Novel effects of ectoine, a bacteria-derived natural tetrahydropyrimidine, in experimental colitis

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Phytomedicine 20 (2013) 585–591

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Novel effects of ectoine, a bacteria-derived natural tetrahydropyrimidine, in experimental colitis Heba Abdel-Aziz a,∗ , Walaa Wadie b , Dalaal M. Abdallah b , Georg Lentzen c , Mohamed T. Khayyal b a b c

Heliopolis University, 3 Cairo-Belbeis Desert Road, Cairo, Egypt Department of Pharmacology, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, Egypt Research and Development, bitop AG, Stockumer Straße 28, Witten, Germany

a r t i c l e

i n f o

Keywords: Inflammatory bowel disease Trinitrobenzenesulfonic acid Compatible solutes Ectoines Intestinal barrier

a b s t r a c t Evidence suggests an important role of intestinal barrier dysfunction in the etiology of inflammatory bowel disease (IBD). Therefore stabilizing mucosal barrier function constitutes a new therapeutic approach in its management. Ectoine is a compatible solute produced by aerobic chemoheterotrophic and halophilic/halotolerant bacteria, where it acts as osmoprotectant and effective biomembrane stabilizer, protecting the producing cells from extreme environmental stress. Since this natural compound was also shown to prevent inflammatory responses associated with IBD, its potential usefulness was studied in a model of colitis. Groups of rats were treated orally with different doses of ectoine (30–300 mg/kg) or sulfasalazine (reference drug) daily for 11 days. On day 8 colitis was induced by intracolonic instillation of 2,4,6trinitrobenzenesulfonic acid, when overt signs of lesions develop within the next 3 days. On day 12, blood was withdrawn from the retro-orbital plexus of the rats and the animals were sacrificed. The colon was excised and examined macroscopically and microscopically. Relevant parameters of oxidative stress and inflammation were measured in serum and colon homogenates. Induction of colitis led to marked weight loss, significant histopathological changes of the colon, and variable changes in levels of myeloperoxidase, reduced glutathione, malondialdehyde, and all inflammatory markers tested. Treatment with ectoine ameliorated the inflammatory changes in TNBS-induced colitis. This effect was associated with reduction in the levels of TNF-␣, IL-1␤, ICAM-1, PGE2 and LTB4 . The findings suggest that intestinal barrier stabilizers from natural sources could offer new therapeutic measures for the management of IBD. © 2013 Elsevier GmbH. All rights reserved.

Introduction Inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, are severe chronic inflammatory disorders of the gastrointestinal tract. Although their etiology and pathophysiology are still unclear, an impaired intestinal barrier function, dysbiosis (an imbalance in the intestinal bacterial ecosystem) and immunologic mechanisms have been advocated an important role (Dotan and Mayer, 2002; Melmed and Abreu, 2004; Hanauer, 2006; Shaw et al., 2011; Kiesslich et al., 2012). IBD affects adversely the quality of life and necessitates longterm dependence on effective drugs (Carty and Rampton, 2003). Mesalazine, sulfasalazine and other 5-aminosalicylic acid (ASA) derivatives are considered currently drugs of choice for the

∗ Corresponding author at: Scientific Department, Steigerwald Arzneimittelwerk, Havelstr. 5, D-64295 Darmstadt, Germany. Tel.: +49 6151 3305 202; fax: +49 6151 3305 471. E-mail address: [email protected] (H. Abdel-Aziz). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.

management of most cases, while corticosteroids and immunosuppressants are retained for more severe forms of the disease. Although these drugs are effective, the risk of adverse effects is high, especially considering the chronic and relapsing nature of this condition. Therefore, the search for new safer therapies continues. Due to recent evidence suggesting an important role of intestinal barrier dysfunction in the etiology of IBD, the use of compounds able to stabilize mucosal barrier function constitutes a new therapeutic approach to the disease, especially for the prevention of flares. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, Fig. 1) is a natural zwitterionic, low molecular weight, strong water binding organic molecule, which was first isolated from Ectothiorhodospira halochloris. Later it was also found in several aerobic chemoheterotrophic and halophilic/halotolerant bacteria. Nowadays, commercially available ectoine is produced biotechnologically in high purity (≥95%) using the so-called “bacterial milking” fermentation technology followed by downstream purification (Pastor et al., 2010). Like other compatible solutes (CS), ectoine confers resistance towards environmental stress conditions such as high temperature,


H. Abdel-Aziz et al. / Phytomedicine 20 (2013) 585–591


delivering the required dose of TNBS/ethanol solution, the catheter was left in place for 30 s and then removed gently. Rats were kept for another 30 s in this position to avoid leakage of the instillate.

NH N Fig. 1. The chemical structure of ectoine (1,4,5,6-tetrahydro-2-methyl-4pyrimidinecarboxylic acid).

freezing, extreme dryness and high salinity (Lippert and Galinski, 1992; Galinski, 1993). In addition to their function as osmoprotectants, CSs are characterized by being effective stabilizers of biomolecules, including proteins, nucleic acids and biomembranes. These properties, in addition to the fact that CSs are biologically inert and do not interfere with overall cellular functions, even though they accumulate in high concentration in the cytoplasm, make them potential candidates as safe treatment options for IBD. Since ectoine was also shown to prevent inflammatory responses relevant to IBD (Nielsen et al., 1994; Goke et al., 1997; Danese et al., 2003; Bünger and Driller, 2004; Buommino et al., 2005), the aim of the present study was to investigate its potential usefulness in an animal model of colitis. Methods Drugs 2,4,6-Trinitrobenzene sulfonic acid (TNBS) was purchased from Sigma-Aldrich (Schnelldorf, Germany). Ectoine was provided by bitop AG (Witten, Germany) and administered as an aqueous solution. Sulfasalazine was a gift from El-Kahira Pharmaceutical Company (Cairo, Egypt) and was used as a suspension in 1% methylcellulose. Animals Adult male Wistar rats, weighing 150–200 g each, were obtained from ‘The Modern Veterinary Office for Laboratory Animals’, Cairo, Egypt and left to acclimatize at the animal facility of the Faculty of Pharmacy, Cairo University, for one week, before subjecting them to experimentation. They were provided with a standard pellet diet and given water ad libitum. The animals were kept at a temperature of 22 ± 3 ◦ C and a 12-h light/dark cycle as well as a constant relative humidity throughout the experimental period. The study was carried out according to The European Communities Council Directive of 1986 (86/609/EEC) and approved by the Ethical Committee for Animal Experimentation at the Faculty of Pharmacy, Cairo University. Induction of colitis Colitis was induced using 2,4,6-trinitrobenzene sulfonic acid (TNBS) as described by Morris et al. (1989). In brief, rats that had been deprived of food but not water for 36 h were lightly anesthetized with diethyl ether. A polyethylene catheter (3 mm diameter) fitted onto a 1-ml syringe was inserted rectally into the colon so that the tip was 8 cm proximal to the anus. In a headdown position, 0.25 ml of 50% (v/v) ethanol containing 50 mg/kg TNBS was then slowly instilled into the lumen of the colon. After

Experimental design Determination of the potential protective effect of ectoine Rats were randomly allocated to 8 groups of 8 animals each. Two groups were given distilled water and were designated as normal control and TNBS control groups. The other groups were treated either with ectoine (30, 50, 100, 200 and 300 mg/kg) or sulfasalazine (300 mg/kg) as a reference drug. The drugs were administered by oral gavage once daily throughout the experimental period (11 days). On day 8, colitis was induced by intra-colonic injection of TNBS in all groups except the normal control group which received vehicle instead. Animals were weighed immediately before colitis induction and just before autopsy to determine the effect of colitis on body weight. The rats were sacrificed on day 12 by cervical dislocation and the distal 8 cm portion of the colon was excised, opened longitudinally and thoroughly rinsed in ice-cold normal saline. The colon segments were placed on an ice-cold dissecting surface, cleaned of fat and mesentery, blotted on filter paper and weighed. Mucosal damage was assessed by measuring the ulcerative area (cm2 ) in the mucosa. The colon mass index (ratio of colon weight to total body weight) was calculated in terms of mg/g. The index was taken as a measure of the degree of colonic edema and the severity of inflammation. The excised portion of the colon was then homogenized in ice-cold bi-distilled water to obtain a 10% (w/v) homogenate and divided into two aliquots which were used for spectrophotometeric estimation of reduced glutathione (GSH) (Beutler et al., 1963) and myeloperoxidase (MPO) activity (Krawisz et al., 1984).

Mechanisms underlying the protective action of ectoine A further four groups of animals, each consisting of 8 rats were designated as normal control, TNBS control, ectoine treated (100 mg/kg) and sulfasalazine treated (300 mg/kg). The groups were subjected to the same protocol of treatment mentioned above. On day 12 (day of sacrifice), blood samples were withdrawn from the retro-orbital plexus of each rat under light ether anesthesia. The separated sera were divided into several aliquots. One aliquot was used for the spectrophotometric estimation of total nitrate/nitrite (NOx ) (Miranda et al., 2001). The remaining aliquots were used to determine tumor necrosis factor alpha (TNF-␣), interleukin-1␤ (IL-1␤), interleukin-10 (IL-10), intercellular adhesion molecule-1 (ICAM-1), leukotriene B4 (LTB4 ) and prostaglandin E2 (PGE2 ) using specific ELISA kits (R&D Systems GmbH, Wiesbaden, Germany). After withdrawal of blood, the rats were sacrificed and the distal 8 cm section of the colon was excised and homogenized. The homogenate was divided into two aliquots:one was used for the estimation of malondialdehyde (MDA) (Mihara and Uchiyama, 1978), while the other was centrifuged at 13,000 rpm for 30 min at 4 ◦ C and the resultant supernatant was frozen at −20 ◦ C for assaying TNF-␣, IL-1␤, IL-10, ICAM-1, PGE2 , LTB4 and NOx . In addition, the spleen was excised, blotted on filter paper and weighed. The spleen mass index (ratio of spleen weight in mg to animal weight in g) was taken as a further marker of inflammation (Cuzzocrea et al., 2003). The colon segments from 2 randomly chosen animals of each group were fixed in 10% (v/v) formalin and preserved for histological examination.

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Fig. 2. Effect of pre-treatment with ectoine on the change in body weight of animals with TNBS-induced colitis. Ectoine and sulfasalazine were administered orally to rats for 11 days. On day 8, colitis was induced in all groups except normal control group. The change in body weight was taken as the difference between the weight before induction of colitis and that immediately before sacrifice on day 12. Each column represents the mean of at least 6 animals ± s.e.m. *denotes statistical significance at p ≤ 0.05 vs TNBS control, # denotes statistical significance at p ≤ 0.05 vs normal control.

Statistical analysis Values were computed as means ± s.e.m. Comparisons between means were carried out using one way analysis of variance (ANOVA), followed by Student–Newman–Keuls multiple comparisons test. A probability (p) level of 0.05 or less was considered as being significant in all types of statistical tests. Fig. 3. Effect of pre-treatment with ectoine on mucosal damage assessed as ulcerative area (A) and colon mass index (B). Ectoine and sulfasalazine were administered orally to rats for 11 days. On day 8, colitis was induced in all groups except normal control group. On day 12, the rats were sacrificed, the distal 8 cm of the colon excised, and the ulcerative area was then measured in cm2 . The ratio of colon weight (mg) to body weight (g) was taken as the colon mass index. Each column represents the mean of at least 6 animals ± s.e.m. *denotes statistical significance at p ≤ 0.05 vs TNBS control, # denotes statistical significance at p ≤ 0.05 vs normal control.

Results Body weight, macroscopic damage and colon mass index Induction of colitis with TNBS led to marked weight loss of the animals, an effect which was offset partially by sulfasalazine. Ectoine tended to normalize the rate of weight gain, particularly in a dose of 100 mg/kg. Smaller and higher doses had a lesser effect (Fig. 2). Ectoine also significantly reduced the ulcerative area (Fig. 3A) and colon mass index (Fig. 3B) in TNBS treated animals, again showing the optimal effect in a dose of 100 mg/kg, an effect which was comparable to that of 300 mg/kg sulfasalazine.

TNBS controls. The effect of ectoine (100 mg/kg) was nearly similar in magnitude to the reference dose of sulfasalazine (300 mg/kg). Neither TNBS nor pre-treatment with any of the drugs had any observed effect on MDA levels (data not shown), indicating a lack of effect on lipid peroxidation.

Tissue GSH content, MDA and colonic MPO activity

Inflammation markers

The favorable effect of ectoine on macroscopic changes was reflected in an improvement of colonic GSH level (Fig. 4A) and MPO activity (Fig. 4B). The best beneficial effect of ectoine was exerted by the dose of 100 mg/kg. This dose increased the colonic GSH level to 162% and reduced colonic MPO activity to 41% as compared to

TNBS induced a rise in the level of all the pro-inflammatory mediators measured in both colon and serum, namely TNF-␣, IL1␤, PGE2 , LTB4 and ICAM-1 (Table 1). The effect of ectoine was in all cases comparable to that of sulfasalazine. Both compounds were effective in tending to normalize to different extents the deranged

Table 1 Effect of pre-treatment with ectoine or sulfasalazine (sulfasal) on colonic and serum levels of various mediators in rats with TNBS-induced colitis. Colonic tissue concentrations Control TNF-␣ IL-1␤ ICAM-1 LT-B4 PGE2 IL-10 NOx * #

(pg/g) (ng/g) (ng/g) (ng/g) (ng/g) (ng/g) (␮M/g)

566.9 23.8 153.7 2.9 43.7 1.5 281.5

± ± ± ± ± ± ±

11.4 1.0 4.1 0.2 4.6 0.05 20.1

TNBS 693.0 114.7 187.0 5.2 79.4 1.7 153.5

± ± ± ± ± ± ±

24.3# 5.1# 10.4# 0.3# 2.7# 0.07# 14.4#

Serum concentrations

Ectoine 599.0 80.7 118.2 3.2 55.5 1.2 281.1

Denotes statistical significance at p < 0.05 vs TNBS control. Denotes statistical significance at p < 0.05 vs normal control.

± ± ± ± ± ± ±

9.4* 5.6*# 10.4* 0.7* 4.8* 0.02* 20.0*



565.3 90.9 125.7 3.9 59.6 1.4 264.9

± ± ± ± ± ± ±

17.2* 7.0* # 8.2* 0.7 5.4* 0.04* 37.3*

Control (pg/ml) (pg/ml) (ng/ml) (pg/ml) (ng/ml) (pg/ml) (␮M)

33.0 130 61.5 180.3 6.6 78.1 28.5

± ± ± ± ± ± ±

0.3 3.7 2.2 35.2 0.3 1.8 2.6

TNBS 35.3 170.6 95.2 839.3 7.8 99.2 46.8

± ± ± ± ± ± ±

0.7# 13.5# 5.2# 73.1# 0.1# 4.5# 3.8#

Ectoine 34.0 136.2 59.0 698.4 7.1 82.9 44.3

± ± ± ± ± ± ±

0.3 3.7* 1.4* 42.1# 0.1* 1.3* 4.8

Sulfasal 34.2 153.4 69.4 706.9 7.0 82.2 36.6

± ± ± ± ± ± ±

0.8 9.5 4.2* 46.3# 0.1* 3.2* 4.8


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Fig. 5. Effect of pre-treatment with ectoine on spleen index of rats with TNBSinduced colitis. Ectoine and sulfasalazine were administered orally to rats for 11 days. On day 8, colitis was induced in all groups except normal control group. On day 12, the rats were sacrificed and the spleen was weighed. The ratio of spleen weight (mg) to body weight (g) was taken as the spleen index. Each column represents the mean of 8–10 animals ± s.e.m. *denotes statistical significance at p ≤ 0.05 vs TNBS control, # denotes statistical significance at p ≤ 0.05 vs normal control.

After pre-treatment with ectoine, the colon of animals showed only few areas of ulceration with no remaining necrosis and moderate lymphocytic infiltration of the submucosa but not extending to the musculosa (Fig. 6C). The colon of animals pre-treated with sulfasalazine showed no necrosis of the mucosa with moderate edema of the submucosa and few neutrophilic infiltrations (Fig. 6D). Discussion Fig. 4. Effect of pre-treatment with ectoine on reduced glutathione levels (A) and MPO activity (B) in colonic tissue of rats with TNBS-induced colitis. Ectoine and sulfasalazine were administered orally to rats for 11 days. On day 8, colitis was induced in all groups except normal control group. On day 12, the rats were sacrificed and GSH and MPO activity were determined in colon homogenates. Each column represents the mean of at least 6 animals ± s.e.m. *denotes statistical significance at p ≤ 0.05 vs TNBS control, # denotes statistical significance at p ≤ 0.05 vs normal control.

mediator levels, particularly in the colonic tissue. With IL-1␤, however, the effect was more pronounced in the serum than in colonic tissue, leading to complete normalization of the former. The level of the anti-inflammatory cytokine, IL-10, was significantly elevated by TNBS in both colon and serum samples to 116% and 127%, respectively (Table 1) but the effect was reversed back to nearly normal levels after treatment with either ectoine or sulfasalazine. The protective effect of ectoine was further evidenced by its effect in limiting the marked rise in spleen index induced by TNBS (Fig. 5). Furthermore, TNBS administration resulted in a reduction in the colonic levels of NOx , but led to marked elevation of its serum levels (Table 1). Ectoine was effective in completely normalizing colon but not serum NOx levels in a manner comparable to that of sulfasalazine. Histological changes The colon of the animals subjected to TNBS showed large confluent areas of complete necrosis of the mucosa with intervening skip areas which were nearly normal. Marked edema with dense transmural infiltration of neutrophils extending from under the necrotic areas through the musculosa and the serosa was also seen. Arteries and arterioles under the necrotic areas showed fibrinoid necrosis of the vascular walls. The rest of the wall showed edema and moderate infiltration of lamina propria and submucosa with lymphocytes, plasma cells and neutrophils (Fig. 6B).

Defects in intestinal barrier function have been associated with the inflammatory condition in IBD (Meddings, 2008; Marchiando et al., 2010). Increased intestinal permeability has been observed in patients with Crohn’s disease (May et al., 1993; Peeters et al., 1994, 1997) and has been shown to be a prognostic indicator of relapse in both Crohn’s disease and ulcerative colitis (Wyatt et al., 1993; Kiesslich et al., 2012). These findings make intestinal barrier stabilization an interesting new therapeutic target in the management of IBD, especially for the maintenance of remission. One group of naturally occurring compounds that could be potential candidates for intestinal barrier stabilization are the compatible solutes. Ectoine is a CS synthesized in response to extreme environmental conditions and acts to protect biopolymers (e.g. proteins, nucleic acids and lipids) from dehydration and other stress conditions, which could lead to conformational changes and loss of biological activity. When added to mammalian cells, ectoine was shown to possess many protective effects including stabilization of cell membranes (Harishchandra et al., 2010), cytoprotection of human keratinocytes (Buommino et al., 2005) and protection of ileal mucosa and muscularis against ischemia and reperfusion injury (Wei et al., 2009). In the present study, its potential to protect rats from intestinal damage induced by TNBS/ethanol as a model of experimental IBD was investigated. This model shares many histopathological and clinical features of human IBD and has been used by several authors to study the pathogenesis of colonic inflammation as well as to assess the therapeutic potential of several agents in IBD. Intra-colonic administration of TNBS resulted in extensive inflammation and necrosis as well as loss of body weight. The inflammatory changes of the colon were associated with marked increase in colon mass index, an indicator of the degree of colonic edema (Wang et al., 2001) and in spleen index, a marker of inflammation (Cuzzocrea et al., 2003). Colitis was also associated with massive neutrophilic infiltration which was shown histologically and further evidenced by a rise in colonic MPO activity. Several

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Fig. 6. Effect of pre-treatment with ectoine on TNBS- induced histological changes of colonic samples. (A) Normal control group showing the normal histology of the rat colon (H & E; magnification, ×60). (B) TNBS control group showing large confluent areas of complete necrosis of the mucosa with intervening skip areas which were nearly normal (H & E; magnification, ×60). (C) Ectoine (100 mg/kg) pre-treated rats showing few areas of ulceration as well as moderate lymphocytic infiltration of submucosa (H & E; magnification, ×100). (D) Sulfasalazine (300 mg/kg) pre-treated rat showing no necrosis of mucosa as well as moderate edema and cellular infiltration of the submucosa (H & E; magnification, ×100).

studies have shown that neutrophils play an important role in the pathogenesis of IBD as they release a variety of inflammatory mediators (Nielsen and Rask-Madsen, 1996; Sartor, 1997; Kolios et al., 1998). In the present study, the levels of many inflammatory mediators, such as TNF-␣, IL-1␤, LTB4 and PGE2 were also increased. TNF-␣ also acts as a potent chemo-attractant (Stallmach et al., 2004), and may account, in part, for neutrophil infiltration in the inflamed colonic tissue. Treatment with ectoine was found to protect against the loss in body weight and to reduce the area of colonic lesions in a bellshaped manner: there was a gradual increase in beneficial effect with increasing doses of ectoine, reaching a peak using a dose of 100 mg/kg, then the beneficial effect started to decline as the dose was further increased. The reduction in the severity of the colonic lesions induced by a dose of 100 mg/kg ectoine was comparable to that induced by 300 mg/kg sulfasalazine. The bell-shaped dose response relationship was extended to the effect of ectoine in reducing the colon mass index and MPO activity and in elevating the reduced GSH levels. A similar bell-shaped dose–response relationship was also reported by Buommino et al. (2005), who showed that a prominent expression of Hsp70B gene was observed in keratinocytes in vitro at a concentration of 100 ␮g/ml ectoine, whereas smaller and larger concentrations produced lesser effects. Ectoine, and other CSs, are believed to exert their protective effects by stabilizing the native conformation of biological macromolecules through a so-called “preferential exclusion” phenomenon, whereby these agents are excluded as such from the immediate hydration shell of biomolecules, such as proteins, and preferentially hydrate the protein surface through their property of being strong water structure formers (Arakawa and Timasheff, 1985; Collins and Washabaugh, 1985; Yu et al., 2007; Roychoudhury et al., 2012).

They are biologically inert and do not directly interact with the protein surface, but they slow down the diffusion of solvent molecules by strongly interacting with water molecules and consequently enhance the stability by making denaturation thermodynamically less favourable. A recent study shows that ectoines increase the hydration of a model biological membrane resulting in higher membrane fluidity (Harishchandra et al., 2010). The increased hydration and fluidization of the cell membrane may help to withstand membrane-damaging stressors and might also accelerate repair mechanisms. The type of dose–response relationship shown indicates that there is an optimal concentration of the solute that will yield maximal stabilization of cellular macromolecules and cell membranes, and hence maximal intestinal barrier stabilization. This optimal concentration seems to have been reached in the present experimental setting with a dose of 100 mg/kg. The reduction in MPO activity, spleen mass index and the measured pro-inflammatory mediators, point to an additional antiinflammatory effect, which could be secondary to the intestinal barrier stabilizing activity. Yet, earlier studies suggest that ectoine might interfere with pro-inflammatory signaling. For instance, treatment with ectoine was shown to protect against nanoparticleinduced neutrophilic lung inflammation in rats (Sydlik et al., 2009) and to restore apoptosis rates in neutrophils obtained from healthy individuals and COPD patients (Sydlik et al., 2012). In vitro, pretreatment with ectoine was reported to reduce the expression of the adhesion molecule, ICAM-1 (Bünger and Driller, 2004). Furthermore, ectoine was found to block nuclear translocation of NF-␬B, to down-regulate the expression of the pro-inflammatory cytokines IL-1␣, IL-6, IL-8 and TNF-␣ and to inhibit MAP-kinase activation (Buommino et al., 2005; Sydlik et al., 2009). These effects might


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be partially mediated by ectoine’s impact on membrane fluidity leading to interference with membrane-coupled pro-inflammatory signaling (Peuschel et al., 2010). Another important mediator of inflammation, PGE2 , was found to be markedly elevated after colitis induction, but effectively reduced to nearly normal levels by ectoine. PGE2 is intricately associated with the formation of inflammatory edema and hyperemia (Sánchez-Fidalgo et al., 2007) on one hand, and with modulation of the intestinal immune response and the production of proinflammatory cytokines such as TNF-␣ and IL-1␤ on the other (Newberry et al., 1999). PGE2 also promotes intestinal cell chloride and water secretion (Lanza et al., 1991) and could therefore play a contributing role to the occurrence of diarrhea in IBD patients (treated animals showed normal fecal consistency: unpublished observation). This may partly account for the observed loss of weight in IBD patients and TNBS treated animals. Another factor contributing to the etiology of diarrhea is IL-1␤ (Siegmund, 2002). The ability of ectoine to reduce the elevated levels of PGE2 and IL1␤ may help to explain the fact that treated animals did not lose as much body weight as those subjected only to TNBS. Yet another important mediator which is usually implicated in the pathogenesis of intestinal inflammation is NO (Grisham and Yamada, 1992; Middleton et al., 1993; Ribbons et al., 1995). However, small quantities of NO are essential for the maintenance of mucosal integrity of the gastrointestinal tract (Pfeiffer and Qiu, 1995). TNBS administration resulted in a significant decrease in colonic NO levels, an effect which may have been due to the increase in arginase expression. This was shown to happen in the microvessels and submucosal gut tissues of IBD patients (Horowitz et al., 2007). Arginase competes enzymatically with NOS for the common substrate l-arginine, hence the loss of NO production. Treatment with ectoine normalized the levels of NO, thereby helping to maintain mucosal integrity. On the other hand, TNBS led to marked elevation of serum NO level, which was not affected by ectoine, supporting the hypothesis of a more local effect of the CS. With regards to the oxidative stress induced by TNBS colitis, the present findings showed marked reduction in GSH. It would have been expected that this would be associated with an increase in lipid peroxidation and a rise in MDA level, but this was not the case. This is in line with other investigators who showed that the plasma level of MDA was not affected in IBD patients (Bhaskar et al., 1995; Tüzün et al., 2002). Accordingly, neither ectoine nor sulfasalazine had any effect on colonic MDA levels. In conclusion, the results of this study showed that the natural compatible solute ectoine could ameliorate the colonic inflammatory changes in the TNBS model of rat colitis. This effect was associated with a reduction in the levels of some of the mediators involved in the inflammatory response of the intestine, such as TNF-␣, IL-1␤ and PGE2 . Although it is difficult to extrapolate findings from animal models to the clinical situation, the present study suggests that ectoine may pave the way for developing intestinal barrier stabilizers from natural sources as a new class of therapeutic agents for the management of human IBD.

Disclosures Georg Lentzen was formerly employed by bitop AG, Witten Germany. All other authors declare no conflict of interest.

Acknowledgements The presented work received funding from bitop AG, Witten, Germany.

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