The traditional production method of Dextran involves bacterial fermentation and alcohol precipitation, which is low in efficiency and costly. Herbon International’s proprietary enzymatice synthesis technology can synthesize the required molecular weight Dextran products. This technology effectively avoids the bacterial pollution caused by the traditional bacterial fermentation method, and simplify the production process. No organic solvents are used during the process. We use a nano filtration purification system to remove impurities, which guarantees high-purity products, ensuring our product quality achieves international standards, also provide high-quality substrate raw materials for our derivative products.

Based on our competitive advantage in Dextran products, we can accommodate customisationstailormake Dextran Sulfate Sodium Salt products according to the needs of client’s needs, including molecular weight and other quality requirements.

Dextran Sulfate Sodium Product List

Product Catalog

Molecular Weight

DS-T1

1,000

DS-T3

3,000

DS-T5

5,000

DS-T8

8,000

DS-T10

10,000

DS-T20

20,000

DS-T40

40,000

DS-T500

500,000


Dextran Sulfate Sodium Product Specifications

Items

Requirement

Identity

To pass test

Solubility (10% solution)

Clear to light yellow solution

Loss on Drying

≤ 10%

pH (10% solution)

5.0-7.5

Sulphur

16.0%-20.0%

Free Sulfate

≤ 0.2%

Chloride

≤ 0.1%

Heavy Metals (lead)

≤ 8ppm


Dextran sulfate sodium salt is an anionic derivative of dextran, which has good physical and chemical properties as well as many special biological functions and is an important member within the polysaccharide biotechnology area. In the past, it was widely used in Japan as a therapeutic agent for stomach and duodenal ulcer. In the early 1980s, it was used as a hypolipidemic drug for the prevention and treatment of hyperlipidemia and atherosclerosis. Dextran sulfate sodium salt was also found to have anticoagulant and anti-HIV effects. Reports on it have been widely published and it was widely used in clinical treatments. With further research, researchers began to be aware that the anti-HIV effect of dextran sulfate sodium salt was related to its molecular weight. Therefore, in recent years, more attention was paid to products with low molecular weight. Apart from medical treatment and prevention, dextran sulfate sodium salt also plays an important role in the field of the separation and purification of biological substances, including cell, virus and gene engineering.

Structure, property and function of dextran sulfate sodium salt

1. Molecular structure
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As shown in the figure, each glucose unit in the dextran molecule has three free hydroxyl  groups which are likely to combine with sulfate radical under suitable conditions. So the molecular structure of the dextran sulfate sodium salt will vary with the different molecular weight and sulphur content of the compound. The sulphur content of dextran sulfate sodium salt ranges between 10%-20%.

2. Physical and chemical properties

(1) Water-solubility: Dextran sulfate sodium salt exists in the form of sodium salt and has good water solubility.
(2) Adhesiveness: Dextran sulfate sodium salt solution’s adhesiveness will increase as its molecular weight increases. This characteristic is not related to its sulphur contents.
(3) Good water absorption and water-retaining property
(4) Stability: The powder form of dextran sulfate sodium salt is very stable at room temperature under dry conditions. The liquid form of dextran sulfate sodium salt is relatively stable in neutral pH at room temperature, but may be partly hydrolyzed under acidic and high temperature environment.

3. Biological properties and functions

(1) Heparinoid properties: Similar to heparin, dextran sulfate sodium salt has anticoagulant properties. However, its anticoagulant strength is 1/6 - 1/20 of heparin and the anticoagulant mechanism is also not same. In addition, the anticoagulant property decreases with the decrease of the compound’s sulphur content, and this indicates that dextran sulfate sodium salt is safer and more controllable than heparin.
(2) Polyanion properties: Dextran sulfate sodium salt is a negative ion biopolymer and may directly combine with various substances with the positive charges through a bridging action, in particular, the living substances (for example, protein and lipoprotein, etc.), so as to provide a new approach for clinical diagnosis and treatment.
(3) Targetability: Researchers on anti-HIV drugs found that dextran sulfate sodium salt can selectively combine with cell surface receptors and can competitively inhibit human immunodeficiency virus to enter into the human body’s cells. In addition, reports also indicate that dextran sulfate sodium salt can prevent the combination and fusion of HIV’s outer membrane protein gp120 with CD4 positive cells to prevent HIV from entering into CD4 cells and inhibit the virus’ activity.
(4) Anti-inflammation: Dextran sulfate sodium salt has anti-inflammatory effects and can be used to treat many inflammatory diseases, including skin disease, venereal disease, HIV, tumor and so on.
(5) Biocompatibility: Dextran sulfate sodium salt is compatible with many organic and inorganic substances, including living matter and drugs. For example, AIDS cocktail therapy programmes.
(6) Biological degradability: Dextran sulfate sodium salt can be naturally degraded in both the human body and ecological environment.

Application of dextran sulfate sodium salt

1. Treatment of peptic ulcer

In 1969, patents on the treatment of peptic ulcer using dextran sulfate sodium salt were published. The patents indicated that dextran sulfate sodium salt of 40000Da (from 8,000Da to 2,000,000Da) molecular weight and 13% of sulphur content were effective via ingestion. The therapeutic mechanism might be related to the full inhibition of pepsinogen. This treatment method was very popular in Japan and has no obvious side effects.

2. Reducing blood lipid and anti-atherosclerosis

Dextran sulfate sodium salt has been proven to be effective in reducing serum triglyceride and cholesterol content in both animal experiments and clinical studies. This effect is related to its effects on lipoprotein lipase activity in blood. Lipoprotein LDL and VLDL in blood both causes atherosclerosis. Dextran sulfate sodium salt can combine with those two elements, deceasing their concentration in blood and increasing the concentration of lipoprotein HDL, which has anti- atherosclerosis properties.

3. Anticoagulation

A large number of clinical and experimental researches have indicated that dextran sulfate sodium salt has anticoagulation effect similar to heparinoid, and this effect is closely related to its molecular weight and sulphur contents. In Japan, dextran sulfate sodium salt with 5,000Da molecular weight and 17%-20% of sulphur content had been widely used as anticoagulant and lipid-lowering drugs in clinical treatment. It was noted that dextran sulfate sodium salt possessed different properties from heparin and had great potential as an anticoagulant. The desirable properties are outlined below:
(1) Heparin’s anticoagulant property derives from its ability to activate antithrombase III. Dextran sulfate sodium salt achieves anticoagulation via activating heparin cofactor II, and its effect is more gentle and durable. Hence dextran sulfate sodium salt is suitable for hyperlipidemia, cardiovascular and cerebrovascular diseases patients with hypercoagulable conditions.
(2) Heparin is extracted from animals. It is a biological agent with antigenicity and can be easily polluted during the extraction process. Thus, heparin easily causes anaphylactic reactions. On the other hand, dextran sulfate sodium salt is not of animal origin and is synthesized, purified under strictly controlled conditions, thus has a lower risk for anaphylactic reactions.
(3) Heparin may only be used in intravenous injection, but dextran sulfate sodium salt may be taken orally.
(4) The extraction process of heparin is complex, its molecular weight distribution is not uniform, and its sulphur content is not controllable. However, dextran sulfate sodium salt’s molecular weight and sulphur content can be manipulated via technology. Products with ultralow molecular weight, for example, maltooligosaccharide (or isomaltooligosaccharide) sulphate, will certainly become strong performers in the area of anticoagulation and antithrombus.

4.Medical diagnosis

The polyanion properties of dextran sulfate sodium salt is important to the separation of lipoprotein. For example, in the presence of divalent cation, dextran sulfate sodium salt can deposit lipoprotein within blood serum which causes atherosis, including chylomicron emulsion, low density lipoprotein (LDL) and very low density lipoprotein (VLDL), so as to separate out anti- atherosis lipoprotein (HDL) and measure high-quality cholesterol content.

5. Anti-AIDS

A great number of studies have indicated that dextran sulfate sodium salt has anti-virus properties. The reports revealed that dextran sulfate sodium salt has a significant inhibiting effect on herpes simplex virus, human cytomegalovirus, herpetic stomatitis virus and human immunodeficiency virus (HIV). In vitro experiments showed that dextran sulfate sodium salt can fully protect T-lymphocytes when they were exposed to HIV. Since dextran sulfate sodium salt has been widely tested in clinical treatments for more than 20 years and has no significant toxic and side effect, Japan and the United States introduced dextran sulfate sodium salt related treatment to AIDS patients since the 1980s.

6. Cosmetics

Dextran sulfate sodium salt possesses many physicochemical properties and physiological functions that are attractive in the field of cosmetics. It has good hygroscopic properties and can help moisture penetrate into tissues, which is critical to maintaining the integrity and mechanical fuctioning of cells and protecting cells from damages induced by free radicals. Studies also showed that dextran sulfate sodium salt is superior than acid mucopolysaccharide, heparin and unsulfonated dextran in terms of maintaining osmotic pressure of colloid. As for skin care, experts considered that dextran sulfate sodium salt can keep skin smooth, soft and bright, with no sticky feeling. More importantly, dextran sulfate sodium salt also has anti-virus, anti-inflammation, antianaphylaxis, anti-wrinkling and anti-aging effects as well as can prevent and treat chapped skin. In a pharmaceutical assessment of the skin preparations containing dextran sulfate sodium salt and betamethasone, experts found that the anti-inflammation effects are far greater when the two drugs are applied in combination than when they are used seperately. It is also known that the combination of dextran sulfate sodium salt with saponin may treat skin redness and allergy, and is particularly effective for redness, pouchiness and dark circles for the eyes area.

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Clinicopathologic study of dextran sulfate sodium experimental murine colitis


We undertook this study in order to fully characterize the clinical and histopathology features of the dextran sulfate sodium (DSS) model of experimental murine colitis and to discover the earliest histopathologic changes that lead to colitis. EXPERIMENTAL DESIGN: Acute colitis was induced in Swiss-Webster mice by 7 days of oral dextran sulfate sodium with animals sacrificed daily. Chronic colitis was induced by: (a) 7 days of oral dextran sulfate sodium followed by 7 days of H2O (for 1, 2, and 3 cycles) and (b) 7 days of oral dextran sulfate sodium followed by 14 and 21 days of H2O. In each experimental group, the entire colons were examined histologically and correlated with clinical symptoms. RESULTS: Acute clinical symptoms (diarrhea and/or grossly bloody stool) were associated with the presence of erosions and inflammation. More importantly, the earliest histologic changes which predated clinical colitis was loss of the basal one-third of the crypt (day 3), which progressed with time to loss of the entire crypt resulting in erosions on day 5. The earliest changes were very focal and not associated with inflammation. Inflammation was a secondary phenomena and only became significant after erosions appeared. Animals treated with only 7 days of dextran sulfate sodium followed by 14 and 21 days of H2O developed a chronic colitis with the following histologic features: areas of activity (erosions and inflammation), inactivity, crypt distortion, florid epithelial proliferation and possible dysplasia. These changes were similar to animals given 3 cycles of dextran sulfate sodium. The clinical disease activity index correlated significantly with pathologic changes in both the acute and chronic phases of the disease. CONCLUSIONS: The mechanism of dextran sulfate sodium colitis is presently unknown. However, the finding of crypt loss without proceeding or accompanying inflammation suggests that the initial insult is at the level of the epithelial cell with inflammation being a secondary phenomena. This may be a good model to study how early mucosal changes lead to inflammation and the biology of the colonic enterocyte. Chronic colitis induced after only 7 days of dextran sulfate sodium may serve as a useful model to study the effects of pharmacologic agents in human inflammatory disease and mechanisms of perpetuation of inflammation. Finally, we believe that this model has the potential to study the dysplasia cancer sequence in inflammatory disease.

Dextran sulfate sodium

Low molecular mass polypeptide 2 (LMP2) is an inducible proteasome subunit. Our goals were to examine LMP2 expression in mice with dextran sulfate sodium (DSS)-induced colitis and to evaluate colitis in LMP2 knockout (LMP2-/-) mice. Mice were given 2.5% dextran sulfate sodium in the drinking water. On day 0, 2, 4, or 6 after dextran sulfate sodium treatment, LMP2 expression was determined in the distal colon by western blot and immunohistochemistry. Parameters of colitis were measured in LMP2-/- mice or wild-type mice. LMP2 expression was enhanced in the colon of DSS-treated mice at all time points. Symptoms of DSS-induced colitis were always lower in LMP2-/- mice. Normalized histology scores and colonic IL-1ss levels increased over the 6-day study period in wild-type mice. These parameters were significantly reduced in LMP2-/- mice that consumed dextran sulfate sodium for 6 days. Enhanced LMP2 expression contributes to the pathogenesis of dextran sulfate sodium-induced colitis in mice.

Dextran sulfate sodium-induced colitis occurs in severe combined immunodeficient mice

Oral administration of dextran sulfate sodium (DSS) has been reported to induce colitis in mice. The purpose of this study was to determine whether the possible pathogenic mechanism involved the acquired immune system. METHODS: Normal BALB/c and related C.B17 severe combined immunodeficient mice were fed 5% dextran sulfate sodium (40 kilodaltons) in their drinking water for 7 days; controls were fed only water. Colons were scored for histological activity at various times. Cytokine production by cultures of colon and of draining lymph node cell was measured. The effect of dextran sulfate sodium on the proliferation of the MCA-38 colonic epithelial cell line was assessed. RESULTS: dextran sulfate sodium feeding resulted in a very reproducible acute distal colitis in both BALB/c and C.B17 severe combined immunodeficient mice. The lesions of BALB/c mice had an increased production of macrophage-derived cytokines, such as interleukin (IL) 1 beta, IL-6, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor, but not the T-cell cytokines IL-3 or interferon gamma. Draining lymph node cells produced these cytokines plus interferon gamma and IL-3. dextran sulfate sodium inhibited MCA-38 cells at doses that would be easily achieved in the distal colon. CONCLUSIONS: Acute DSS-induced colitis does not require the presence of T cells or B cells because it occurred in C.B17 severe combined immunodeficient mice that lack these cells. Its induction may result from a toxicity of dextran sulfate sodium for colonic epithelial cells.

Neutralization of interleukin-17 aggravates dextran sulfate sodium-induced colitis in mice

We evaluated the effects of rat anti-mouse IL-17 neutralizing monoclonal antibody (mAb) on the development of dextran sulfate sodium (DSS)-induced colitis. Tissue samples were evaluated by standard immunohistochemical procedure. The mucosal mRNA expression of cytokines was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). In the mice treated with the anti-IL-17 mAb, the body weight was significantly lower, and anal prolapse and colon shortening were apparent. A histological analysis indicated that the anti-IL-17 mAb markedly enhanced the severity of colitis. The mucosal infiltration of CD4-positive helper T cells and CD11b-positive granulocytes-monocytes was increased in the anti-IL-17 mAb-treated mice. Treatment with the anti-IL-17 mAb increased the mucosal expression of mRNAs of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, IL-6, RANTES, and IP-10. Blocking of IL-17 activity in vivo using the anti-IL-17 mAb enhanced the development of dextran sulfate sodium-colitis in mice. This suggests an inhibitory role for IL-17 in the development of dextran sulfate sodium-colitis.

Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin

The use of oral and intravenous cyclosporin represents a significant advance in the therapy of refractory inflammatory bowel diseases (IBD). However, oral administration of cyclosporin is fraught with improper delivery of cyclosporin to the colon for its topical action. Because of unpredictable metabolism by cytochrome P-450 IIIA, the targeted blood level for systemic effect is not reached at low doses. Furthermore, the doses that have been used for therapy of IBD have been shown to induce several adverse side effects. Thus, an alternate method of delivering cyclosporin to the colon is desirable. In this study, the effect of intracolonically administered cyclosporin was tested for its efficacy to heal mucosal erosions in dextran sulfate sodium (DSS)-induced colitis in mice. Both acute and chronic colitis was induced by feeding female Swiss-Webster mice with 5% dextran sulfate sodium for five or seven days, respectively. Therapy was advocated prophylactically, prophylaxis plus therapy and therapeutically during the acute and chronic phase of the disease and therapeutically during the chronic phase of the disease. Intracolonic cyclosporin given prophylactically showed adverse effects by increasing the damage to the colonic mucosa. However, intracolonic cyclosporin given therapeutically in 2.5, 5, and 10 mg/kg after the induction of colitis resulted in dramatic responses in terms of reducing the disease activity and histologic scores, corroborated by complete histological resolution compared to oral cyclosporin given at identical doses. Intracolonic cyclosporin (5 mg/kg) was also very effective in reducing the chronic inflammation. The results of this study highlight the application of this animal model for therapeutic research. Furthermore, cyclosporin administered as an enema provides a new stratagem for the therapy of IBD because of its rapid onset of action at very low doses without the risk inherent in oral or systemic administration.

Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines

Oral administration of dextran sulfate sodium has been reported to induce an acute and chronic colitis in mice. The aim of our study was to evaluate if the chronic phase of dextran sulfate sodium-induced colitis was characterized by a Th1/Th2 response and how this would relate to mucosal regeneration. Swiss Webster mice were fed 5% dextran sulfate sodium in their drinking water for 7 days, followed by 2-5 weeks consumption of water. Control mice received only water. The animals were killed at 3 and 6 weeks after induction. Their colons were isolated for histology and immunohistochemistry, using specific MoAbs for T and B cells, macrophages, interferon-gamma (IFN-gamma), IL-4 and IL-5. Colons were scored for inflammation, damage and regeneration. Two weeks after stopping dextran sulfate sodium the colonic epithelium had only partially healed. Total colitis scores were still increased, especially in the distal colon, which was due to more inflammation, damage and less regeneration. In areas of incomplete colonic healing the basal parts of the lamina propria contained macrophages and CD4+ T cells. These CD4+ T cells showed a focal increase of IFN-gamma and IL-4 staining compared with control animals. These findings were still observed 5 weeks after stopping dextran sulfate sodium in some mice, albeit less extensive. Chronic dextran sulfate sodium-induced colitis is characterized by focal epithelial regeneration and a Th1 as well as Th2 cytokine profile. We postulate that chronic immune activation mediated by both populations of Th cells can interfere with colonic healing and can play a role in the pathogenesis of chronic colitis.
Neutralization of interleukin-17 aggravates dextran sulfate sodium-induced colitis in mice.

We evaluated the effects of rat anti-mouse IL-17 neutralizing monoclonal antibody (mAb) on the development of dextran sulfate sodium (dextran sulphate sodium)-induced colitis. Tissue samples were evaluated by standard immunohistochemical procedure. The mucosal mRNA expression of cytokines was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). In the mice treated with the anti-IL-17 mAb, the body weight was significantly lower, and anal prolapse and colon shortening were apparent. A histological analysis indicated that the anti-IL-17 mAb markedly enhanced the severity of colitis. The mucosal infiltration of CD4-positive helper T cells and CD11b-positive granulocytes-monocytes was increased in the anti-IL-17 mAb-treated mice. Treatment with the anti-IL-17 mAb increased the mucosal expression of mRNAs of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, IL-6, RANTES, and IP-10. Blocking of IL-17 activity in vivo using the anti-IL-17 mAb enhanced the development of dextran sulphate sodium-colitis in mice. This suggests an inhibitory role for IL-17 in the development of dextran sulphate sodium-colitis.

Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin

The use of oral and intravenous cyclosporin represents a significant advance in the therapy of refractory inflammatory bowel diseases (IBD). However, oral administration of cyclosporin is fraught with improper delivery of cyclosporin to the colon for its topical action. Because of unpredictable metabolism by cytochrome P-450 IIIA, the targeted blood level for systemic effect is not reached at low doses. Furthermore, the doses that have been used for therapy of IBD have been shown to induce several adverse side effects. Thus, an alternate method of delivering cyclosporin to the colon is desirable. In this study, the effect of intracolonically administered cyclosporin was tested for its efficacy to heal mucosal erosions in dextran sulfate sodium (DSS)-induced colitis in mice. Both acute and chronic colitis was induced by feeding female Swiss-Webster mice with 5% dextran sulphate sodium for five or seven days, respectively. Therapy was advocated prophylactically, prophylaxis plus therapy and therapeutically during the acute and chronic phase of the disease and therapeutically during the chronic phase of the disease. Intracolonic cyclosporin given prophylactically showed adverse effects by increasing the damage to the colonic mucosa. However, intracolonic cyclosporin given therapeutically in 2.5, 5, and 10 mg/kg after the induction of colitis resulted in dramatic responses in terms of reducing the disease activity and histologic scores, corroborated by complete histological resolution compared to oral cyclosporin given at identical doses. Intracolonic cyclosporin (5 mg/kg) was also very effective in reducing the chronic inflammation. The results of this study highlight the application of this animal model for therapeutic research. Furthermore, cyclosporin administered as an enema provides a new stratagem for the therapy of IBD because of its rapid onset of action at very low doses without the risk inherent in oral or systemic administration.

Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis

Dextran sulfate sodium (DSS)-induced murine colitis represents an experimental model for human inflammatory bowel disease. The aim of this study was to screen various inbred strains of mice for genetically determined differences in susceptibility to dextran sulphate sodium-induced colitis. Mice of strains C3H/HeJ, C3H/HeJBir, C57BL/6J, DBA/2J, NOD/LtJ, NOD/LtSz-Prkdc(scid)/Prkdc(scid), 129/SvPas, NON/LtJ, and NON.NOD-H2g7 were fed 3.5% dextran sulphate sodium in drinking water for 5 days and necropsied 16 days later. Ceca and colons were scored for histological lesions based on severity, ulceration, hyperplasia, and area involved. Image analysis was used to quantitate the proportion of cecum ulcerated. Histological examination revealed significant differences among inbred strains for all parameters scored. In both cecum and colon, C3H/HeJ and a recently selected substrain, C3H/HeJBir, were highly dextran sulphate sodium susceptible. NOD/LtJ, an autoimmune-prone strain, and NOD/LtSz-Prkdc(scid)/Prkdc(scid), a stock with multiple defects in innate and adoptive immunity, were also highly dextran sulphate sodium susceptible. NON/LtJ, a strain closely related to NOD, was quite dextran sulphate sodium resistant. The major histocompatibility (MHC) haplotype of NOD mice (H2g7), a major component of the NOD autoimmune susceptibility, was not crucial in determining dextran sulphate sodium susceptibility, since NON mice congenic for this MHC haplotype retained resistance. C57BL/6J, 129/SvPas, and DBA/2J mice showed various degrees of susceptibility, depending upon the anatomical site. A greater male susceptibility to dextran sulphate sodium-induced colonic but not cecal lesions was observed. In summary, this study demonstrates major differences in genetic susceptibility to dextran sulphate sodium-induced colitis among inbred strains of mice. Knowledge of these strain differences in genetic responsiveness to acute inflammatory stress in the large intestine will permit design of genetic crosses to elucidate the genes involved.

Dextran sulfate sodium (DSS) induced experimental colitis in immunodeficient mice: Effects in CD4 + -cell depleted, athymic and NK-cell depleted SCID mice

Administration of dextran sulfate to mice, given in the drinking water results in acute or subacute colonic inflammation, depending on the administration protocol. This colonic inflammation exhibits ulceration, healing and repair, and a therapeutic response that makes it valuable for the study of mechanisms that could act in the pathogenesis of human ulcerative colitis, a disease thought to have an immunologically dependent pathogenesis. To investigate if immunological mechanisms were involved in the induction of colonic inflammation in this model, mice with different degrees of immunodeficiency were used. It was shown that dextran sulfate induced colitis could be induced in Balb/c mice depleted of CD4 + helper T cells by treatment with monoclonal antibodies preceded by adult thymectomy. The depletion of CD4 + was verified by flow cytometric analysis. Furthermore, the colonic inflammation could equally be induced in athymic CD-1 nu/nu mice lacking thymusderived T cells, in T and B-cell deficient SCID mice, and also in SCID mice depleted of NK cells by treatment with anti-asialo GM1 antibodies. The NK-cell depletion was verified by measuring spleen NK-cell activity. The resulting colonic inflammation in all these types of deficient mice was qualitatively comparable, as shown by clinical and histological appearance. These results indicate that the presence of functional T, B and NK cells is not crucial for the induction of dextran sulfate colitis in mice.

An ICAM-1 antisense oligonucleotide prevents and reverses dextran sulfate sodium-induced colitis in mice

Mice treated p.o. with 5% dextran sodium sulfate develop a mild to moderate colitis characterized by focal areas of inflammation and crypt abscesses. Immunohistological analysis of colons from dextran sodium sulfate-treated mice revealed an increased expression of intercellular adhesion molecule 1 (ICAM-1) and infiltration of lymphocyte function antigen 1-positive cells. A murine-specific antisense oligonucleotide, ISIS 3082, was used to determine the role of ICAM-1 expression in the development of colitis. Prophylactic treatment of dextran sodium sulfate-treated mice with ISIS 3082 reduced the clinical signs of colitis in a dose-dependent manner, with maximal effects occurring at a dose of 1 mg/kg/day. Reductions in ICAM-1 immunostaining and infiltrating leukocytes were observed in colons of animals treated with 1 mg/kg ISIS 3082. Scrambled control oligonucleotides failed to modify the course of the disease. The ICAM-1 oligonucleotide also diminished the clinical severity of colitis in mice with established colitis. The toxicity of ISIS 3082 was assessed in normal CD-1 mice by administering the oligonucleotide intravenously every other day for 2 weeks. At pharmacologically relevant doses of ISIS 3082 (1 and 10 mg/kg), there were no signs of toxicity with respect to body and organ weights, clinical chemistry or hematology. At a dose of oligonucleotide 20- to 100-fold greater than maximal pharmacological doses, the oligonucleotide produced an increase in liver and spleen weights; a mild chronic inflammation in liver, lung and lymph nodes; monocytosis and an elevation of serum liver transaminases. These data suggest that an antisense oligonucleotide that reduces ICAM-1 expression could be effective in the therapy of inflammatory bowel disease in humans and that such an oligonucleotide would be safe at pharmacologically relevant doses.

Dextran Sulfate Sodium (DSS) Colitis in Rats (Clinical, Structural, and Ultrastructural Aspects)

Aim of this study was to assess the structural,ultrastructural, immunohistochemical, and clinicalaspects in Sprague-Dawley rats with dextrane sulfate sodium (DSS)-induced colitis. Colitis was induced in Sprague-Dawley rats by seven days of Dextran sulfate sodium oral administration followed by seven days of tap wateronly (for one, two and three cycles). Controls were fedwith water only. Segments of proximal, mid-, and distal colon of each animal were adequatelyprepared for light and scanning electron microscopeobservations. The severity of the lesions was scoredhistologically. For immunohistochemical study, acocktail of S-100, NSE, and antineurofilament antibodieswas used. Symptoms such as weight, feces consistency,diarrhea, hematochezia were recorded daily. From aclinical point of view symptoms appeared significantly later after the first cycle than after thesecond and third cycles and lasted significantly longerin the second and third cycles. Treated rats showed aslower weight gain rate by 20% compared to controls, and the whole colon length appeared to besignificantly shorter after colitis induction comparedto controls. Structural observations by light microscopyshowed prominent involvement of the distal colon. Immunohistochemical study of both submucosaland myoenteric nerve plexuses was similar to controls.Scanning electron microscope observations of the colonicmucosal surface in colitis rats showed a complete subversion of its architecture, characterizedby dilatations of gland crypt openings, dropout ofgoblet cells, and inhomogeneous distribution or lack ofmicrovilli. These were most evident after the third cycle. In conclusion, experimental Dextran sulfate sodium colitisin SD rats appeared to be highly reproducible and sharedmost features with human UC, not only from a structuraland clinical but also from an ultrastructural point of view.

Loss of the tight junction protein ZO-1 in dextran sulfate sodium induced colitis

BACKGROUND:
Inflammatory bowel disease (IBD) is associated with increased intestinal permeability and decreased expression of tight junction (TJ) proteins in the inflamed mucosa. Whether this alteration in TJ expression is a prerequisite for the development of intestinal inflammation or a secondary result of that inflammation is unknown. This study looked at the expression of the TJ protein ZO-1 and the corresponding permeability changes in dextran sulfate sodium (DSS) induced colitis in a mouse model.
MATERIALS AND METHODS:
BALB/c mice were fed 3% Dextran sulfate sodium or water for 1, 3, 5, or 7 days. The animals were weighed, stool was checked for blood, and the colon length measured. Segments of the colon were used for histology, immunohistochemistry for ZO-1, or Western blot for TJ proteins. Colonic permeability was measured using Evan's Blue dye.
RESULTS:
Dextran sulfate sodium treated animals had heme positive stools, colitis by histology, significant weight loss, and colon shortening. There was an absence of ZO-1 by Western blot in the 7-day Dextran sulfate sodium treated animals, double the amount of claudin-1 and normal cytokeratin. The loss of ZO-1 started after 1 d of Dextran sulfate sodium treatment and was followed by a significant increase in permeability to Evan's blue by day 3.
CONCLUSIONS:
The loss of ZO-1 and increased permeability preceded the development of significant intestinal inflammation suggesting that in Dextran sulfate sodium colitis alterations in the TJ complex occur before the intestinal inflammation and not as a consequence of it. These changes in the TJ complex may facilitate the development of the inflammatory infiltrate seen in colitis.

Dysplasia and carcinoma development in a repeated dextran sulfate sodium-induced colitis model

As an important mechanism underlying the increased risk of colorectal carcinoma development in patients with long-standing ulcerative colitis, promotion as a result of the regenerative process has been proposed. In the present study, a dysplasia-carcinoma sequence in a novel repeated colitis model in mice is documented.Repeated colitis was induced by nine administration cycles of 3% dextran sulfate sodium (DSS; molecular weight, 54 000): each administration cycle comprised 3% Dextran sulfate sodium for 7 days followed by distilled water for the subsequent 14 days, to give conditions similar to the clinically observed active and remission phases in humans.Multiple colorectal tumors (nine low- and four high-grade dysplasias and two carcinomas) developed in 25 mice. These neoplastic lesions consisted of tubular structures, presenting as various types of elevated, flat and depressed tumor, similar to those in ulcerative colitis patients. A time-course study with assessment of the severity of colitis and in vivo bromodeoxyuridine uptake during a single 3% Dextran sulfate sodium administration cycle revealed a high level of regenerative activity in the colitis-affected mucosal epithelia.Thus, with the present repeated colitis model, regeneration and neoplastic lesions were apparent, the biological features of which provide evidence of a colorectal dysplasia-invasive carcinoma sequence in ulcerative colitis.

Iron supplementation increases disease activity and vitamin E ameliorates the effect in rats with dextran sulfate sodium-induced colitis

Inflammatory bowel disease is often associated with iron deficiency anemia and oral iron supplementation may be required. However, iron may increase oxidative stress through the Fenton reaction and thus exacerbate the disease. This study was designed to determine in rats with dextran sulfate sodium (DSS)-induced colitis whether oral iron supplementation increases intestinal inflammation and oxidative stress and whether the addition of an antioxidant, vitamin E, would reduce this detrimental effect. Four groups of rats that consumed 50 g/L Dextran sulfate sodium in drinking water were studied for 7 d and were fed: a control, nonpurified diet (iron, 270 mg, and dl-alpha-tocopherol acetate, 49 mg/kg); diet + iron (iron, 3000 mg/kg); diet + vitamin E (dl-alpha-tocopherol acetate, 2000 mg/kg) and the diet + both iron and vitamin E, each at the same concentrations as above. Body weight change, rectal bleeding, histological scores, plasma and colonic lipid peroxides (LPO), plasma 8-isoprostane, colonic glutathione peroxidase (GPx) and plasma vitamin E were measured. Iron supplementation increased disease activity as demonstrated by higher histological scores and heavier rectal bleeding. This was associated with an increase in colonic and plasma LPO and plasma 8-isoprostane as well as a decrease in colonic GPx. Vitamin E supplementation decreased colonic inflammation and rectal bleeding but did not affect oxidative stress, suggesting another mechanism for reducing inflammation. In conclusion, oral iron supplementation resulted in an increase in disease activity in this model of colitis. This detrimental effect on disease activity was reduced by vitamin E. Therefore, the addition of vitamin E to oral iron supplementation may be beneficial.

Dietary rutin, but not its aglycone quercetin, ameliorates dextran sulfate sodium-induced experimental colitis in mice

Oxidative stress has been shown to play a pivotal role in the onset of inflammatory bowel disease (IBD) and carcinogenesis. We evaluated the effects of two dietary anti-oxidants, rutin and its aglycone quercetin, on dextran sulfate sodium (DSS)-induced experimental colitis in mice. Female ICR mice were fed a diet containing 0.1% rutin or 0.1% quercetin for 2 weeks, and given 5% Dextran sulfate sodium in drinking water during the second week to induce colitis. We also examined the dose-dependency of rutin and quercetin (0.01% and 0.001% each) as well as their therapeutic efficacy, which was evaluated following Dextran sulfate sodium administration, on DSS-induced colitis. The protein level of interleukin (IL)-1β in both colonic mucosa and peritoneal macrophages was quantified by enzyme-linked immunosorbent assay. Further, mRNA expression levels of IL-1β, tumor necrosis factor-α , IL-6 , granulocyte macrophage-colony stimulating factor , inducible nitric oxide synthase , and cyclooxygenase (COX)-1 and COX-2 in colonic mucosa were determined by reverse transcription-polymerase chain reaction. A diet containing 0.1% rutin, but not quercetin, attenuated DSS-induced body weight loss and shortening of the colorectum ( P 02<020.01 and <0.05, respectively), and dramatically improved colitis histological scores. Further, DSS-induced increases in colonic mucosal IL-1β levels were blunted significantly in rutin-, but not quercetin-, fed mice ( P 02<020.01), while dietary rutin attenuated the expressions of IL-1β and IL-6 mRNA in colonic mucosa (each, P 02<020.01). As for dose dependency, 0.01%, but not 0.001%, dietary rutin significantly reduced mucosal IL-1β levels ( P 02<020.01). Notably, a 0.1% rutin diet given 3 days after Dextran sulfate sodium treatment significantly suppressed both colorectal shortening and IL-1β production ( P 02<020.05 and <0.01, respectively). Dietary rutin ameliorates DSS-induced colitis, presumably by suppressing the induction of pro-inflammatory cytokines. Our results suggest that rutin may be useful for the prevention and treatment of IBD and colorectal carcinogenesis via attenuation of pro-inflammatory cytokine production.

Agonists of cannabinoid receptor 1 and 2 inhibit experimental colitis induced by oil of mustard and by dextran sulfate sodium.

Abstract Oil of mustard (OM) is a potent neuronal activator that is known to elicit visceral hyperalgesia when given intracolonically, but the full extent to which OM is also proinflammatory in the gastrointestinal tract is not known. We have previously shown that male CD-1 mice given a single administration of 0.5% OM develop a severe colitis that is maximum at day 3 and that gradually lessens until essentially absent by day 14. OM-induced neuronal stimulation is reported to be reduced by cannabinoid agonists, and cannabinoid receptor 1 (CB1R)-/- mice have exacerbated experimental colitis. Therefore, we examined the role of cannabinoids in this OM-induced 3-day model of colitis in CD-1 mice and in a 7-day dextran sulfate sodium (DSS) colitis model in BALB/c mice. In OM colitis, the CB1R-selective agonist ACEA and the CB2R-selective agonist JWH-133 reduced (P < 0.05) colon weight gain (means +/- SE; 82 +/- 13% and 47 +/- 15% inhibition, respectively), colon shrinkage (98 +/- 24% and 42 +/- 12%, respectively), colon inflammatory damage score (49 +/- 11% and 40 +/- 12%, respectively), and diarrhea (58 +/- 12% and 43 +/- 11%, respectively). Histological damage was similarly reduced by these treatments. Likewise, CBR agonists attenuated Dextran sulfate sodium colitis, albeit at higher doses; ACEA at 10 mg/kg, twice daily, inhibited (P < 0.05) macroscopic and microscopic scores (46 +/- 9% and 63 +/- 7%, respectively); whereas 20 mg/kg, twice daily, of JWH-133 was required to diminish (P < 0.05) macroscopic and microscopic scores (29 +/- 7% and 43 +/- 5%, respectively). CB1R and CB2R immunostaining of colon sections revealed that CB1R in enteric neurons was more intense in colitic vs. control mice; however, CB1R was also increased in the endothelial layer in OM colitis only. CB2R immunostaining was more marked in infiltrated immune cells in OM colitis. These findings validate the OM colitis model with respect to the Dextran sulfate sodium model and provide strong support to the emerging idea that cannabinoid receptor activation mediates protective mechanisms in experimental colitis. The demonstration of CB1R agonist effects in colitis support the neurogenic nature of the OM-induced colitis model and reinforce the importance of neuronal activation in intestinal inflammation.

Uridine Ameliorates Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice
Abstract
Uridine, one of the four components that comprise RNA, has attracted attention as a novel therapeutic modulator of inflammation. However, very little is known about its effect on intestinal inflammation. The aim of the present study was to investigate the potential protective effect of intracolonic administered uridine against Dextran Sulfate Sodium induced colitis in male C57BL/6 mice. Intracolonic instillation of 3 doses of uridine 1?mg/Kg (lower dose), 5?mg/Kg (medium dose), and 10?mg/Kg (higher dose) in saline was performed daily. Uridine at medium and high dose significantly reduced the severity of colitis (DAI score) and alleviated the macroscopic and microscopic signs of the disease. The levels of proinflammatory cytokines IL-6, IL-1β and TNF in serum as well as mRNA expression in colon were significantly reduced in the uridine treated groups. Moreover, colon tissue myloperoxidase activities, protein expression of IL-6, TNF- α, COX-2, P-NFkB and P-Ikk-βα in the colon tissues were significantly reduced in medium and high dose groups. These findings demonstrated that local administration of uridine alleviated experimental colitis in male C57BL/6 mice accompanied by the inhibition of neutrophil infiltration and NF-κB signaling. Thus, Uridine may be a promising candidate for future use in the treatment of inflammatory bowel disease.
Introduction
Inflammatory Bowel Disease (IBD) is a broad term referring to conditions with chronic inflammation of the gastrointestinal tract. Ulcerative colitis (UC) and Crohn’s disease are two common forms of IBD that share some characteristics, but also exhibit distinct differences in risk factors, genetic predisposition and clinical and histological features1.
Crohn’s disease can affect the entire gastrointestinal tract and presents frequently with abdominal pain, fever, and clinical signs of bowel obstruction or diarrhoea with passage of blood and mucus. It has been postulated that the pathophysiology Crohn’s disease is mediated and perpetuated by an imbalance of effector Th-1 or Th17 cells responsible for secretion of interferon RecNNFα, and interleukins 17 and 22, versus naturally regulatory T cells responsible for secretion of interleukin 10 and transforming growth factor [TGF]2. Rapid influx and retention of leukocytes, a known feature of Crohn’s disease, is mediated by chemokines, selectins, integrins and their respective ligands (immunoglobulin superfamily, ICAM-1, MAdCam-1)3, 4.
Inflammation in UC occurs typically in the colon and rectum5. Symptoms include the development of bloody diarrhoea with or without mucus, rectal urgency, tenesmus, abdominal pain, weight loss, fatigue and extraintestinal manifestations6. It has been suggested that ulcerative colitis is associated with an atypical Th2 response mediated by non-classic natural killer T-cells producing interleukins 5 and 13, the latter being highly cytotoxic to epithelial cells which further increases intestinal permeability7. Tumor necrosis factor ?α (TNF), which is elevated in the blood, stool samples and mucosa of patients with ulcerative colitis, also induces apoptosis in intestinal epithelium and is an important and effective target for controlling the disease8. E and P-selectin are up-regulated on the vascular endothelium of ileum and colon and control migration of leukocytes especially lymphocytes to the intestinal lamina propria9. Further, the mucosal vascular addressin cell adhesion molecule (MAdCAM-1), expressed on HEV’s of the intestinal lamina propria, binds to α4β7 integrin and controls T-cell traffic to the gut-associated lymphoid tissue10.
It is now well documented that commensal bacteria are involved in the pathophysiology and disease progression of IBD11. The trigger for the initial leakage is unknown, but, once the epithelial barrier is dysfunctional, bacterial components activate production of cytokines like TNF, IL-6, IL-1β9, 12. This leads to further recruitment of neutrophils and macrophages that are more susceptible to bacterial stimulation than the resident macrophages and epithelial cells, on which the bacterial recognition receptors such as TLR or CD14 are down regulated8, 13. Modern treatments of IBD, including suppression of inflammation using 5-aminosalicylic acid, corticosteroids, immunomodulators and biological agents have proven to be efficacious2. However, these therapies are associated with major adverse effects14. Since none of the existing therapeutic modalities are able to provide complete or long lasting disease remission, there is still a pressing need to find better therapies for inflammatory bowel disease.
The Dextran Sulfate Sodium induced colitis model is a relevant model for translation of mice data to human disease and the model has been validated by using different therapeutic agents for human IBD15. Several studies have demonstrated that Dextran Sulfate Sodium induces breakdown of the mucosal epithelial barrier, allowing entry of luminal microorganisms into the mucosa, resulting in an overwhelming inflammatory response including NF-κB activation, over expression of pro-inflammatory cytokines and clinical symptoms of colitis16,17,18.
Uridine, a small and inexpensive pyrimidine nucleoside, is essential for synthesis of RNA and biomembranes. Uridine has attracted attention due to its anti-inflammatory effect in a rabbit dry eye model and an animal model of asthma19, 20. We have previously shown that uridine inhibits leukocyte adhesion in vitro and is a potent inhibitor of leukocyte extravasation in a model of sephadex induced lung inflammation21. Also, local administration of uridine into knee joints was able to protect against antigen induced arthritis in mice22. Although the exact mechanism of action of uridine is still unclear, it has been hypothesised that uridine could inhibit selectin-mediated adhesion through direct binding to selectins23. Another mechanism of action could be through generation of uridine-5′diphosphate (UDP) and uridine-5′-triphosphate (UTP) that can bind and activate the P2Y2, P2Y4 and P2Y6 receptors20.
Since, IBD including UC and Crohn’s disease are also chronic inflammatory diseases, we hypothesized that uridine could be effective in the treatment of IBD. Hence, the present study was undertaken to investigate the anti-inflammatory potential of locally administered uridine in Dextran Sulfate Sodium induced colitis in mice.
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Article | OPEN

Uridine Ameliorates Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice
Manish Kumar Jeengar, Dinesh Thummuri, Mattias Magnusson, V. G. M. Naidu & Srinivas Uppugunduri
Scientific Reportsvolume 7, Article number: 3924 (2017)
doi:10.1038/s41598-017-04041-9
Download Citation
Chronic inflammationUlcerative colitis
Received:
01 February 2017
Accepted:
08 May 2017
Published online:
20 June 2017
Abstract
Uridine, one of the four components that comprise RNA, has attracted attention as a novel therapeutic modulator of inflammation. However, very little is known about its effect on intestinal inflammation. The aim of the present study was to investigate the potential protective effect of intracolonic administered uridine against Dextran Sulfate Sodium induced colitis in male C57BL/6 mice. Intracolonic instillation of 3 doses of uridine 1?mg/Kg (lower dose), 5?mg/Kg (medium dose), and 10?mg/Kg (higher dose) in saline was performed daily. Uridine at medium and high dose significantly reduced the severity of colitis (DAI score) and alleviated the macroscopic and microscopic signs of the disease. The levels of proinflammatory cytokines IL-6, IL-1β and TNF in serum as well as mRNA expression in colon were significantly reduced in the uridine treated groups. Moreover, colon tissue myloperoxidase activities, protein expression of IL-6, TNF- α, COX-2, P-NFkB and P-Ikk-βα in the colon tissues were significantly reduced in medium and high dose groups. These findings demonstrated that local administration of uridine alleviated experimental colitis in male C57BL/6 mice accompanied by the inhibition of neutrophil infiltration and NF-κB signaling. Thus, Uridine may be a promising candidate for future use in the treatment of inflammatory bowel disease.

Introduction
Inflammatory Bowel Disease (IBD) is a broad term referring to conditions with chronic inflammation of the gastrointestinal tract. Ulcerative colitis (UC) and Crohn’s disease are two common forms of IBD that share some characteristics, but also exhibit distinct differences in risk factors, genetic predisposition and clinical and histological features1.

Crohn’s disease can affect the entire gastrointestinal tract and presents frequently with abdominal pain, fever, and clinical signs of bowel obstruction or diarrhoea with passage of blood and mucus. It has been postulated that the pathophysiology Crohn’s disease is mediated and perpetuated by an imbalance of effector Th-1 or Th17 cells responsible for secretion of interferon RecNNFα, and interleukins 17 and 22, versus naturally regulatory T cells responsible for secretion of interleukin 10 and transforming growth factor [TGF]2. Rapid influx and retention of leukocytes, a known feature of Crohn’s disease, is mediated by chemokines, selectins, integrins and their respective ligands (immunoglobulin superfamily, ICAM-1, MAdCam-1)3, 4.

Inflammation in UC occurs typically in the colon and rectum5. Symptoms include the development of bloody diarrhoea with or without mucus, rectal urgency, tenesmus, abdominal pain, weight loss, fatigue and extraintestinal manifestations6. It has been suggested that ulcerative colitis is associated with an atypical Th2 response mediated by non-classic natural killer T-cells producing interleukins 5 and 13, the latter being highly cytotoxic to epithelial cells which further increases intestinal permeability7. Tumor necrosis factor ?α (TNF), which is elevated in the blood, stool samples and mucosa of patients with ulcerative colitis, also induces apoptosis in intestinal epithelium and is an important and effective target for controlling the disease8. E and P-selectin are up-regulated on the vascular endothelium of ileum and colon and control migration of leukocytes especially lymphocytes to the intestinal lamina propria9. Further, the mucosal vascular addressin cell adhesion molecule (MAdCAM-1), expressed on HEV’s of the intestinal lamina propria, binds to α4β7 integrin and controls T-cell traffic to the gut-associated lymphoid tissue10.

It is now well documented that commensal bacteria are involved in the pathophysiology and disease progression of IBD11. The trigger for the initial leakage is unknown, but, once the epithelial barrier is dysfunctional, bacterial components activate production of cytokines like TNF, IL-6, IL-1β9, 12. This leads to further recruitment of neutrophils and macrophages that are more susceptible to bacterial stimulation than the resident macrophages and epithelial cells, on which the bacterial recognition receptors such as TLR or CD14 are down regulated8, 13. Modern treatments of IBD, including suppression of inflammation using 5-aminosalicylic acid, corticosteroids, immunomodulators and biological agents have proven to be efficacious2. However, these therapies are associated with major adverse effects14. Since none of the existing therapeutic modalities are able to provide complete or long lasting disease remission, there is still a pressing need to find better therapies for inflammatory bowel disease.

The Dextran Sulfate Sodium induced colitis model is a relevant model for translation of mice data to human disease and the model has been validated by using different therapeutic agents for human IBD15. Several studies have demonstrated that Dextran Sulfate Sodium induces breakdown of the mucosal epithelial barrier, allowing entry of luminal microorganisms into the mucosa, resulting in an overwhelming inflammatory response including NF-κB activation, over expression of pro-inflammatory cytokines and clinical symptoms of colitis16,17,18.

Uridine, a small and inexpensive pyrimidine nucleoside, is essential for synthesis of RNA and biomembranes. Uridine has attracted attention due to its anti-inflammatory effect in a rabbit dry eye model and an animal model of asthma19, 20. We have previously shown that uridine inhibits leukocyte adhesion in vitro and is a potent inhibitor of leukocyte extravasation in a model of sephadex induced lung inflammation21. Also, local administration of uridine into knee joints was able to protect against antigen induced arthritis in mice22. Although the exact mechanism of action of uridine is still unclear, it has been hypothesised that uridine could inhibit selectin-mediated adhesion through direct binding to selectins23. Another mechanism of action could be through generation of uridine-5′diphosphate (UDP) and uridine-5′-triphosphate (UTP) that can bind and activate the P2Y2, P2Y4 and P2Y6 receptors20.

Since, IBD including UC and Crohn’s disease are also chronic inflammatory diseases, we hypothesized that uridine could be effective in the treatment of IBD. Hence, the present study was undertaken to investigate the anti-inflammatory potential of locally administered uridine in Dextran Sulfate Sodium induced colitis in mice.

Results
We used a higher concentration of Dextran Sulfate Sodium (4% w/v concentration) in order to study the effect of uridine on overall mortality of animals (Fig. 1A). A lower concentration of Dextran Sulfate Sodium (3.5% w/v) was used in all subsequent experiments to study the dose dependent anti-inflammatory effect of uridine (Fig. 1B). It was found that the survival rate was significantly prolonged in the uridine 10?mg/kg +Dextran Sulfate Sodium group (Dextran Sulfate Sodium+UH) compared to Dextran Sulfate Sodium control mice (Fig. 2). Only 10% of the Dextran Sulfate Sodium control mice survived while 60% of the Dextran Sulfate Sodium+UH treated mice survived up to 15 days.
Schematic diagram to illustrate the experimental design. (A) Survival study: Mice were divided into two groups Dextran Sulfate Sodium control & Dextran Sulfate Sodium+ 10?mg/kg uridine treated group and challenged with 4% w/v of Dextran Sulfate Sodium with drinking water for 5 days, and were further assessed for survival upto day15. (B) Evaluation of dose dependent anti-inflammatory effect of intracolonic administered uridine on Dextran Sulfate Sodium (DSS)-induced experimental colitis in Mice; Experimental Colitis was induced by administration of 3.5% of w/v Dextran Sulfate Sodium with drinking water for 5 days and later on normal drinking water for next 7 days. Uridine treatment group received intracolonic uridine solution once daily from day 0 to day 12 while remaining group mice received normal saline. Body weight, health status were measured daily. Colon length measurement, colon histopathology were performed after necropsy. Dextran Sulfate Sodium+UL, Low dose uridine 1?mg/kg; Dextran Sulfate Sodium+UM, medium dose uridine 5?mg/kg; V, Dextran Sulfate Sodium+UH, higher dose uridine 10?mg/kg.
Body weight
All mice treated with 3.5% of Dextran Sulfate Sodium showed body weight loss starting from day 4 post Dextran Sulfate Sodium administration (Fig. 3A). Uridine treatment showed a protective effect on colitis-induced body weight loss. The uridine higher dose (10?mg/kg) group (Dextran Sulfate Sodium+UH) showed a significant improvement in body weight from day 9 to day 12 (p??0.05).
Effect of uridine treatment on the clinical signs of colitis and macroscopic signs of inflammation in colon tissue. Experimental colitis was induced by 3.5% w/v of Dextran Sulfate Sodium in drinking water (ad libidum) for 5 days. (A) Percentage change in body weight. (B) Disease activity index, a composite measure of weight loss, stool consistency and blood in stool. (C) Representative photographs showing colon tissue from I, Normal; II, Dextran Sulfate Sodium control; III, Dextran Sulfate Sodium+UL; IV, Dextran Sulfate Sodium+UM; V, Dextran Sulfate Sodium+UH (D) Changes in colon length. Data presented indicate the mean?±?SEM (n?=?6). +++p? Disease Activity Index (DAI)
DAI, calculated as a composite of body weight loss, stool consistency and stool blood, was scored on alternate days to analyze the anti-inflammatory potential of uridine. Dextran Sulfate Sodium (3.5%) administration was associated with significant clinical changes including weight loss, appearance of occult faecal blood and diarrhoea in Dextran Sulfate Sodium control mice. The DAI of Dextran Sulfate Sodium control group was significantly elevated on day 4 compared to baseline and reached its maximum on day 8. Treatment with 5 and 10?mg/kg of uridine markedly reduced the DAI score from day 6 and onwards (Fig. 3B). Uridine treatment delayed or reduced the appearance of the colitis symptoms like appearance of hemoccult and diarrhoea which resulted in a significant reduction of DAI in Dextran Sulfate Sodium+UM and Dextran Sulfate Sodium+UH groups. The representative photos of Dextran Sulfate Sodium control mice showing rectal bleeding compared to Dextran Sulfate Sodium+UH mice and comparison of stool samples from each group on day 12 are shown in Supplementary Figs S1 and S2.

Colon length
All colon tissues were collected and measured at the end of study, to study the effect of uridine on inflammation induced decrease in colon length, a classical symptom of colonic inflammation. Severe signs of inflammation and bleeding clearly observed in colon tissue of Dextran Sulfate Sodium control (Fig. 3C) were eliminated in Dextran Sulfate Sodium+UM and Dextran Sulfate Sodium+UH groups. The colon tissues of mice exposed to Dextran Sulfate Sodium exhibited a marked decrease in colon length compared with the normal group (Fig. 3D, p?
Histopathology
Histological analysis (H&E staining) of the distal colon tissue revealed that treatment of mice with Dextran Sulfate Sodium leads to destruction of crypt structure with goblet cell loss, a disturbed epithelial layer and massive infiltration of inflammatory cells in the colon tissue when compared to the normal morphology of colon tissue (Fig. 4A). Colon tissue from Dextran Sulfate Sodium+UH treated mice exhibited predominantly intact colon histology, with reduced signs of inflammation, preserved epithelial layer and crypt structure when compared to Dextran Sulfate Sodium control group. The Dextran Sulfate Sodium+UL and Dextran Sulfate Sodium+UM treated mice showed moderate to mild signs of inflammation in colon histopathology.
Effect of uridine treatment on the histopathological changes and myloperoxidase (MPO) activity of the colon tissue. (A) Representative images of hematoxylin and eosin staining of colon tissue from each group taken at 100x magnification. Colon tissue from Normal group did not show any pathological modification, Dextran Sulfate Sodium-induced colon tissue injury was associated with partial destruction of the epithelial architecture such as loss of crypts and epithelial integrity, submucosal edema and intense infiltration of inflammatory cells. Treatment with different dosages attenuated the injury of colon tissue in dose dependent manner. L, Lumen; GC, Goblet cells; M, Mucosa; SM, Submucosa; I, Inflammation; asterisk (*) indicates area of goblet cell depletion and distortion of crypt architecture; number sign (#) indicates cellular infiltration. (B) The myloperoxidase (MPO) activity in colon tissue, values are the mean?±?SEM (n?=?6). +++p? Myeloperoxidase (MPO) activity assay
Figure 4B depicts the MPO activity in colon tissue of mice of different groups. There was a significant (p?
Serum cytokine levels
Serum levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β were estimated by flow cytometry and significantly elevated levels of these cytokines were found in Dextran Sulfate Sodium control group (p? Cytokine/chemokine mRNA expression in the colon
As shown in Fig. 5B, a significant (p?
Estimation of protein expression in the colon
Protein expression of the proinflammatory cytokines TNF, IL-6, and COX-2 enzyme was estimated in the colon tissue using immunoblotting (Fig. 6A). There was a significantly (p? Effect of uridine treatment on the protein expression of pro-inflammatory cytokines, enzyme levels in colon tissue. (A) Representative western blots showing changes in the protein expression of pro-inflammatory cytokines and enzyme, TNF, IL-6, and COX-2 in colon tissue (B) Graphical depiction of western blotting analysis showing quantitative results. Band intensities were quantified using NIH Image J software. The relative protein levels were normalized to the b-actin level. Each value was calculated on the basis of the data obtained from three independent experiments. Values are the mean?±?SEM (n?=?3). +++p? Full size ima
Treatment with 5?mg/kg and 10?mg/kg of uridine significantly reduced the levels of COX-2 compared to Dextran Sulfate Sodium control (P? Uridine treatment inhibits Dextran Sulfate Sodium induced NF-κB activity in colon tissue. (A) Representative western blotting bands showing the expression levels of phospho-NF-κB-p65, NFκB and phospho-IKKα/β (176/180), IKKα/β (176/180). β-actin was used for equal loading of protein. (B) Bar diagrams showing the relative ratio of each protein. All the values were expressed as mean mean?±?SEM (n?=?3). ++p?
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Article | OPEN

Uridine Ameliorates Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice
Manish Kumar Jeengar, Dinesh Thummuri, Mattias Magnusson, V. G. M. Naidu & Srinivas Uppugunduri
Scientific Reportsvolume 7, Article number: 3924 (2017)
doi:10.1038/s41598-017-04041-9
Download Citation
Chronic inflammationUlcerative colitis
Received:
01 February 2017
Accepted:
08 May 2017
Published online:
20 June 2017
Abstract
Uridine, one of the four components that comprise RNA, has attracted attention as a novel therapeutic modulator of inflammation. However, very little is known about its effect on intestinal inflammation. The aim of the present study was to investigate the potential protective effect of intracolonic administered uridine against Dextran Sulfate Sodium induced colitis in male C57BL/6 mice. Intracolonic instillation of 3 doses of uridine 1?mg/Kg (lower dose), 5?mg/Kg (medium dose), and 10?mg/Kg (higher dose) in saline was performed daily. Uridine at medium and high dose significantly reduced the severity of colitis (DAI score) and alleviated the macroscopic and microscopic signs of the disease. The levels of proinflammatory cytokines IL-6, IL-1β and TNF in serum as well as mRNA expression in colon were significantly reduced in the uridine treated groups. Moreover, colon tissue myloperoxidase activities, protein expression of IL-6, TNF- α, COX-2, P-NFkB and P-Ikk-βα in the colon tissues were significantly reduced in medium and high dose groups. These findings demonstrated that local administration of uridine alleviated experimental colitis in male C57BL/6 mice accompanied by the inhibition of neutrophil infiltration and NF-κB signaling. Thus, Uridine may be a promising candidate for future use in the treatment of inflammatory bowel disease.

Introduction
Inflammatory Bowel Disease (IBD) is a broad term referring to conditions with chronic inflammation of the gastrointestinal tract. Ulcerative colitis (UC) and Crohn’s disease are two common forms of IBD that share some characteristics, but also exhibit distinct differences in risk factors, genetic predisposition and clinical and histological features1.

Crohn’s disease can affect the entire gastrointestinal tract and presents frequently with abdominal pain, fever, and clinical signs of bowel obstruction or diarrhoea with passage of blood and mucus. It has been postulated that the pathophysiology Crohn’s disease is mediated and perpetuated by an imbalance of effector Th-1 or Th17 cells responsible for secretion of interferon RecNNFα, and interleukins 17 and 22, versus naturally regulatory T cells responsible for secretion of interleukin 10 and transforming growth factor [TGF]2. Rapid influx and retention of leukocytes, a known feature of Crohn’s disease, is mediated by chemokines, selectins, integrins and their respective ligands (immunoglobulin superfamily, ICAM-1, MAdCam-1)3, 4.

Inflammation in UC occurs typically in the colon and rectum5. Symptoms include the development of bloody diarrhoea with or without mucus, rectal urgency, tenesmus, abdominal pain, weight loss, fatigue and extraintestinal manifestations6. It has been suggested that ulcerative colitis is associated with an atypical Th2 response mediated by non-classic natural killer T-cells producing interleukins 5 and 13, the latter being highly cytotoxic to epithelial cells which further increases intestinal permeability7. Tumor necrosis factor ?α (TNF), which is elevated in the blood, stool samples and mucosa of patients with ulcerative colitis, also induces apoptosis in intestinal epithelium and is an important and effective target for controlling the disease8. E and P-selectin are up-regulated on the vascular endothelium of ileum and colon and control migration of leukocytes especially lymphocytes to the intestinal lamina propria9. Further, the mucosal vascular addressin cell adhesion molecule (MAdCAM-1), expressed on HEV’s of the intestinal lamina propria, binds to α4β7 integrin and controls T-cell traffic to the gut-associated lymphoid tissue10.

It is now well documented that commensal bacteria are involved in the pathophysiology and disease progression of IBD11. The trigger for the initial leakage is unknown, but, once the epithelial barrier is dysfunctional, bacterial components activate production of cytokines like TNF, IL-6, IL-1β9, 12. This leads to further recruitment of neutrophils and macrophages that are more susceptible to bacterial stimulation than the resident macrophages and epithelial cells, on which the bacterial recognition receptors such as TLR or CD14 are down regulated8, 13. Modern treatments of IBD, including suppression of inflammation using 5-aminosalicylic acid, corticosteroids, immunomodulators and biological agents have proven to be efficacious2. However, these therapies are associated with major adverse effects14. Since none of the existing therapeutic modalities are able to provide complete or long lasting disease remission, there is still a pressing need to find better therapies for inflammatory bowel disease.

The Dextran Sulfate Sodium induced colitis model is a relevant model for translation of mice data to human disease and the model has been validated by using different therapeutic agents for human IBD15. Several studies have demonstrated that Dextran Sulfate Sodium induces breakdown of the mucosal epithelial barrier, allowing entry of luminal microorganisms into the mucosa, resulting in an overwhelming inflammatory response including NF-κB activation, over expression of pro-inflammatory cytokines and clinical symptoms of colitis16,17,18.

Uridine, a small and inexpensive pyrimidine nucleoside, is essential for synthesis of RNA and biomembranes. Uridine has attracted attention due to its anti-inflammatory effect in a rabbit dry eye model and an animal model of asthma19, 20. We have previously shown that uridine inhibits leukocyte adhesion in vitro and is a potent inhibitor of leukocyte extravasation in a model of sephadex induced lung inflammation21. Also, local administration of uridine into knee joints was able to protect against antigen induced arthritis in mice22. Although the exact mechanism of action of uridine is still unclear, it has been hypothesised that uridine could inhibit selectin-mediated adhesion through direct binding to selectins23. Another mechanism of action could be through generation of uridine-5′diphosphate (UDP) and uridine-5′-triphosphate (UTP) that can bind and activate the P2Y2, P2Y4 and P2Y6 receptors20.

Since, IBD including UC and Crohn’s disease are also chronic inflammatory diseases, we hypothesized that uridine could be effective in the treatment of IBD. Hence, the present study was undertaken to investigate the anti-inflammatory potential of locally administered uridine in Dextran Sulfate Sodium induced colitis in mice.

Results
We used a higher concentration of Dextran Sulfate Sodium (4% w/v concentration) in order to study the effect of uridine on overall mortality of animals (Fig. 1A). A lower concentration of Dextran Sulfate Sodium (3.5% w/v) was used in all subsequent experiments to study the dose dependent anti-inflammatory effect of uridine (Fig. 1B). It was found that the survival rate was significantly prolonged in the uridine 10?mg/kg +Dextran Sulfate Sodium group (Dextran Sulfate Sodium+UH) compared to Dextran Sulfate Sodium control mice (Fig. 2). Only 10% of the Dextran Sulfate Sodium control mice survived while 60% of the Dextran Sulfate Sodium+UH treated mice survived up to 15 days.

Figure 1

Schematic diagram to illustrate the experimental design. (A) Survival study: Mice were divided into two groups Dextran Sulfate Sodium control & Dextran Sulfate Sodium+ 10?mg/kg uridine treated group and challenged with 4% w/v of Dextran Sulfate Sodium with drinking water for 5 days, and were further assessed for survival upto day15. (B) Evaluation of dose dependent anti-inflammatory effect of intracolonic administered uridine on Dextran Sulfate Sodium (DSS)-induced experimental colitis in Mice; Experimental Colitis was induced by administration of 3.5% of w/v Dextran Sulfate Sodium with drinking water for 5 days and later on normal drinking water for next 7 days. Uridine treatment group received intracolonic uridine solution once daily from day 0 to day 12 while remaining group mice received normal saline. Body weight, health status were measured daily. Colon length measurement, colon histopathology were performed after necropsy. Dextran Sulfate Sodium+UL, Low dose uridine 1?mg/kg; Dextran Sulfate Sodium+UM, medium dose uridine 5?mg/kg; V, Dextran Sulfate Sodium+UH, higher dose uridine 10?mg/kg.
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Figure 2

Survival study. Comparison of survival rate was done with mice challenged with 4% of Dextran Sulfate Sodium and Dextran Sulfate Sodium+ Uridine 10?mg/kg. Percentage survival data of n?=?10 animals.
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Body weight
All mice treated with 3.5% of Dextran Sulfate Sodium showed body weight loss starting from day 4 post Dextran Sulfate Sodium administration (Fig. 3A). Uridine treatment showed a protective effect on colitis-induced body weight loss. The uridine higher dose (10?mg/kg) group (Dextran Sulfate Sodium+UH) showed a significant improvement in body weight from day 9 to day 12 (p??0.05).

Figure 3

Effect of uridine treatment on the clinical signs of colitis and macroscopic signs of inflammation in colon tissue. Experimental colitis was induced by 3.5% w/v of Dextran Sulfate Sodium in drinking water (ad libidum) for 5 days. (A) Percentage change in body weight. (B) Disease activity index, a composite measure of weight loss, stool consistency and blood in stool. (C) Representative photographs showing colon tissue from I, Normal; II, Dextran Sulfate Sodium control; III, Dextran Sulfate Sodium+UL; IV, Dextran Sulfate Sodium+UM; V, Dextran Sulfate Sodium+UH (D) Changes in colon length. Data presented indicate the mean?±?SEM (n?=?6). +++p? Full size image
Disease Activity Index (DAI)
DAI, calculated as a composite of body weight loss, stool consistency and stool blood, was scored on alternate days to analyze the anti-inflammatory potential of uridine. Dextran Sulfate Sodium (3.5%) administration was associated with significant clinical changes including weight loss, appearance of occult faecal blood and diarrhoea in Dextran Sulfate Sodium control mice. The DAI of Dextran Sulfate Sodium control group was significantly elevated on day 4 compared to baseline and reached its maximum on day 8. Treatment with 5 and 10?mg/kg of uridine markedly reduced the DAI score from day 6 and onwards (Fig. 3B). Uridine treatment delayed or reduced the appearance of the colitis symptoms like appearance of hemoccult and diarrhoea which resulted in a significant reduction of DAI in Dextran Sulfate Sodium+UM and Dextran Sulfate Sodium+UH groups. The representative photos of Dextran Sulfate Sodium control mice showing rectal bleeding compared to Dextran Sulfate Sodium+UH mice and comparison of stool samples from each group on day 12 are shown in Supplementary Figs S1 and S2.

Colon length
All colon tissues were collected and measured at the end of study, to study the effect of uridine on inflammation induced decrease in colon length, a classical symptom of colonic inflammation. Severe signs of inflammation and bleeding clearly observed in colon tissue of Dextran Sulfate Sodium control (Fig. 3C) were eliminated in Dextran Sulfate Sodium+UM and Dextran Sulfate Sodium+UH groups. The colon tissues of mice exposed to Dextran Sulfate Sodium exhibited a marked decrease in colon length compared with the normal group (Fig. 3D, p?
Histopathology
Histological analysis (H&E staining) of the distal colon tissue revealed that treatment of mice with Dextran Sulfate Sodium leads to destruction of crypt structure with goblet cell loss, a disturbed epithelial layer and massive infiltration of inflammatory cells in the colon tissue when compared to the normal morphology of colon tissue (Fig. 4A). Colon tissue from Dextran Sulfate Sodium+UH treated mice exhibited predominantly intact colon histology, with reduced signs of inflammation, preserved epithelial layer and crypt structure when compared to Dextran Sulfate Sodium control group. The Dextran Sulfate Sodium+UL and Dextran Sulfate Sodium+UM treated mice showed moderate to mild signs of inflammation in colon histopathology.

Figure 4

Effect of uridine treatment on the histopathological changes and myloperoxidase (MPO) activity of the colon tissue. (A) Representative images of hematoxylin and eosin staining of colon tissue from each group taken at 100x magnification. Colon tissue from Normal group did not show any pathological modification, Dextran Sulfate Sodium-induced colon tissue injury was associated with partial destruction of the epithelial architecture such as loss of crypts and epithelial integrity, submucosal edema and intense infiltration of inflammatory cells. Treatment with different dosages attenuated the injury of colon tissue in dose dependent manner. L, Lumen; GC, Goblet cells; M, Mucosa; SM, Submucosa; I, Inflammation; asterisk (*) indicates area of goblet cell depletion and distortion of crypt architecture; number sign (#) indicates cellular infiltration. (B) The myloperoxidase (MPO) activity in colon tissue, values are the mean?±?SEM (n?=?6). +++p? Full size image
Myeloperoxidase (MPO) activity assay
Figure 4B depicts the MPO activity in colon tissue of mice of different groups. There was a significant (p?
Serum cytokine levels
Serum levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β were estimated by flow cytometry and significantly elevated levels of these cytokines were found in Dextran Sulfate Sodium control group (p?
Figure 5

Effect of uridine treatment on the inflammatory biomarkers of Dextran Sulfate Sodium induced UC. (A) Serum proinflammatory cytokines level TNF, IL-6 and IL-1β measured by cytometric bead array assay. (B) m-RNA expression of pro-inflammatory cytokines and enzyme in colon tissue measured by RT-PCR. All the values were expressed as mean mean?±?SEM (n?=?5). +++p? Full size image
Cytokine/chemokine mRNA expression in the colon
As shown in Fig. 5B, a significant (p?
Estimation of protein expression in the colon
Protein expression of the proinflammatory cytokines TNF, IL-6, and COX-2 enzyme was estimated in the colon tissue using immunoblotting (Fig. 6A). There was a significantly (p?
Figure 6

Effect of uridine treatment on the protein expression of pro-inflammatory cytokines, enzyme levels in colon tissue. (A) Representative western blots showing changes in the protein expression of pro-inflammatory cytokines and enzyme, TNF, IL-6, and COX-2 in colon tissue (B) Graphical depiction of western blotting analysis showing quantitative results. Band intensities were quantified using NIH Image J software. The relative protein levels were normalized to the b-actin level. Each value was calculated on the basis of the data obtained from three independent experiments. Values are the mean?±?SEM (n?=?3). +++p? Full size image
Treatment with 5?mg/kg and 10?mg/kg of uridine significantly reduced the levels of COX-2 compared to Dextran Sulfate Sodium control (P?
Figure 7

Uridine treatment inhibits Dextran Sulfate Sodium induced NF-κB activity in colon tissue. (A) Representative western blotting bands showing the expression levels of phospho-NF-κB-p65, NFκB and phospho-IKKα/β (176/180), IKKα/β (176/180). β-actin was used for equal loading of protein. (B) Bar diagrams showing the relative ratio of each protein. All the values were expressed as mean mean?±?SEM (n?=?3). ++p? Full size image
Discussion
Uridine has attracted attention as a novel therapeutic modulator of inflammation due to proven efficacy in various animal models of inflammation combined with a good safety profile21, 24. However, poor oral bioavailability has limited the use of uridine as an effective oral therapeutic agent25. We have previously reported that locally administered uridine has potent anti-inflammatory effects in animal models of lung inflammation and arthritis21, 22. The present study is the first of its kind demonstrating the efficacy of intracolonic administration of uridine in experimental Dextran Sulfate Sodium induced ulcerative colitis. Administration of 3.5% Dextran Sulfate Sodium to mice for 5 days elicited the predominant clinical symptoms of colitis including weight loss, diarrhoea, bloody faeces, crypt distortion, epithelial injury, reduced colon length and inflammatory cell infiltration. These clinical symptoms of IBD were efficiently relieved by the treatment of uridine in a dose dependent manner. The higher and medium dose of uridine significantly reduced the DAI score, macroscopically visible damage and reversed inflammation induced reduction in colon length. In the survival study the 10?mg/kg uridine treatment prolonged the survival rate of mice compared to Dextran Sulfate Sodium control group. These observations clearly suggested that uridine at 10?mg/kg is efficient in suppressing overt clinical features of Dextran Sulfate Sodium induced colitis.

Uridine treatment resulted in a dose-dependent protection of histological integrity in the colon tissue. Since Dextran Sulfate Sodium-induced mice developed immunological abnormalities, such as prominent shortening of the colon, thickening of the muscular layer and crypt damage in the inflamed areas and inflammatory cell infiltration in the lamina propria and mucosa of the colon and that was noticeably reduced by the uridine treatment. In this study increased MPO levels, an accepted measure of neutrophil infiltration, were substantially reduced by uridine treatment demonstrating a significant reduction in the infiltration of neutrophils. These results are in line with our previous studies where we have shown that uridine inhibits leukocyte adhesion in vitro26 and local administration of uridine reduced neutrophil influx in BAL fluid in animal model of lung inflammation21 and almost abrogated neutrophil and macrophage influx in synovium of antigen induced arthritic mice22.

The pro-inflammatory cytokines such as TNF, IL-6 and IL-1β have a pivotal role in the pathogenesis of IBD9, 27. Several studies have also reported that interaction between pro-inflammatory cytokines and the intestinal mucosal immune system can lead to the disruption of tight junction proteins and affect intestinal homeostasis28. Increased serum and tissue levels of pro-inflammatory cytokines such as IL-6, IL-1β and TNF-α are characteristic features of colitis and many other chronic inflammatory diseases29. Secretion of IL-6, IL-1β and TNF was significantly elevated in the serum by Dextran Sulfate Sodium treatment and was remarkably suppressed by the uridine treatment at a higher dose. This is noteworthy because a single intraarticular injection of uridine that protected mice from antigen-induced arthritis did not inhibit the rise of proinflammatory cytokines in serum22.

We have previously shown that uridine inhibited the extravasation of inflammatory cells into the synovium and inhibited expression of TNF, IL-6 and adhesion molecules in the synovium of arthritic mice22. Uridine treatment inhibited the increased mRNA expression for TNF, IL-1, IL-6 and protein levels of TNF and IL-6 in a dose dependent manner. Taken together, our results suggest that uridine was able to decrease the levels of these pro-inflammatory cytokines and the inhibitory effect of uridine is already at the mRNA level affecting both local and circulating cytokine levels.

Upregulation of inducible pro-inflammatory enzyme COX-2 in the intestinal epithelium of IBD patients is well documented and it plays an important role in the amplification of mucosal inflammation in IBD30, 31. Our results indicated that increased expression of COX-2 was attenuated at both mRNA and protein level by uridine treatment. It could be speculated that uridine might also exert an analgesic effect due to the observed effect on COX-2 expression. However, this needs to be studied in detail in specific models of pain and analgesia.

The synthesis of COX-2 as well as production of proinflammatory cytokines including TNF and IL-6 is directly modulated by the pleiotropic transcription factor NF-κB32, 33. Phosphorylation of IKKα/β (ser 176/180) is a critical step in NF-κB activation and the expression of P-IKKα/β (ser 176/180) was found to be markedly increased in Dextran Sulfate Sodium control group31. Our results clearly indicated that treatment of uridine at medium and high dose was able to inhibit phosphorylation of IKKα/β without affecting the expression of IKKα. Further, reduced levels of P-NF-κB p65 in colon tissue of uridine treated groups (Dextran Sulfate Sodium+UM & Dextran Sulfate Sodium+UH) confirmed that uridine attenuates NF-kB activation. Thus, our data strongly suggests that the anti-ulcerative colitis effect of uridine may be associated with the suppression of NF-κB signaling activation.

In summary, the findings in our study suggest that intra-colonic administration of uridine effectively prevented the development and progression of the Dextran Sulfate Sodium induced colitis symptoms in C57 BL6 mice. We highlighted that the plausible underlying mechanism of uridine might associated with alleviating inflammatory responses, inhibiting NF-κB signaling activation and reducing the neutrophils infiltration. Collectively, our results suggest that uridine has the potential to serve an as effective anti-IBD therapy. Further it would be of great interest to investigate therapeutic benefit of uridine treatment in already established IBD.

Materials
Dextran sulphate sodium (Dextran Sulfate Sodium; product code# 02160110; Mw: 36000–50000) and uridine (product code# 02103216) was purchased from MP Biomedicals, Santa Ana, CA, USA.

Animals
Male C57BL/6 mice, 7–8 weeks of age were purchased from Sanzyme P. Ltd. Banjara Hills, Hyderabad, India. All mice were housed and fed in a dedicated pathogen-free facility and maintained at standard laboratory conditions of 23?±?2?°C under 12-hour day and night cycles throughout the experiment. All procedures described were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), NIPER Hyderabad, India. The animal experiments were conducted in accordance with the CPCSEA guidelines (IAEC approval number: NIP/12/2015/PC/162).

Induction of colitis and treatment protocol
The Dextran Sulfate Sodium model is a robust, well established model and has been used to screen potential drug candidates and to investigate their mechanism of action26. Mice were randomly divided into five groups (n?=?6), comprising a normal group (Normal), Dextran Sulfate Sodium control, Dextran Sulfate Sodium and low dose uridine (1?mg/kg body weight; Dextran Sulfate Sodium+Dextran Sulfate Sodium+UL), Dextran Sulfate Sodium and medium dose uridine (5?mg/kg; Dextran Sulfate Sodium+UM), Dextran Sulfate Sodium and high dose uridine (10?mg/kg; Dextran Sulfate Sodium+UH;). Experimental colitis was induced in mice by administration of 3.5% w/v Dextran Sulfate Sodium in drinking water for 5 days, followed by a regime of 7 days of fresh water (reflecting acute inflammation), whereas the normal group received only normal drinking water throughout the experiment34. In present study, uridine was administered intra-colonially, thus avoiding bioavailability problems associated with oral or intravenous administration. Daily instillation of 0.1?ml intracolonic saline in the control group or appropriate dose of uridine in the treatment group was performed on anesthetized mice by using flexible polyethylene tubing positioned in the midcolon after lubrication (4?cm proximal to the anus). Uridine treatment group received intracolonic uridine solution once daily for 12 days starting on day 0 of the Dextran Sulfate Sodium treatment. At day 12, mice were euthanized under CO2 asphyxiation and the colon was removed; length was measured and stored for subsequent analysis.

For the survival study, 10 mice each in the Dextran Sulfate Sodium and Dextran Sulfate Sodium+UH groups were subjected to 4% w/v of Dextran Sulfate Sodium until day 5, when they were returned to normal distilled water and monitored for survival upto day 1535.

Evaluation of Colitis
Body weight change, stool consistency and gross bleeding were assessed daily. Collection of feces was done by placing a single mouse in an empty cage without bedding material for 15–30?min and fecal pellet was used to monitor hemoccult. The criteria to calculate disease activity index (DAI) score described previously36 are shown in Table 1.
Histology
Colon tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin. 5?μm thick sections were prepared from each block, stained with haematoxylin & eosin and observed under phase contrast microscope (100x magnification, Nikon, Japan).

Myeloperoxidase (MPO) activity assay
MPO activity as a measure of neutrophil infiltration is a well established biomaffigrker of ulcerative colitis37, 38. Tissue MPO activity was performed as reported previously with some modifications39. Briefly, colon tissue was thawed and homogenized in 50?mM phosphate buffer (pH 6) containing 0.5% hexadecyltrimethylammonium bromide (HTAB). The tissue homogenates were subjected to a brief sonication for 10?s, one cycle of freezing, thawing and sonicated for a further 10?s. After sonication, suspensions were centrifuged at 13,000?rpm for 20?min at 4?°C. The supernatant (0.1?mL) was mixed in 2.9?ml of in 50?mM phosphate buffer (pH 6) containing 0.53?mM of o-dianisidine hydrochloride and 0.15?mM hydrogen peroxide and the change in absorbance was measured every 15?s for, 5?min at 460?nm. The results were expressed in units (U) of MPO/ mg of protein. The protein concentrations of each sample were evaluated by using the Bradford methodusing bovine serum albumin as the standard.

Flow Cytometry
Serum samples were processed for cytokine measurement using Cytometric Bead Array (CBA Flex Sets BD Biosciences, UK). Three key cytokines were assessed: IL-1β, IL-6 and TNF. The detection limit for IL-1β, IL-6 and TNF was 1.9?pg/ml, 1.4?pg/ml and 2.8?pg/ml respectively.

RT-PCR analysis
For quantitative real time PCR measurements, colon tissue samples were cut and frozen immediately in liquid nitrogen with TRI reagent (Sigma Aldrich) and stored at ?80?°C until RNA extraction. Total RNA isolation was performed as reported earlier40. Sample quality control and the quantitative analysis were carried out by NanoDrop (Thermo Scientific). Reverse-transcription to cDNA was performed using Verso cDNA synthesis kit according to the manufacturer’s instructions. The Real-Time PCR reaction was performed on the ABS 7500 fast instrument with SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) with an initial denaturation step at 95?°C for 10?min, followed by 40 cycles of 95?°C for 10?s annealing temperature with extension step for 45?s at 55?°C by using respective following primers of various target genes: TNF-Left primer (L): 5′-GAACTGGCAGAAGAGGCACT-3′, TNF-Right primer (R): 5′-AGGGTCTGGGCCATAGAACT-3′, IL-1β (L): 5′-GCCCATCCTCTGTGACTCAT-3′, IL-1β(R): 5′-AGGCCACAGGTATTTTGTCG-3′, IL-6(L): 5′-AGTTGCCTTCTTGGGACTGA-3′, IL-6 (R): 5′-CAGAATTGCCATTGCACAAC-3′, β-Actin(L): 5′-AGCCATGTACGTAGCCATCC-3′ and β-Actin(R): 5′-CTCTCAGCTGTGGTGGTGAA-3′. The relative changes in TNF-α, IL-1 β, and IL-6 and COX-2 with respect to β-Actin expression were examined using 2?ΔΔCt method using normal mice as reference.

Western blot analysis
Colon tissues were cut and frozen immediately in liquid nitrogen and stored at ?80?°C. Protein lyaste were prepared as described previously41. Briefly, Colon tissue was homogenized in RIPA buffer with 1% protease and phoshphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and incubated on ice for 1?h. Lysates were then centrifuged for 15?min at 13000?rpm at 4?°C. Proteins were separated using SDS–PAGE and transferred onto PVDF (poly-vinylidene difluoride) membrane. The blots were blocked with 3.5% BSA (Bovine Serum Albumin) in TBST (20?mM Tris–HCl, pH 7.4, 137?mM NaCl, and 0.05%Tween-20) at room temperature for 1?h and incubated overnight with the appropriate primary antibody at 4?°C. After washing with TBST, the blots were incubated with peroxidase conjugated secondary antibody for 1?h. β-actin was used as an internal control to ensure equal protein loading. Bands were monitored using enhanced chemiluminescence reagent (Millipore, U.S.A). The strength of western blotting bands was determined by Image J density measurement program.

Antibodies used for western blot analysis were PNF-κB (p65), NF-κB P65, phospho-Iκβα, IκBα, COX-2, TNF-α, IL-6, IL-10 and MPO. All the primary and secondary antibodies were obtained from Cell Signaling Technology, Baverly, USA.

Statistical analysis
All data are represented as mean?±?SEM. Statistical analysis was performed using GraphPad Prism 5.0 software, utilizing One-way ANOVA and Dunnett’s multiple comparison test and considered significant if p values were <0.05.

Additional information
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