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Synergistic effect of κ-carrageenan on oxazolone-induced inflammation in BALB/c mice
© Wu et al. 2016
Received: 22 August 2015
Accepted: 19 March 2016
Published: 25 March 2016
Carrageenan is a traditional ingredient that has been widely used in the food industry. In the present study, we propose a hypothesis that carrageenan is a conditional inflammatory agent. When the intestinal tract is in an “unhealthy” state such as that during bacterial infection or acute inflammation, carrageenan can synergistically enhance the inflammatory response.
BALB/C mice received κ-carrageenan via intragastric administration prior to the induction of oxazolone colitis. Weight changes, survival rate, histologic change, secretion of inflammatory cytokines, ratio of regulatory T cells (Tregs) in peripheral blood, and expression of genes and proteins involved in inflammation and cell proliferation in the colonic mucosa were examined.
Intragastric administration of κ-carrageenan to BALB/c mice prior to the induction of oxazolone colitis resulted in an aggravation of body weight loss, a decrease in the survival ratio, aggravation of colonic inflammation, and decrease in the ratio of CD4 + CD25+/CD4+. The secretion of interleukin-4 (IL-4), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) also significantly increased after κ-carrageenan administration. κ-Carrageenan, together with oxazolone, suppressed the expression of forkhead box p3 (FOXp3) and increased the expression of Toll-like receptor 4 (TLR4), Nuclear factor-κB (NF-κB), and proliferating cell nuclear antigen in the colonic mucosa. These results were confirmed by qRT-PCR and western blot analyses at the molecular and protein levels, respectively.
κ-Carrageenan aggravated oxazolone-induced intestinal inflammation in BALB/c mice. This effect is associated with an activation of the TLR4-NF-κB pathway, a decreased ratio of Tregs, and the induction of Th2-dependent immune responses.
Carrageenan, a high-molecular weight saccharide, is used widely as a thickener, stabilizer, emulsifier, and texturizer in a variety of processed foods (including infant formula, whipped cream, cottage cheese, ice cream, and nutritional supplements) and other products (i.e., pharmaceuticals, cosmetics, and toothpaste) [1–3]. Several international agencies have earlier reported that carrageenan is safe for human consumption [4, 5]. However, investigators later demonstrated that carrageenan induces gastrointestinal ulcerations and cancer in animal models [2, 4]. Carrageenan has been widely used in experimental models of inflammation to assess the activity of anti-inflammatory drugs and to study mediators of inflammation . Therefore, the concept of safety of carrageenan has remained highly controversial.
Current research studies on carrageenan have mainly focused on its effects at the organismal and cellular levels. Animal models such as rats , guinea pigs , pig , mouse , and rhesus monkeys  have demonstrated that carrageenan induces colorectal tumors and ulcerative colitis. However, several international agencies have concluded that carrageenan is harmless [4, 5]. The US Food and Drug Administration (FDA) has declared food-grade carrageenan as generally recognized as safe (GRAS) for use as a food ingredient . Tobacman et al. conducted extensive research studies to evaluate the safety of carrageenan at the cellular level [2, 12–19]. They observed that exposure of human intestinal epithelial cells to food-grade carrageenan triggered a distinct inflammatory response via the activation of Bcl10 in the NF-κB pathway , as well as up regulation of interleukin-8 (IL-8) secretion [16, 20], and identified that the Toll-like receptor 4 (TLR4) is the surface membrane receptor for carrageenan in human colonic epithelial cells. Bernard et al. observed that carrageenan induces tumor necrosis factor alpha (TNF-α) production in human monocytes . Although an upregulation of inflammatory cytokines in human cells occurs following food-grade carrageenan exposure, we found that the increase was actually low, only 2–3-fold higher than that observed in the blank control [16, 20]. Is this difference in expression level enough to cause a strong inflammatory reaction in vivo? Although lipopolysaccharides, which are highly proinflammatory molecules, can increase the secretion of IL-8 and TNF-α in macrophages by a dozen- to a hundred-fold , the inflammatory mechanism of carrageenan remains elusive.
In our previous studies, we observed that λ-carrageenan enhances the LPS-induced production of IL-8 at the cellular level . Moreover, when differentiated human colonic epithelial cells were co-cultured with macrophage cells, the epithelial monolayers were significantly and easily damaged by carrageenan. Based on these earlier findings, we hereby propose the hypothesis that carrageenan is a conditional inflammatory agent. When the intestinal tract is in an “unhealthy” state such as that during bacterial infection or acute inflammation, carrageenan can synergistically enhance the inflammatory response.
Therefore, in the present study, an oxazolone (OXA)-induced mild intestinal inflammation model was developed to investigate whether κ-carrageenan can enhance the inflammatory responses of the gut with acute inflammation. Furthermore, the mechanism underlying this inflammatory response was also investigated in relation to the TLR4 pathway.
Seven-week-old male and female BALB/c mice with weights ranging from 20-25 g were purchased (SCXK 2012–0001, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), housed in the animal center of Ningbo University Medicine College (Ningbo, China), and used in the present study. Five same-sex mice were housed in a cage and maintained under a 12-h light/12-h dark cycle (08:00 AM lights on) with food and water provided ad libitum. Housing and experimental environments were temperature- and humidity-controlled (21 ± 2 °C and approximately 60 %, respectively). All mice were allowed to habituate to the housing environment for 3 days prior to gavage administration. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approved by the Ethical Committee of Animal Use and Protection of Ningbo University Health Science Center.
Animal experimental procedure
Eighty mice were divided into eight groups and placed in clear plastic cages with a mesh top under specific pathogen-free conditions, with each experimental group consisting of four males and four females. The experimental groups were designed as follows: the blank group only received a saline solution; the OXA group was treated with 150 μL of 1 % OXA; the carrageenan groups were treated with different concentrations of carrageenan, namely, the low-dose group (1.7 mg/kg, LOW), medium-dose group (8.3 mg/kg, MED), and high-dose group (41.7 mg/kg, HIG); carrageenan + OXA groups received low, medial, and high doses of carrageenan following by the administration of OXA and marked as “LOW + OXA,” “MED + OXA,” and “HIG+ OXA.”
κ-Carrageenan (Sigma Chemical Co., St Louis, MO, USA) was dissolved in saline with vigorous stirring at 37 °C and administered at three different doses, 1.7 mg/kg, 8.3 mg/kg, and 41.7 mg/kg by gavage administration. These doses were decided according to the data of preliminary experiments based on related study . To assess the synergistic effect of carrageenan on OXA-induced inflammation, different doses of carrageenan were gavage administered for two weeks prior to OXA administration.
OXA-induced intestinal inflammation was established as previously described by Heller et al., with minor modifications . To presensitize mice, a 2 × 2 cm area of the abdominal skin was shaved, and 200 μL of OXA [a 3 % (w/v) solution in 100 % ethanol, Sigma-Aldrich, St. Louis, MO] was smeared 5 days before OXA was administered. Five days after presensitization (day 0), the mice were rechallenged intrarectally with 100 μL of 1 % OXA in 50 % ethanol or only 50 % ethanol (vehicle, Blank group) under general anesthesia with isoflurane (Baxter, Deerfield, IL, USA). Intrarectal injection was administered with a polyurethane umbilical catheter. To ensure distribution of the OXA throughout the entire colon, the mice were held head down in a vertical position for 60 s after injection.
The mice were then monitored for symptoms of diarrhea, loss of body weight, and death the next day (day 1). The mice underwent exsanguination by cardiac puncture and the collected blood was used to determine the CD4+CD25+ Treg ratio and cytokine levels by flow cytometry. The colon was also isolated; one portion was used in macroscopic evaluation, whereas the rest was fixed in paraformaldehyde (Merck, Darmstadt, Germany) or glutaraldehyde solution for immunohistochemical and scanning electron microscopy analysis.
Macroscopic evaluation of colonic tissues used the following criteria: 0, normal; 1, erythema only; 2, erythema, slight edema, and small erosions; 3, two or more bleeding ulcers and/or inflammation and/or moderate adhesions; and 4, severe ulceration and/or stenosis with dilations and/or severe adhesions.
The colonic tissues were dissected and washed with Hank's balanced salt solution (containing 10 μg/mL of gentamicin, 100 U/mL of penicillin, and 100 μg/mL of streptomycin). The tissues were then fixed in 10 % natural buffered formalin, embedded in paraffin, cut into tissue sections (5-μm-thick), and stained with hematoxylin and eosin (H&E).
Histological examination was evaluated as previously described . The degree of histologic damage and inflammation was graded in a blinded fashion by expert histologists. The following manifestations were included in the evaluation: the amount of inflammation (0, none; 1, mild; 2, moderate; 3, severe; and 4, accumulation of inflammatory cells in the gut lumen), distribution of lesions (0, none; 1, focal; 2, multifocal; 3, nearly diffuse; and 4, diffuse), depth of inflammation and layers involved (0, none; 1, mucosa only; 2, mucosa and submucosa; 3, limited transmural involvement; and 4, transmural), and nature of mucosal changes (0, none; 1, minimal degeneration; 2, more degeneration; and 3, more necrosis). The overall histologic score was the sum of the four manifestations (maximum score: 15).
Intestinal mucosal morphology
The processing of intestinal segments for scanning electron microscopy was conducted as described elsewhere . Briefly, one segment of the tissue sample was fixed with 2.5 % glutaraldehyde. The segments were washed twice in phosphate buffer (pH = 7.2) and then post-fixed in osmium tetroxide (1 % in phosphate buffer, pH = 7.2, 2 h). After a series of dehydration steps in alcohol from 30 % to 100 %, the segments were critical point-dried (CPD030; BAL-TEC; Balzers; Liechtenstein), mounted on Al stubs, sputter-coated with gold by using a high-resolution fine coater (SCD005; BAL-TEC; Balzers; Liechtenstein), and then examined under a Jeol JSM-6301 scanning microscope (JEOL Ltd., Musashino, Tokyo, Japan). Morphological evaluation of the intestinal mucosal and microvilli was then performed. Scanning electron microscopic images (×200 magnification) were analyzed to measure the histological changes on the epithelial cell surface.
Determination of CD4 + CD25 + Treg ratio by flow cytometry
A total of 2.5 μL of anti-mouse CD3-eFluor450, 0.625 μL of anti-mouse CD4-PE, 0.625 μL of anti-mouse CD25-APC (all from eBioscience, California, USA) were added to 100 μL of peripheral blood. Following incubation at room temperature in the dark for 15–30 min, 1 mL of erythrocyte lysis solution was added to the samples and incubated for 15–20 min. The cells were washed with 2 mL phosphate-buffered saline (PBS) and resuspended in 500 μL PBS. The cells were then analyzed using a Beckman Gallios flow cytometer (Beckman Coulter, Inc., California, USA).
For immunocytochemical analysis, the mouse colonic tissues were fixed with 4 % paraformaldehyde in PBS, embedded in paraffin, and cut into tissue sections (5-μm-thick). The tissues were blocked with 1 % bovine serum albumin (BSA, Nacalai tesque) for 60 min at room temperature. The primary antibodies used in the present study included anti-TLR4 antibody (1:200, ab22048, Abcam, Cambridge, UK), anti-NF-κB antibody (1:200, 8242, CST, Danvers, MA, USA), anti-FOXp3 antibody (1:200, ab54501, Abcam, Cambridge, UK), and anti-PCNA antibody (1:100, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies included goat anti-rabbit IgG (PV-9001, ZSGB-BIO, Peking, China) and rabbit anti-mouse IgG (PV-9005, ZSGB-BIO, Peking, China). Negative controls substituted with non-immunized mouse IgG at the same concentrations as that for each primary antibody were employed. Images were captured using a Leitz Dialux 22 Microscope (Leica Microsystems, Wetzlar, Germany) equipped with a QIcam Fast 1394 camera (QImaging, Surrey, BC, Canada). The images were analyzed using the software, Image Pro Plus 6.0 (Media Cybernetics UK, Marlow, UK). The levels of expression of TLR4, NF-κB, FOXp3, and PCNA were determined by measuring the positively labeled areas in relation to the total area.
Analysis of cytokine levels
Serum samples were collected after centrifugation of blood and stored at −80 °C. The levels of proinflammatory cytokines, including interleukin IL-6, tumor necrosis factor (TNF), Th1 (IL-2, interferon IFN-γ), Th2 (IL-4, IL-10), and Th17 (IL-17) cytokines in each serum sample were determined by using the BD Cytometric Bead Array mouse Th1/Th2/Th17 7 cytokine kit (BD BioSciences, NJ, USA) according to the manufacturer’s instructions. Samples and standards were analyzed on a FACS Calibur flow cytometer (BD BioSciences, NJ, USA). The concentrations were assessed by using FCAP Array software.
RNA extraction and real-time quantitative reverse transcription-PCR (RT-qPCR) analysis
Oligonucleotide primers used in this work
Preparation of nuclear extracts
To prepare a whole lysate solution, the collected tissues were washed twice with ice-cold PBS and lysed in a lysis buffer (Beyotime, Shanghai, China) that contained 1 mM of phenylmethylsulfonyl fluoride (PMSF) and a protein phosphatase inhibitor cocktail (Roche, Basel, Switzerland) for 10 min on ice. The homogenates were centrifuged at 12,000 rpm at 4 °C for 10 min. Total protein was extracted from the scraped colonic mucosa of the experimental mice using a NE-PER nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA), following the manufacturer's recommendations. Protein concentration was determined using a Bio-Rad DC Protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA), following the manufacturer's instructions.
Western blot analysis
Proteins (30 μg) in the nuclear extracts or in the whole lysate were separated by 10 % sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes, and probed with antibodies against TLR4, NF-κB, and p65 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by appropriate horseradish peroxidase (HRP)-linked secondary antibodies (CST). The immunoreactive proteins were detected by using WesternBright ECL (Advansta Inc., Menlo Park, CA, USA). The results were quantified by measuring the band intensity relative to that of β-actin using the AlphaView™ Software (Alpha Innotech, San Leandro, CA, USA), and expressing as the relative intensities.
Statistical analyses were performed using the SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA). The results were expressed as means ± SEM. P < 0.05 was considered to indicate statistically significant differences. Macroscopic and histological score analyses were performed by using the student’s t-test. Differences in weight loss, cytokine levels, ratio of Treg (CD4+CD25+CD127dim), mRNA expression, western blot data, and average integral optical density of expression of various inflammatory proteins in mouse colonic mucosa among different groups were analyzed by using one-way ANOVA after post hoc testing.
κ-Carrageenan aggravates weight loss and decrease in the survival ratio of mice with OXA-induced inflammation
Consistent with the observed difference in weight change, the Blank and carrageenan-treated groups showed similar survival rates, whereas the OXA-treated mice showed an 87.5 % survival ratio. The survival ratio of the carrageenan + OXA group was lower than that of the OXA-treated groups. Among these, the group that received a medial-dose (8.3 mg/kg) κ-carrageenan for 14 days showed a 37.5 % mortality rate, which was the highest (Fig. 1b), suggesting that medial-dose (8.3 mg/kg) κ-carrageenan significantly aggravated weight loss and decreased the survival ratio due to OXA exposure.
Effects of κ-carrageenan on the severity of colonic damage
Carrageenan alone showed no impact on the colons of animals, wherein the colonic mucosa was apparently normal, and symptoms of congestion, edema, and erosion were not detected (Fig. 2b–d). No significant differences in colonic macroscopic scores of mice from the Blank group mice and those of the LOW, MED, and HIG treatment groups were observed. Figure 2f–h reveals that OXA-induced colonic damage was markedly augmented in mice pretreated with different concentration of carrageenan. The colons were more reddish due to the production of a bloody stool compared to those of the OXA-treated mice, suggesting that a more severe intestinal inflammation had occurred in the LOW + OXA, MED + OXA, and HIG-OXA-treated mice (Fig. 2f–g). The colonic damage scores of the LOW + OXA, MED + OXA, and HIG + OXA groups were higher than that of the OXA-induced group (P < 0.05, Fig. 2i). Among these, the MED + OXA group showed the highest score of 3.75. A 1.55-fold higher colonic damage score was observed in the MED + OXA group compared to the OXA-treated group (P < 0.01). These results indicate that carrageenan can synergistically enhance OXA-induced inflammation.
Effects of κ-carrageenan on the histological changes of OXA-induced colonic damage
Histological analysis showed that carrageenan was effective in aggravating OXA-induced colitis (Fig. 3f–h). The colonic sections from the LOW + OXA, MED + OXA, and HIG + OXA-treated mice revealed mucosal inflammation, which was characterized by lymphocyte cell infiltration, as well as a significant reduction in the number of goblet cells. A 1.22-fold augmentation in histologic score was obtained in the LOW + OXA group (P < 0.05), a 1.32-fold increase in the MED + OXA group (P < 0.01), and a 1.25-fold increase in the HIG + OXA group relative to the OXA-treated group (P < 0.05) (Fig. 3i).
Analysis of histological changes of the colon by using scanning electron microscopy
κ-Carrageenan administration promoted the OXA-induced decrease in the ratio of CD4+CD25+ regulatory T cells (Tregs)
κ-Carrageenan promotes cytokine production in OXA-induced mice colonic inflammation
The levels of seven cytokines in the serum were determined by flow cytometry by using the Mouse Inflammation Cytometric Bead Array (CBA) assay. Of these cytokines, TNF-α and IL-6 as classified as pro-inflammatory cytokines; IFN-γ and IL-2 are Th1 cytokines; IL-4, IL-10 are Th2 cytokines; and IL-17A is a Th17 cytokine.
The levels of the inflammatory cytokines IL-10, IL-17A, TNF, IFN-γ, IL-6, IL-4, and IL-2 in mouse serum (pg/ml, mean ± SEM)
1.73 ± 0.15
2.31 ± 0.35
3.71 ± 0.20
1.87 ± 0.08
2.61 ± 0.15
1.79 ± 0.11
1.80 ± 0.32
6.48 ± 1.19 **
2.94 ± 0.99
10.98 ± 0.78**
1.81 ± 0.38
47.6 ± 2.64 **
2.34 ± 0.43
2.43 ± 0.09
3.42 ± 0.45
2.91 ± 1.00
6.18 ± 0.70
2.64 ± 0.44
3.57 ± 0.50
2.33 ± 0.33
2.14 ± 0.28
LOW + OXA
11.34 ± 2.4**#
3.07 ± 0.83
26.62 ± 3.07**##
1.69 ± 0.27
92.32 ± 3.36**#
2.70 ± 0.27*
2.28 ± 0.27
2.27 ± 0.78
2.34 ± 2.08
6.40 ± 0.35
2.58 ± 0.91
4.31 ± 0.55
2.20 ± 0.60
2.08 ± 0.21
MED + OXA
19.67 ± 2.35**##
2.56 ± 0.44
35.0 ± 3.13**##
1.88 ± 0.21
182.17 ± 7.85**##
2.49 ± 0.12**
2.57 ± 0.18
2.80 ± 0.74
2.07 ± 0.17
5.95 ± 0.75
1.90 ± 0.14
3.64 ± 0.39
2.36 ± 0.68
2.60 ± 0.24
HIG + OXA
12.32 ± 2.57**#
2.56 ± 0.34
30.63 ± 2.83**##
1.61 ± 0.08
107.17 ± 3.74**#
2.66 ± 0.11**
2.16 ± 0.45
The expression levels of TLR4, NF-κB, PCNA, and FOXp3 mRNA in the colonic mucosa of experimental mice
Synergistic effects of κ-carrageenan on OXA-induced protein expression in colonic mucosa of experimental mice
Impact of carrageenan on the expression of various proteins in mouse colonic mucosa (Au, mean ± SEM)
1.57 ± 0.48
6.24 ± 0.48
7.01 ± 0.73
25.97 ± 0.38
5.30 ± 0.24
1.84 ± 0.31
13.83 ± 0.21**
18.07 ± 0.82**
16.51 ± 0.72**
13.54 ± 0.43**
2.65 ± 0.19
6.45 ± 0.32
7.8 ± 1.22
24.23 ± 0.58
5.86 ± 0.20
LOW + OXA
2.74 ± 0.23
15.25 ± 0.23**#
20.26 ± 1.40**#
14.24 ± 0.65**#
15.96 ± 0.69**#
2.53 ± 0.28
6.63 ± 0.25
7.95 ± 1.26
24.50 ± 0.75
6.19 ± 0.18
MED + OXA
2.36 ± 0.29
17.61 ± 0.45**##
26.68 ± 0.83**##
6.48 ± 0.68**##
24.41 ± 0.27**##
2.59 ± 0.23
6.56 ± 0.20
7.33 ± 1.16
24.56 ± 0.75
5.99 ± 0.28
HIG + OXA
77.6 ± 15.3
15.07 ± 0.78**#
21.33 ± 1.90**#
14.18 ± 0.86**#
16.98 ± 1.70**##
Table 3 shows that the expression levels of Foxp3 expression in the colonic epithelium decreased after OXA injection (P < 0.01), whereas carrageenan treatment significantly decreased the level of OXA-induced expression of FOXp3 (Fig. 6; Table 3; P < 0.05).
κ-Carrageenan enhances oxazolone-induced activation of TLR4 and NF-κB in experimental mice
Although the FDA and JECFA consider carrageenan as a safe saccharide [1, 5, 11], studies have shown that it induces ulcerations in animal models, suggests that it poses a carcinogenic risk to humans [1–3, 27]. This finding has sparked controversy and intense debate, and the mechanism underlying this pathological condition has remained uncertain. The present study presents a possible mechanism of carrageenan that involves an inflammatory response.
BALB/c is a mouse strain that is susceptible to OXA-induced inflammation [24, 28]. In this research, we used OXA to establish an acute gut inflammation BALB/c mouse model, which resembles ulcerative colitis in pathology and the immune response inof human. It has been reported that the initial toxic effects of OXA leads to a flooding of the lamina propria and inducing an increase in the secretion of Th2 cytokines IL-4 and IL-5, which in turn leads to further inflammation. [24, 29]. Moreover, rectal instillation of 150 μL of 1 % OXA is enough to lead to an accelerated inflammation lasting for 1–2 days by a Th2 response in the distal half of the mouse colon of the mice, thereby resulting in either rapid recovery or death . Therefore, one day after injection of OXA was decided as the experimental time to detect the aggravating effect of carrageenan on the inflammatory response.
The average daily intake of carrageenan in the infant formula is 0.3 g/L , amounting to 240 mg/5.8 kg/day (≈41.7 mg/kg/day). In the typical Western diet, the daily intake of carrageenan is considerably more than 500 mg/60 kg/day (≈8.3 mg/kg/day) . Bhattacharyya et al. reported that a dose of 50 μg/30 g/day (≈1.7 mg/kg/day) carrageenan induces colonic inflammation in IL-10-deficient mice . Therefore, in the present study, we used these concentrations to investigate the effect of carrageenan on OXA-induced inflammation.
Here, we first discovered that κ-carrageenan aggravates OXA-induced inflammation in BALB/c mice by significantly increasing weight loss, lethality rate, and degree of colonic injury. Of particular interest is our finding that mice pretreated with a medial-dose (8.3 mg/kg) of κ-carrageenan for 14 days resulted in a 37.5 % of mortality rate, whereas that of OXA-treated control mice was 12.5 % (Fig. 2). Histological and microscopic evaluation also confirmed that intragastric administration of κ-carrageenan aggravated OXA-induced inflammation in BABL/c mice. Of the three doses of κ-carrageenan used in the present study, the medium dose showed highest score histological scores. The survival ratio of the OXA + MED group was also the lowest. It is possible that the high concentration of carrageenan induces its formation into a gel in the gut of mice, which in turn reduces its contact without other gut content. In addition, Weiner et al. also observed that intragastric administration of a high dose of food-grade carrageen an just directly affected theresulted in the production of soft stools or development of diarrhea, which are common effects of non-digestible high-molecular weight compounds . Therefore, the aggravated effects of carrageenan do not appear to respond to a traditional dose–response curve.
OXA-induced inflammation is often employed in research investigations on the contribution of the Th2-dependent immune response to intestinal inflammation . The present study showed that OXA-induced inflammation is accompanied by the release of various inflammatory cytokines, not only Th2 type cytokines IL-10, but also pro-inflammatory cytokines, TNF-α and IL-6 [24, 28]. Previous reports have revealed that OXA-induced inflammation is characterized by an upregulation of IL-4 ; however, in the present study, although an increased level of IL-4 expression was observed in the OXA group, this was not statistically significant. No increase in the levels of Th1 cytokines IFN-γ, IL-2, and IL-17 was observed with OXA and κ-carrageenan treatment. The synergistic effect of κ-carrageenan on OXA-induced changes is also reflected in the secretion levels of inflammatory cytokines. The secretion of pro-inflammatory cytokines TNF-α and IL-6 in OXA-treated mice were all significantly augmented by κ-carrageenan pretreatment. These findings suggest that carrageenan aggravated the OXA-induced inflammation of intestinal tissues.
CD4+CD25+ regulatory T cells (Tregs) can regulate and suppress the immune response, thus; therefore, these play an important role in the maintenance of peripheral immune tolerance. The decrease in the number of Tregs often results in a development of various kinds of autoimmune disease such as inflammatory bowel disease (IBD) . The present study showed that the number of Tregs in the peripheral blood of OXA-treated group was lower than that of the Blank group, which further decreased after κ-carrageenan treatment, demonstrating that OXA can cause an imbalance in the immune system of mice, and carrageenan augments this imbalance.
FOXp3 is a transcription factor that plays a major role in the development and maintenance of the immunosuppressive function of Tregs . Galitovskiy et al. observed that the expression of FOXp3 is decreased in the inflamed areas of ulcerative colitis compared to that of the normal colon . This finding is consistentin agreement with the observations of the present study, wherein the intragastric administration of κ-carrageenan significantly decreased the OXA-induced mRNA expression of FOXp3 in the colonic mucosa, indicating that the suppressor activity of FOXp3 might have been abrogated in vivo or was insufficient to counterbalance the acute mucosal inflammation in OXA-treated mice.
Mice pretreated with different doses of carrageenan showed a significant increase in the levels of OXA-induced expression of NF-κB and TLR4. Some studies have demonstrated that carrageenan upregulated interleukin-8 (IL-8) secretion through TLR4, and activated NF-κB in human colonic epithelial cells [16, 20]. Benard et al. also observed that κ-carrageenan induced TNF production via NF-κB activation . Recently, we have beenare exploring the mechanism of underlying the synergistic effect of κ-carrageenan on lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) secretion in HT-29 cells. We found that κ-carrageenan pretreatment increased LPS-stimulated IL-8 secretion. The TLR4-NF-κB pathway was activated, as evidenced by the enhancement of Bcl10 (B-cell CLL/lymphoma 10) expression, IκBα phosphorylation, and nuclear NF-κB localization (Wu, firstname.lastname@example.org). These findings demonstrate that intragastric administration with κ-carrageenan significantly activated intestinal inflammation by upregulating TLR4 and NF-κB in colonic mucosa.
In conclusion, intrarectal injection of OXA in mice causes colonic damage and inflammation. Treatment of mice with κ-carrageenan significantly aggravated OXA-induced inflammation in BALB/c mice. The results of the present study suggest that the synergistic effect of κ-carrageenan might be linked to the induction of an imbalance in the expression of intestinal inflammatory cytokines, which then magnified the existing intestinal inflammation. Therefore, under the intestinal “unhealthy” state, carrageenans may serve as potential inflammatory agents that impart certain harmful effects.
This work was supported by grants from the NSFC project (81370532), the outstanding (postgraduate) dissertation growth foundation of Ningbo University (py2014002), the Ocean Public Welfare Scientific Research Project, State Oceanic Administration of China (201505033), the K.C. Wong Magna Fund in Ningbo University, and the 151 Talents Project.
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- Marcus R, Watt J. Danger of carrageenan in foods and slimming recipes. Lancet. 1981;1(8215):338.View ArticlePubMedGoogle Scholar
- Tobacman JK. Review of harmful gastrointestinal effects of carrageenan in animal experiments. Environ Health Perspect. 2001;109(10):983–94.View ArticlePubMedPubMed CentralGoogle Scholar
- Watt J, Marcus R. Harmful effects of carrageenan fed to animals. Cancer Detect Prev. 1981;4(1):129–34.PubMedGoogle Scholar
- Cohen SM, Ito N. A critical review of the toxicological effects of carrageenan and processed eucheuma seaweed on the gastrointestinal tract. Crit Rev Toxicol. 2002;32(5):413–44.View ArticlePubMedGoogle Scholar
- Shahrul ZA,Wong WY,Intan ZA, Rohaya AW, Zaidah ZA, Sahidan S. Cytotoxicity effect of degraded andundegraded kappa and iota carrageenan in human intestine and liver cell lines. BMC Complement AlternMed.2014;14:508.Google Scholar
- Kitsukawa Y, Saito H, Suzuki Y, Kasanuki J, Tamura Y, Yoshida S. Effect of ingestion of eicosapentaenoic acid ethyl ester on carrageenan induced colitis in guinea pigs. Gastroenterology. 1992;102(6):1859–66.PubMedGoogle Scholar
- Sharratt M, Grasso P, Carpanini F, Gangolli SD. Carrageenan ulceration as a model for human ulcerative colitis. Lancet. 1970;2(7679):932.View ArticlePubMedGoogle Scholar
- Poulsen E. Short-term peroral toxicity of undegraded carrageenan in pigs. Food Cosmet Toxicol. 1973;11(2):219–27.View ArticlePubMedGoogle Scholar
- Maillet M, Bonfils S, Lister RE. Carrageenan: effects in animals. Lancet. 1970;2(7669):414–5.View ArticlePubMedGoogle Scholar
- Benitz KF, Golberg L, Coulston F. Intestinal effects of carrageenans in the rhesus monkey (Macaca mulatta). Food Cosmet Toxicol. 1973;11(4):565–75.View ArticlePubMedGoogle Scholar
- Friedman SD. Taking responsibility for scientific discourse. Science. 2008;321(5892):1039–40.View ArticlePubMedPubMed CentralGoogle Scholar
- Tobacman JK, Bhattacharyya S, Borthakur A, Dudeja PK. The carrageenan diet: not recommended. Science. 2008;321(5892):1040–1.View ArticlePubMedGoogle Scholar
- Bhattacharyya S, Dudeja PK, Tobacman JK. Lipopolysaccharide activates NF-kappaB by TLR4-Bcl10-dependent and independent pathways in colonic epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2008;295(4):G784–790.View ArticlePubMedGoogle Scholar
- Bhattacharyya S, Borthakur A, Dudeja PK, Tobacman JK. Carrageenan induces cell cycle arrest in human intestinal epithelial cells in vitro. J Nutr. 2008;138(3):469–75.PubMedGoogle Scholar
- Bhattacharyya S, Kotlo K, Shukla S, Danziger RS, Tobacman JK. Distinct effects of N-acetylgalactosamine-4-sulfatase and galactose-6-sulfatase expression on chondroitin sulfates. J Biol Chem. 2008;283(15):9523–30.View ArticlePubMedGoogle Scholar
- Bhattacharyya S, Gill R, Chen ML, Zhang F, Linhardt RJ, Dudeja PK, Tobacman JK. Toll-like receptor 4 mediates induction of the Bcl10-NFkappaB-interleukin-8 inflammatory pathway by carrageenan in human intestinal epithelial cells. J Biol Chem. 2008;283(16):10550–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Bhattacharyya S, Feferman L, Borthakur S, Tobacman JK. Common food additive carrageenan stimulates Wnt/beta-catenin signaling in colonic epithelium by inhibition of nucleoredoxin reduction. Nutr Cancer. 2014;66(1):117–27.View ArticlePubMedGoogle Scholar
- Bhattacharyya S, Xue L, Devkota S, Chang E, Morris S, Tobacman JK. Carrageenan-induced colonic inflammation is reduced in Bcl10 null mice and increased in IL-10-deficient mice. Mediators Inflamm. 2013;2013(10):397642.PubMedPubMed CentralGoogle Scholar
- Borthakur A, Bhattacharyya S, Anbazhagan AN, Kumar A, Dudeja PK, Tobacman JK. Prolongation of carrageenan-induced inflammation in human colonic epithelial cells by activation of an NFkappaB-BCL10 loop. Biochim Biophys Acta. 2012;1822(8):1300–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Borthakur A, Bhattacharyya S, Dudeja PK, Tobacman JK. Carrageenan induces interleukin-8 production through distinct Bcl10 pathway in normal human colonic epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2007;292(3):G829–838.View ArticlePubMedGoogle Scholar
- Benard C, Cultrone A, Michel C, Rosales C, Segain JP, Lahaye M, Galmiche JP, Cherbut C, Blottiere HM. Degraded carrageenan causing colitis in rats induces TNF secretion and ICAM-1 upregulation in monocytes through NF-kappaB activation. PLoS One. 2010;5(1):e8666.View ArticlePubMedPubMed CentralGoogle Scholar
- Ulevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol. 1995;13:437–57.View ArticlePubMedGoogle Scholar
- Chen H, Wang F, Mao H, Yan X. Degraded lambda-carrageenan activates NF kappaB and AP 1 pathways in macrophages and enhances LPS-induced TNF alpha secretion through AP 1. Biochim Biophys Acta. 2014;1840(7):2162–70.View ArticlePubMedGoogle Scholar
- Heller F, Fuss IJ, Nieuwenhuis EE, Blumberg RS, Strober W. Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity. 2002;17(5):629–38.View ArticlePubMedGoogle Scholar
- Mudter J, Amoussina L, Schenk M, Yu J, Brustle A, Weigmann B, Atreya R, Wirtz S, Becker C, Hoffman A. The transcription factor IFN regulatory factor-4 controls experimental colitis in mice via T cell-derived IL-6. J Clin Invest. 2008;118(7):2415–26.PubMedPubMed CentralGoogle Scholar
- Fang PA, Verdelis K, Yang X, Lukashova L, Boskey AL, Beniash E. Ultrastructural organization of dentin in mice lacking dentin sialo-phosphoprotein. Connect Tissue Res. 2014;55 Suppl 1:92–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Sharratt M, Grasso P, Carpanini F, Gangolli SD. Carrageenan ulceration as a model for human ulcerative colitis. Lancet. 1971;1(7691):192–3.View ArticlePubMedGoogle Scholar
- Onitake T, Ueno Y, Tanaka S, Sagami S, Hayashi R, Nagai K, Hide M, Chayama K. Pulverized konjac glucomannan ameliorates oxazolone-induced colitis in mice. Eur J Nutr. 2015;54(6):959–69.View ArticlePubMedGoogle Scholar
- Boirivant M, Fuss IJ, Chu A, Strober W. Oxazolone colitis: A murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4. J Exp Med. 1998;188(10):1929–39.View ArticlePubMedPubMed CentralGoogle Scholar
- Tobacman JK. Toxic considerations related to ingestion of carrageenan. Food Nutr Toxic. 2003;3(1):204–29.View ArticleGoogle Scholar
- Weiner ML. Food additive carrageenan: Part II: A critical review of carrageenan in vivo safety studies. Crit Rev Toxicol. 2014;44(3):244–69.View ArticlePubMedGoogle Scholar
- Billiard F, Litvinova E, Saadoun D, Djelti F, Klatzmann D, Cohen JL, Marodon G, Salomon BL. Regulatory and effector T cell activation levels are prime determinants of in vivo immune regulation. J Immunol. 2006;177(4):2167–74.View ArticlePubMedGoogle Scholar
- van Loosdregt J, Coffer PJ. Post-translational modification networks regulating FOXP3 function. Trends Immunol. 2014;35(8):368–78.View ArticlePubMedGoogle Scholar
- Galitovskiy V, Qian J, Chernyavsky AI, Marchenko S, Gindi V, Edwards RA, Grando SA. Cytokine-induced alterations of alpha7 nicotinic receptor in colonic CD4 T cells mediate dichotomous response to nicotine in murine models of Th1/Th17- versus Th2-mediated colitis. J Immunol. 2011;187(5):2677–87.View ArticlePubMedPubMed CentralGoogle Scholar