin, another TJ protein, was also significantly higher in animals in the HGS group compared with those in the CD group (Fig. 6).
Discussion Glucosamine has been widely used as a complementary therapeutic supplement to suppress osteoarthritis. Results from a recent cohort study suggest that the use of glucosamine supplements is
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Figure 5 Effect of glucosamine sulfate supplementation on colonic TNF-α and IL-1β mRNA expression. Values are presented as a mean ± SD of 10 animals per group. Values with different letters are significantly different (P = < 0.0001 for TNF-α and P = 0.0028 for IL-1β) as determined by Duncan’s multiple range test. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.10% glucosamine sulfate diet.
Figure 6 Effects of glucosamine sulfate supplementation on colonic NF-κB, lipocalin 2, ZO-1 and occludin protein expressions. Values are presented as a mean ± SD of 10 animals per group. Values with different letters are significantly different (P = 0.011 for NF-κB, P = 0.046 for lipocalin 2, P = 0.006 for ZO-1 and P = 0.044 for occludin) as determined by Duncan’s multiple range test. CD, control diet; LGS, 0.05% glucosamine sulfate diet; HGS, 0.1% glucosamine sulfate diet.
inversely associated with CRC.20 Based on evidence showing antiinflammatory actions of glucosamine, as well as a close association between chronic inflammation and CRC, it is possible to hypothesize that glucosamine suppresses colonic inflammation, and thereby reduces CRC risk. Therefore, in this study, we used an animal model of colitis to examine the in vivo efficacy of glucosamine supplementation to alleviate colonic inflammation, and elucidated possible mechanisms of action of this effect. DSSinduced colitis is a well-established experimental model with many signs and symptoms that resemble those of human UC, including diarrhea, bloody feces, weight loss, mucosal ulceration, and shortening of the large intestine.26,27 Our study results showed that glucosamine supplementation at a concentration of 0.1% (w/w diet) improved the clinical symptoms and pathological features of colitis, while a lower concentration of glucosamine (0.05%) slightly, but not significantly, improved the DAI. Similarly, glucosamine supplementation reduced blood
markers of inflammation. In this study, we measured serum SAP, a major acute phase reactant in mice,28 as an indicator of inflammation. Our results showed that mean serum SAP concentration was 45% lower in the HGS group as compared with the CD group. Another major acute phase protein synthesized in response to inflammation is CRP. Although CRP is highly induced in humans, it never rises above 2–3 mg/L in mice.29 A recent study reported that CRP was inversely associated with the use of glucosamine supplements in a subgroup of the NHANES population.21 Two small human intervention studies have also evaluated the association between glucosamine supplementation and circulating CRP concentration.30,31 Glucosamine supplementation did not reduce plasma CRP in rheumatoid arthritis patients, but 3-month supplementation of glucosamine hydrochloride significantly decreased serum prostaglandin E2 concentrations. The findings from our study, coupled with other human studies, suggest a potential role of glucosamine in reducing systemic inflammation.
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Glucosamine supplementation was shown to reduce the inflammatory responses of joint cartilage by inhibiting the activation of NF-κB, which lies upstream of inflammatory mediators such as CRP, IL-1β, IL-8,32,33 and TNF-α. Studies have demonstrated that an imbalance of pro-inflammatory and anti-inflammatory cytokines is related to the pathogenic mechanisms of UC.34 Binding of pro-inflammatory cytokines to their respective receptors amplifies the immune response by enhancing the proliferation of T cells, promoting leukocyte infiltration, and facilitating cellcell signaling.35 A previous study in a DSS-induced colitis model indicated that IL-1, which consists of IL-1α and IL-1β, increases the production of IL-8, IL-6, and TNF-α by monocytes and macrophages,36 and IL-8 expression is upregulated by IL-1β and TNF-α stimulation in human epithelial cells via MAPK phosphorylation and NF-κB activation. In our study, glucosamine supplementation led to significant decreases in both circulating IL-8 concentrations and colonic expression of IL-1β and TNF-α, which may be mediated by the observed decrease in NF-κB expression. Therefore, it is reasonable to hypothesize that glucosamine may protect against inflammation-related intestinal tissue damage and related mucosal barrier disruption by suppressing the synthesis of major inflammatory mediators.8 Glucosamine is a precursor for glycosaminoglycans (GAGs) that are major components of joint cartilage. The antiinflammatory activity of glucosamine is thought to be derived from the direct interactions of GAGs with the protein structure of NF-κB due to the presence of charged groups such as sulfate and carboxyl groups in GAGs.37,38 Previous studies reported that certain chemokines, including pro-inflammatory cytokines, require interactions with GAGs for their in vivo function.39,40 These interactions are thought to play a role in the sequestration of chemokines and subsequent presentation to the receptor expressed on the leukocyte cell surface.39–41 We also observed that intestinal TJ protein expression was increased in the animals that received the glucosaminesupplemented diets. A widely accepted hypothesis regarding the mechanism of chronic relapsing intestinal inflammation in IBD is that gut epithelial barrier dysfunction permits luminal antigens to enter the subepithelial tissues, resulting in the recruitment and activation of leukocytes.42,43 Pro-inflammatory cytokines play a key role in the induction of barrier defects in IBD.44 TNF-α and IFN-γ released during mucosal inflammation can induce an increase in epithelial permeability through regulation of the TJ proteins.45,46 Three of the key proteins of the TJ are ZO-1, occludin, and the more recently identified family of claudins. Expression of these TJ proteins has been shown to be decreased in the intestinal inflammation of IBD.47–49 Intestinal membrane barrier disruption due to inflammation evokes the influx of neutrophils into the mucosa, followed by transepithelial migration of neutrophils, which is a first line of defense against inflammationassociated barrier disruption. IL-8 is known to play an essential role in directing the sequential process of neutrophil rolling, adhesion, and transmigration in the inflamed microvasculature. In addition, pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 increase the expression of adhesion molecules on endothelial cells and neutrophils. In this study, Lcn2 was used as a neutrophil infiltration marker in the DSS-induced colitis model. Our results showed that 0.1% glucosamine supplementation significantly decreased colonic protein expression of Lcn2 compared with the 962
CD group as control. Therefore, it is possible to assume that the production of inflammatory mediators induced less damage to the intestinal barrier suppressing neutrophil infiltration. In summary, this study demonstrated that glucosamine sulfate attenuates DSS-induced colitis symptoms, possibly by suppressing the NF-κB-mediated TNF-α and IL-1β production and neutrophil activation that contribute to intestinal barrier disruption in colitic mice. The effective dose of glucosamine to suppress the DAI was 0.1% of the total diet, which corresponds to approximately 0.5 g/ day based on daily calorie consumption. If one uses the body surface area normalization formula to translate the effective dose in animals to humans,50 a dose of glucosamine of 0.1% of the total diet corresponds to approximately 9 mg/d. This study provides insights on possible utilization of glucosamine sulfate in a practical manner to prevent colitis and CRC. Further efficacy studies in humans are required in the future.
Acknowledgment This research was supported by the SRC program (Center for Food & Nutritional Genomics: grant number 2012-0000642) and the Mid-Career Research Program (2012R1A2A2A01046228) of the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Science, and Technology.
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