CHONDROITIN SULFATE AND GLUCOSAMINE SULFATE AS PROTECTIVE AND ANTI-INFLAMMATORY AGENTS IN THE ULCERATIVE COLITIS DSS MODEL IN RATS

Condroitim sulfato e glucosamina sulfatada como agentes protetores e anti-inflamatórios no modelo de colite ulcerativa induzida por DSS em ratos

ABSTRACT

Chondroitin sulfate (CS) and glucosamine (GlcN) are indicated for the treatment of some inflammatory diseases, such as osteoarthritis, mainly because of the anti-inflammatory effects in reducing metalloproteinases activities (MMP), and other inflammatory mediators. Herein, we reported the structure of the CS, the anti-inflammatory and protective effects of the CS, and GlcN administration in ulcerative colitis model induced by dextran sulfate sodium (DSS) in rats. Experimental data indicated that CS disaccharide composition is very similar to the C4S standard, with modal molecular weight at 30.4 kDa. Orally administration of the CS/GlcN improved the severity of acute colitis with reduction the histological score and goblet cells destruction. We also observed a decreasing in NO production, myeloperoxidase and MMP, especially MMP-9, activities. Moreover, CS/GlcN not cytotoxic in the intestinal epithelial cells. These results indicate that combination CS/GlcN showed improvements in intestinal inflammation and protection intestinal barrier, suggesting CS/GlcN might have beneficial effects in treatment of IBD.

Keywords:
Chondroitin sulfate; glucosamine; structure; anti-inflammation; inflammatory bowel disease; dextran sulfate sodium

HIGHLIGHTS

•Chondroitin sulfate and glucosamine (CS/GlcN) preparation was analyzed for structure.

•CS/GlcN reduced histological score and macrophage infiltration.

•CS/GlcN decreased MMP-9 activity and NO production.

•CS/GlcN not cytotoxic in the intestinal epithelial cells.

RESUMO

Contexto:  O condroitim sulfato (CS) e a glucosamina sulfatada (GlcN) são indicados para o tratamento de algumas doenças inflamatórias, como a osteoartrite, principalmente devido aos seus efeitos anti-inflamatórios na redução das atividades de metaloproteinases (MMP) e de outros mediadores inflamatórios. Neste estudo, relatamos a estrutura do CS, assim como os efeitos anti-inflamatórios e protetores da administração de CS e GlcN no modelo de colite ulcerativa induzida por dextran sulfato de sódio (DSS) em ratos. Os dados experimentais indicaram que a composição de dissacarídeos do CS é muito semelhante ao padrão de C4S, com peso molecular modal de 30,4 kDa. A administração oral de CS/GlcN melhorou a gravidade da colite aguda com redução do escore histológico e da destruição das células caliciformes. Também observamos uma redução na produção de NO, nas atividades de mieloperoxidase e MMP, especialmente MMP-9. Além disso, CS/GlcN não apresentou citotoxicidade nas células epiteliais intestinais. Esses resultados indicam que a combinação CS/GlcN apresentou melhorias na inflamação intestinal e na proteção da barreira intestinal, sugerindo que CS/GlcN pode ter efeitos benéficos no tratamento de doenças inflamatórias intestinais (DII).

Palavras-chave:
Sulfato de condroitina; glucosamina; estrutura; anti-inflamatório; doença inflamatória intestinal; sulfato de dextrana sódico

INTRODUCTION

Inflammatory bowel diseases (IBDs) include Crohn’s disease (CD) and ulcerative colitis (UC) and are characterized by chronic inflammation of the gastrointestinal tract^1-3.

Although the pathogenesis and etiology of IBDs have not been completely elucidated, the excessive immune response to some commensal enteric bacteria, especially in genetically susceptible individuals, environmental factors, and the loss of the epithelial barrier may trigger disease onset^4-6.

The excessive activation of immune cells in IBDs increases the release of pro-inflammatory cytokines, which results in tissue damage. Nuclear transcription factor-kB (NF-kB) is one of the main regulators of this immune response in UC⁷, and it regulates the transcription of genes involved in inflammation to increase the expression of cytokines, the inducible form of nitric oxide synthase (iNOS) and metalloproteinases (MMPs), which perpetuate inflammatory processes^8,9.

The dextran sodium sulfate (DSS)-induced model in rats is a widely used chemically induced colitis model due to its morphological similarities to human UC^10,11. DSS damages epithelial integrity and increases colonic mucosal permeability¹².

Several types of clinical treatments are used to control IBDs¹³. These drugs reduce inflammatory processes by attenuating the expression of some pro-inflammatory molecules^14-16. However, the side effects of these drugs severely disrupt the quality of the patient’s life, particularly during long-term treatment¹⁷. Therefore, the discovery of new therapies for IBDs is highly relevant.

Chondroitin sulfate (CS) is a glycosaminoglycan composed of disaccharide units formed by (1→3)-β-N-acetyl-d-galactosamine (GalNAc) and (1→4)-β-d-glucuronic acid (GlcA), which may be sulfated at the C4 and/or C6 positions of GalNAc and/or C2 of GlcA or not sulfated^18,19. CS are hybrid polymers composed of more than one type of disaccharide unit, and this heterogeneity depends on the tissue source. The size, disaccharide unit compositions, and sulfation patterns may vary in CS from cartilage with the animal species, age, position within the tissue and disease state²⁰. Chondroitin sulfate exhibits various pharmacological activities, such as anti-oxidation²¹ and anticoagulation²².

Another compound used in combination with CS, glucosamine (GlcN), is an amino monosaccharide precursor molecule in the synthesis of glycosaminoglycans, glycolipids, and glycosylated proteins and one of the most abundant sugars in the body²³. GlcN reduced NF-kB activity²⁴ and prevented an increase in nitric oxide production and the expression of metalloproteinases in cultured horse cartilage fragments and a model of osteoarthritis using human chondrocytes²⁵.

CS and GlcN show anti-inflammatory activity and are recommended by EULAR (European League Against Rheumatism) for the treatment of osteoarthritis based on a meta-analysis of several clinical studies²⁶. Recent evidence suggests that CS and GlcN have a beneficial effect on the risk of colorectal cancer^27-29.

Therefore, the current study investigated the role of CS-and GlcN as an anti-inflammatory and protective treatment in a DSS-induced colitis model in rats.

METHODS

Materials and reagents

Chondroitin sulfate and glucosamine were obtained from Artroliveâ, Aché. Chondroitin 4-sulfate (from bovine trachea), chondroitin 6-sulfate (from shark cartilage), dermatan sulfate (from porcine mucosa), hexadecyltrimethylammonium bromide, O-dianisidine dihydrochloride, sulfanilamide, N-[1-naphthyl]-etilenodiamine, sodium nitrite, 2-aminoacridone (AMAC), sodium cyanoborohydreto, glycine and gelatin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Heparan sulfates (from bovine pancreas) and chondroitin sulfate lyases AC (from Flavobacterium heparinum) were prepared using previously described methods^30,31. Agarose was purchased from Bio-Rad Laboratories (Richmond, CA, USA). Dextran sulfate sodium (DSS- MW: 36-50 kDa) was purchased from MP Biomedicals (Solon, OH, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), streptomycin and penicillin were acquired from Gibco Life Technologies (Waltham, MA, USA). All other chemicals and solvents used in the experiments were of analytical reagent grade.

Analysis of chondroitin sulfate and glucosamine

Characterization by ¹³C solid state nuclear magnetic resonance (SSNMR)

Solid-state ¹³C NMR experiments were performed in a Bruker Avance III HD 300 (7.04 T) spectrometer operated at a Larmor frequency of 75.00 MHz. The analyses were performed using a MAS probe and ZrO2 rotors (and caps of Kel-F) of 4 mm. The spectra were obtained at a rotation frequency of 10000 Hz, with a relaxation time of 3.5 s and a 90° pulse of 2.0 μs using magical spin-angle and cross-polarization. The chemical shifts were indirectly standardized to a glycine sample, with a carbonyl sign at 176.00 ppm in relation to TMS, which was the primary standard.

Agarose gel electrophoresis

Aliquots of the CS/GlcN sample and the C4S and C6S commercial standards (5 µl containing 5 µg) were subjected to agarose gel electrophoresis in 0.05 M 1,3-diaminopropane-acetate buffer, pH 9 (PDA)^32,33. An aliquot of 5 µL of each sample was subjected to electrophoresis for 1 h at 100 V. GAGs were precipitated in the gel using 0.1% CETAVLON (cetyltrimethylammonium bromide) for 2 h at room temperature. The gel was dried and stained with a 0.1% solution of toluidine blue in acetic acid-ethanol-water (0.1:5:4.9, vol/vol). Quantification was performed using densitometry at 530 nm (QuickScan 2000 WIN version 1.14/00, Copyright 2001, Helena Laboratories) of the toluidine blue-stained electrophoretic slide. After fixation with cetyltrimethylammonium bromide and toluidine blue staining, the glycosaminoglycans were quantified using densitometry of the gel slabs¹⁸.

Fluorophore-assisted carbohydrate electrophoresis

CS was further characterized using enzymatic degradation with chondroitin AC lyase (EC 4.2.2.5) from Flavobacterium heparinum, which was prepared and incubated as previously described^34,35. Briefly, aliquots of CS (50-100 µg) were incubated with 2 x 10^-4 units of chondroitin AC lyase in 0.05 M ethylenediamine-acetate (EDA) buffer (pH 8.0) in a final volume of 20 µl. After 6 h of incubation at 37°C, aliquots of the incubation mixtures were subjected to FACE¹⁸, as previously described.

CS molecular weight determination

The molecular weight of the CS was determined using polyacrylamide gel electrophoresis in a Bio-Rad Mini-Protean System. Samples (10 µg) were mixed with sample buffer, applied to 7.5% gels, and run for 1 h at 13 V/cm. Gels were stained with toluidine blue, as previously described³¹. Molecular weight evaluation was performed via the densitometric acquisition of bands and comparison of their migration times on the calibration curve, which was constructed by plotting the retention times of standards against their logarithm of molecular weight values.

Cell culture

IEC-6 and Caco-2 cell lines were obtained from the Cell Bank of Rio de Janeiro (BCRJ-UFRJ) and cultured at 37°C under 5% CO₂ in DMEM supplemented with antibiotics (100 U/mL penicillin and 100 μg/ml streptomycin) and 10% heat-inactivated fetal bovine serum.

Cell viability assay

The effects of CS/GlcN on the number of viable IEC-6 and Caco-2 cells was assessed using the MTT assay as described previously (Mosmann & Methods, 1983). The cells were detached from culture plates with 5 mM EDTA in EBSS. Detached cells were suspended in DMEM and seeded in 96-well plates (1 x 10⁴ cells/well, 200 mL). After 24 h, the culture medium was replaced with fresh DMEM containing CS/GlcN (1-1000 mg/mL). After 48 h, a stock solution of MTT was prepared in phosphate-buffered saline (PBS), diluted in DMEM, and added to cell-containing wells at a concentration of 0.5 mg/mL after removal of the culture medium. The plates were incubated for 4 h at 37 °C under 5% CO₂. The reduction of MTT to formazan was assessed using an enzyme-linked immunosorbent assay plate reader. The medium was collected, the formazan was solubilized in 200 mL of dimethyl sulfoxide (DMSO), and the OD 540 was measured³⁶.

Animal experiment

Male Wistar rats (6-8 weeks old) were obtained from the Biological Center of Reproduction (BCR), Federal University of Juiz de Fora (UFJF, Juiz de Fora, MG, Brazil) for the induction of colitis using DSS (MW: 36-50 kDa). Animals were housed in the Glycoconjugates Analysis Laboratory of the Department of Biochemistry, Institute of Biological Sciences. During the entire experimental period, animals were housed in individual plastic boxes under controlled environmental conditions (temperature of 23±1˚C; 50% humidity; 12:12 h light:dark cycles) and fed standard laboratory food and water ad libitum. The procedures performed in animals are consistent with the recommendations of the Ethics Committee of Animal UFJF (002/2010-CEA UFJF) and the Guide for the care and use of laboratory animals.

Induction of acute colitis using DSS

Colitis was induced as described by Okayasu et al.³⁷ with some modifications. Rats were randomized into four groups: control group (n=16); control + CS/GlcN group (n=10); DSS group (n=10); and DSS + CS/GlcN group (n=10). For colitis induction (DSS and DSS + CS/GlcN groups), rats were given drinking water containing 5% (w/v) DSS for 7 days. The CS/GlcN daily intake was approximately 40 mL, which corresponded to 125 mg/day of CS and 100 mg/day of GlcN. On the seventh day, the animals were sacrificed using sodium pentobarbital (100 mg/kg), and colons and blood were collected for biochemical and histological studies.

Disease activity index (DAI)

DSS-induced colitis was assessed using the disease activity index (DAI) described by Cooper et al. (Cooper, Murthy, Shah, & Sedergran, 1993): no weight loss was 0 points; weight loss of 1-5%was 1 point; weight loss of 5-10% was 2 points; weight loss of 10-15% was 3 points; and weight loss greater than 15% was 4 points. Stool was characterized as normal (0 points), loose (1 point), very soft (2 points), and diarrheal (4 points). No bleeding was scored 0 points, visible blood in the stool received 2 points, and rectal bleeding was 4 points. The following parameters were considered: weight loss, stool consistency, and bleeding. The scores were added to obtain a DAI that ranged from 0 (healthy) to 12 (the maximal activity of colitis).

Biochemical and hematological analysis

Blood and plasma samples were collected as specified. At the end of the experiment, the animals were anesthetized, blood was drawn via cardiac puncture, and euthanasia was applied.

Red and white blood cells, hematocrit and hemoglobin were analyzed using an automatic cell counter (veterinary hematology analyzer Poch-100i V Diff, Sysmex). Serum analysis was performed using commercial kits, and albumin was measured using Labtest Diagnostic SA (Labmax Progress).

Histology

The distal colon was harvested and dissected between the ileocaecal valve and rectum, and the length and weight were measured. Feces were carefully removed from the intestinal tract lumen, and the tissues were rinsed with saline. The samples were fixed in 10% paraformaldehyde, pH 7.0, embedded in paraffin using a “Swiss roll” technique, and sectioned (5 μm) onto glass slides. After deparaffinization in xylene, the samples were rehydrated in ethanol washes (100%, 90%, 80%, 70%, and 60%), and the sections were stained with hematoxylin and eosin and Alcian blue pH 2.5.

The slides were examined, and histological scoring was performed as described previously³⁸. Briefly, tissue damage was categorized into four grades: grade 0, normal colonic mucosa; grade 1, loss of 1/3 of the crypts; grade 2, loss of 2/3 of the crypts; grade 3, the lamina propria covered with a single layer of epithelium, and mild inflammatory cell infiltration was present; and grade 4, erosions and marked inflammatory cell infiltration were present.

Myeloperoxidase (MPO) activity

Neutrophil infiltration into the colon was quantified by measuring MPO activity as previously described³⁹. Briefly, a portion of the colon was homogenized in 5 mL of 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide in an ice bath. The homogenate was sonicated for 10 s, subjected to three freeze-thaw cycles, and centrifuged at 12000 g and 4°C for 15 min. O-dianisidine dihydrochloride and hydrogen peroxide were added to the supernatant. Changes in absorbance (λ=460 nm) were measured (Thermo Scientific^® - MULTISKAN GO, Waltham, MA, USA). MPO activity was calculated using the molar extinction coefficient (ε=11.48 mM^-1.cm^-1)⁴⁰.

NO quantification

NO was measured as nitrite (NO₂ ^-) accumulation in the colon. A spectrophotometer assay based on the Griess reaction was used. Briefly, Griess reagent (1% sulfanilamide, 0.1% N-[1-naphthyl]-etilenodiamine in H₃PO₄) was added to an equal volume of sample, and the absorbance was measured at 550 nm (Thermo Scientific^® - MULTISKAN GO, Waltham, MA, USA). The NO concentration (µg of NO/mg tissue) was determined from a standard curve of NaNO₂⁴¹.

Metalloproteinase activity assay

The activity of MMP-2 and MMP-9 was measured as previously described in homogenates of colonic tissues^42,43. Colonic samples were homogenized and mixed with electrophoresis loading buffer. The samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with gelatin (2 mg/ml). After electrophoresis, the gels were washed with 2% Triton X-100 for 1 h (3 times, 20 min each) and incubated for 24 h in enzyme assay buffer (50 mM Tris-HCl, pH 8.2, 5 mM CaCl₂ and 1 µM ZnCl₂) for the development of enzyme activity bands. The gels were fixed and stained in 40% methanol, 10% acetic acid, and 0.1% (w/v) Coomassie Blue for 1 h then destained. The gelatinolytic activities were detected as transparent bands against the background of Coomassie brilliant blue-stained gelatin^44,45. MMPs were identified by their molecular weight compared with standards. The intensities of the bands were measured using TotalLab Quant^® (England, UK). The activity values were corrected for protein content.

Statistical analysis

Statistical analyses were performed using GraphPad Prism, version 19. All values are expressed as the means ± SEMs. One-way ANOVA was used to analyze significant differences between groups. P<0.05 was considered statistically significant.

Ethical approval

All procedures performed in studies involving animals were performed in accordance with the ethical standards of the institution where the studies were performed.

RESULTS

Analytical data of CS/GlcN

The CS contents of the preparation were assessed using metachromatic staining with toluidine blue in agarose gel. Commercial C4S and commercial chondroitin 6-sulfate (C6S) were used as standards, and the results indicated that the CS in the sample was C4S (Figure 1B) because the sample migrated as a single band near standard C4S. The molecular weights of CS were assessed using polyacrylamide gel electrophoresis (Figure 1C). The modal molecular weight was 30.4 kDa. Figure 1A shows the ¹³C NMR spectra for the CS/GlcN association, and the spectra were very similar to the standard. This result indicates the absence of atypical structures and significant contaminants.

FIGURE 1
¹³C NMR spectra of CS and GlcN (A), agarose (B) and polyacrylamide (C) gel electrophoresis.

These compounds were subjected to chondroitin AC lyase from F. heparinum, and the products formed were analyzed using FACE (Figure 2A). FACE showed that CS exhibited a disaccharide composition very similar to the C4S standard (Figure 2B).

FIGURE 2
Fluorophore-assisted carbohydrate electrophoresis (FACE) of CS degradation products.

Assessment of disease activity index (DAI) in DSS-induced colitis

Administration of 5% DSS induced rectal bleeding, diarrhea and weight loss in all animals (DSS and DSS+CS/GlcN groups). Significant changes were observed in clinical signs (DAI) from the third to the seventh day of DSS induction (Figure 3).

Although colitis reached higher levels, CS/GlcN attenuated the DAI score on the third, fifth and seventh days compared to the induced group (P<0.05, Figure 3A). Reduction in weight loss (sixth and seventh days, P<0.05), prevention of rectal bleeding onset and minor changes in stool consistency were also noted (Figure 3C-1D). Macroscopically, a reduction in colon length shortening in the treated group was observed (Figure 3F). No significant difference in the length/weight ratio of CS/GlcN treatment was observed (Figure 3E).

FIGURE 3
Effect of CS/GlcN administration on the disease activity index (DAI) in a model of DSS-induced colitis.

Alterations in clinical signs and macroscopic changes were not noted in the control or control+CS/GlcN groups.

Effect of CS/GlcN administration on hematological and biochemical parameters in DSS-induced colitis

Hematological parameters were analyzed as an indicator of rectal bleeding in the DSS experiments. Administration of CS/GlcN attenuates hematological parameter changes (red blood cell count, hemoglobin concentration and hematocrit levels). Analysis of serum albumin concentration was reduced in both induced groups (Table 1).

TABLE 1
Hematological parameters and serum albumin in animals.

	Red blood cells (x 106/mm3)	Hematocrit (%)	Hemoglobin (g/dL)	Albumin (g/dL)
Control	8.88±0.53	47.9±3.2	17.7±1.1	2.9±0.7
Control-CS/GlcN	8.80±0.50	46.8±2.0	17.3±0.7	2.9±0.7
DSS	6.74±1.42	39.2±6.5	13.1±2.5	2.3±0.4
DSS-CS/GlcN	8.35±1.58*	45.9±8.8*	16.68±3.4*	2.3±0.2

• DSS: dextran sulfate sodium. Data are presented as the means ± standard error. *P<0.05 compared to the DSS group.

Histopathological changes and myeloperoxidase (MPO) activity

Histopathological analysis of the colon in the DSS group showed multifocal areas of mucosal erosions with epithelial cell loss, goblet cell depletion, crypt loss, inflammatory cell infiltration, edema and vascular congestion (Figure 4A). Although some histological alterations were present, CS/GlcN significantly reduced these changes and showed a lower shortening of crypts and just a few areas of erosions and ulcerations (Figure 4A). This attenuation was also reflected in the histological score analysis (Figure 4B, P<0.001).

FIGURE 4
Histopathological analysis of the administration of CS/GlcN in DSS-induced colitis.

A significant reduction in the goblet cell number and mucin was noted in the DSS group (Figure 4A). Maintenance/regeneration of the crypts and preservation of the goblet cells were observed in the DSS+CS/GlcN group.

MPO activity was increased in DSS-induced colitis (Figure 4C), and treatment with CS/GlcN reduced this activity (P<0.01) and showed a reduction in neutrophil infiltration in the inflamed area. These results are also consistent with the observed histopathological changes.

Nitric oxide (NO) tissue levels

NO levels were reduced in the colon of animals in the DSS+CS/GlcN group compared to the DSS group (P<0.05). CS/GlcN treatment had no effect on the NO levels in the control group (Figure 5).

FIGURE 5
Administration of CS/GlcN reduces NO levels in the colon in DSS-induced colitis.

Metalloproteinase activity assay

Zymogram analysis showed an increase in MMP-2 and MMP-9 activities in the DSS group. Treatment with CS/GlcN induced a significant reduction in MMP-9 activity (P<0.05), but a slight decrease in MMP-2 activity was also observed (Figure 6A-B).

FIGURE 6
CS/GlcN reduces MMP-9 activity, but not MMP-2, in DSS-induced experimental colitis.

In vitro effects of CS/GlcN in human intestinal epithelial cells

An MTT assay determined that CS/GlcN had no significant cytotoxic effect at concentrations of up to 750 μg/mL in IEC-6 and 100 μg/mL Caco-2 cells (Figure 7).

FIGURE 7
Cytotoxic effect of CS/GlcN in IEC-6 and Caco-2 cells.

DISCUSSION

DSS-induced colitis is a well-established model of intestinal inflammation with histological and biochemical features that mimic human disease^46. We evaluated the composition of CS/GlcN and the therapeutic effects using this model.

CS/GlcN are also used as chondroprotective drugs, with application in the therapy of osteoarthritis of the knee and the articular cartilage. Due to the large heterogeneity of various CSs, an accurate evaluation of their structures and properties, molecular mass, charge density and positions of sulfate groups are of paramount importance primarily for pharmacological applications. The present study showed that, the CS disaccharide composition was very similar to C4S, with a modal molecular weight of 30.4 kDa.

Our results showed that CS/GlcN administration reduced the DAI by slowing the progression of colitis and preventing a reduction in hematological parameters. We noted an improvement in the histopathological changes in the colonic mucosa with decreases in the inflammatory process, ulceration and destruction of goblet cells caused by DSS.

Inflammation is a central pathological process in UC, in which the infiltration of leukocytes into the mucosa leads to tissue damage and mucosal dysfunction^47. The combination of hyaluronic acid (HA) and CS reduced neutrophil infiltration and MPO activity in hepatic experimental models⁴⁸. Our study revealed that CS/GlcN also reduced neutrophil infiltration in the colitis mucosa, as reflected by a marked decrease in MPO activity, which is found in cytoplasmic granules of neutrophils that increases tissue damage⁴⁹ (Prokopowicz, Marcinkiewicz, Katz, & Chain, 2012).

Excessive inflammation damages the colon mucosa and affects intestinal homeostasis via a series of pro-inflammatory cytokines, enzymes, and reactive oxygen species. Studies in humans showed that the concentration of NO in rectal IBD patients were significantly higher than healthy patients, and decreased production of this compound using iNOS inhibitors or ablation of this gene showed improvements in rodent experimental colitis models^50,51. Our findings demonstrated that CS/GlcN treatment reduced NO levels in the DSS group. This result is important in the pathological process because the production of at high levels is associated with dysregulation of signaling events and the immune response, which lead to cell and tissue injury⁵².

Among the many factors involved in the inflammatory progression of IBDs, metalloproteinases play important roles. Two isoforms are commonly involved in the inflammatory process. MMP-2 is expressed constitutively and exerts tissue protection to play an important role in maintaining the intestinal barrier, and MMP-9 is absent in most human tissues^53,54.

^MMP-9 is an important mediator of tissue damage, and it is responsible for the accelerated breakdown of the ECM in colitis⁴². This enzyme is overexpressed in patients with colitis compared to healthy patients^55. The development of a colitis inflammatory response was attenuated in MMP-9 knockout mice⁵³. We showed that MMP-2 and MMP-9 activities were elevated during experimental colitis, which is similar to the results of Garg et al.⁵³. However, CS/GlcN administration significantly reduced MMP-9 activity without a significant reduction in MMP-2 activity. This result is relevant because some drugs used for the inhibition of MMP-2 and MMP-9 exhibit low specificity and reduce both enzyme activities due to their structural similarity.

In addition to the upregulation of pro-inflammatory mediators, disruption of epithelial barrier function is also a well-known pathophysiology of IBDs⁵⁶. Increasing evidence suggests that intestinal permeability may be crucial for IBDs⁵. The present study observed a DSS-induced increase in the destruction of goblet cells. However, CS/Glcn reduced this destruction. Goblet cells specialize in the secretion of mucus, which covers the surface of the intestinal epithelium. Mucus is composed of proteins, carbohydrates, lipids, and a high degree of water, but it also exhibits antimicrobial properties thanks to antimicrobial peptides, primarily defensins produced by Paneth cells, and cell protection⁵⁷.

Intestinal epithelial cell cytotoxicity is one of the main factors contributing to barrier dysfunction. IECs are central to the maintenance of intestinal homeostasis, and inflammation and barrier dysfunction of intestinal epithelial cells contribute to the pathology of IBDs⁵⁸. CS/GlcNlcN at concentrations up to 100 mg/mL and 750 mg/mL did not affect Caco-2 and IEC-6 cells, respectively, which avoided damage to intestinal cells and barrier dysfunction.

CONCLUSION

In summary, our results suggest a new biological effect of CS/GlcN in a DSS-induced colitis model. Studies on CS and GlcN treatment in colitis have not assessed their effects on MMP activity and IEC cells. Our data suggest that CS/GlcN act as a protective and anti-inflammatory agent in the DSS-induced colitis model because it effectively reduced MPO activity, NO in tissues, and histological score. CS/GlcN also reduced MMP-9 activity, which is an important protease involved in tissue damage, but maintained MMP-2 activity to prevent damage in goblet cells. CS/GlcN did not exert cytotoxicity on the IEC cells.

While further experimental and pre-clinical studies are required to establish the efficacy of CS/GlcN as potential therapeutic agents for the treatment of inflammatory bowel disease (IBD) in humans, we posit that their incorporation as adjuvant in conventional treatment regimens may mitigate side effects and enhance patient quality of life, particularly in the context of long-term treatment.

ACKNOWLEDGMENTS

This research was supported by Universidade Federal by Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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Disclosure of funding:

this work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - Number 479701/2013-0

Declaration of use of artificial intelligence:

none

Corresponding author:

Jair Adriano Kopke Aguiar. E-mail: [e-mail]

Editor associado:

Ricardo Guilherme Viebig.

Declared conflict of interest of all authors:

none

Publication Dates

• Publication in this collection
  20 Dec 2024
• Date of issue
  2024

History

• Received
  09 Aug 2024
• Accepted
  15 Oct 2024