Article pubs.acs.org/jnp
Bacteria-Derived Compatible Solutes Ectoine and 5α-Hydroxyectoine Act as Intestinal Barrier Stabilizers to Ameliorate Experimental Inflammatory Bowel Disease Heba Abdel-Aziz,*,†,⊥ Walaa Wadie,‡ Olaf Scherner,§ Thomas Efferth,⊥ and Mohamed T. Khayyal‡ †
Scientific Department, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, Darmstadt, Germany Department of Pharmacology, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, Egypt § Research and Development, Bitop AG, Stockumer Straße 28, Witten, Germany ⊥ Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany ‡
ABSTRACT: Earlier studies showed that the compatible solute ectoine (1) given prophylactically before induction of colitis by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in rats prevented histological changes induced in the colon and the associated rise in inflammatory mediators. This study was therefore conducted to investigate whether ectoine (1) and its 5α-hydroxy derivative (2) would also be effective in treating an already established condition. Two days after inducing colitis in rats by instilling TNBS/alcohol in the colon, animals were treated orally once daily for 1 week with either 1 or 2 (50, 100, 300 mg/kg). Twenty-four hours after the last drug administration rats were sacrificed. Ulcerative lesions and colon mass indices were reduced by 1 and 2 in a bell-shaped manner. Best results were obtained with 100 mg/kg ectoine (1) and 50 mg/kg 5αhydroxyectoine (2). The solutes normalized the rise in myeloperoxidase, TNFα, and IL-1β induced by TNBS but did not affect levels of reduced glutathione or ICAM-1, while reducing the level of fecal calprotectin, an established marker for inflammatory bowel disease. The findings indicate that the naturally occurring compatible solutes ectoine (1) and 5α-hydroxyectoine (2) possess an optimum concentration that affords maximal intestinal barrier stabilization and could therefore prove useful for better management of human inflammatory bowel disease. called “preferential exclusion” or “preferential hydration” phenomenon, whereby these agents are expelled or excluded from the immediate hydration shell of biomolecules, such as proteins or lipid membranes, resulting in the modulation of the solvent characteristics of surrounding water. Thus, compatible solutes are able to form a protective and stabilizing hydrate capsule around the protein and therefore help to protect biomolecules and proteins from irreversible structural modifications by inhibiting dehydration. This indirect effect leads to a more compact and more stable folding of proteins and increases the stability of lipid membranes by increasing their fluidity.11−16 One of these compatible solutes is ectoine (1) (1,4,5,6tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), a natural zwitterionic, low molecular weight, strong water binding organic molecule, which was first isolated from Ectothiorhodospira halochloris. Later it was found to be widely synthesized by chemoheterotrophic and halophilic/halotolerant bacteria.17 1 is currently produced commercially in high purity (>95%) using the so-called “bacterial milking” fermentation technology followed by downstream purification.18
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he current treatments for inflammatory bowel disease (IBD), which commonly manifests itself in the form of Crohn’s disease or as ulcerative colitis, are not satisfactory.1,2 The commonly used drugs include mesalazine, sulfasalazine, and other 5-aminosalicylic acid derivatives, while corticosteroids and immune suppressants are retained for more severe forms of the disease.2 Although these drugs are effective, the risk of adverse effects is high,3 especially since patients have to continue taking them on a long-term basis, considering the chronic and relapsing nature of the condition. Therefore, the search for new, safer therapies continues. Defects in intestinal barrier function play a crucial role in the pathogenesis of inflammatory bowel disease.4−7 Increased intestinal permeability has been observed in patients with Crohn’s disease5,8,9 and has been shown to be a prognostic indicator of relapse in both Crohn’s disease and ulcerative colitis.6,10 These findings make intestinal barrier stabilization an interesting 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 is the group of compatible solutes (CS). CS are biologically inert and are believed to exert their protective effects by stabilizing the native conformation of biological macromolecules through a so© XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 14, 2015
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DOI: 10.1021/acs.jnatprod.5b00115 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Ectoine (1) and the closely related 5α-hydroxyectoine (2) ((4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4carboxylic acid) are synthesized in response to extreme environmental conditions and act to protect biopolymers from dehydration and other stress conditions, which could lead
Figure 1. Effect of treatment with ectoine (1) or 5α-hydroxyectoine (2) on weight gain (A), ulcerative area (B), and colon mass index (C) of animals with TNBS-induced colitis. Colitis was induced by rectal instillation of TNBS/ethanol. Forty-eight hours after induction, 1, 2, or sulfasalazine was administered orally once per day for 1 week at the indicated dose levels [mg/kg]. Each column represents the mean of at least 6 animals ± SEM. * denotes statistical significance at p < 0.05 vs TNBS control (black column); # denotes statistical significance at p < 0.05 vs untreated normal control (white column). B
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Figure 2. Effect of treatment with ectoine (1) or 5α-hydroxyectoine (2) on myeloperoxidase (MPO) activity (A) and reduced glutathione (GSH) levels (B) in colonic tissue homogenates of rats with TNBS-induced colitis. Colitis was induced by rectal instillation of TNBS/ethanol. Forty-eight hours after induction, 1, 2, or sulfasalazine was administered orally once per day for 1 week at the indicated dose levels (mg/kg). Each column represents the mean of at least 6 animals ± SEM. * denotes statistical significance at p < 0.05 vs TNBS control (black column); # denotes statistical significance at p < 0.05 vs untreated normal control (white column).
sulfonic acid (TNBS) in rats, prevented to a large extent the histological changes induced by TNBS in the colon and the associated rise in inflammatory mediators.24 The present study was therefore conducted to investigate whether ectoine (1) would also prove effective in treating an already established colitis by TNBS and to compare its efficacy with 5αhydroxyectoine (2). For this purpose, we again used the wellestablished TNBS/ethanol model of experimental IBD.25,26
to conformational changes and loss of biological activity. When added to mammalian cells, 1 was shown to stabilize cell membranes,19 to act as a cytoprotectant in human keratinocytes,20 and to protect ileal mucosa and muscularis against ischemia and reperfusion injury.11 In studies on lung inflammation induced by carbon nanoparticles or allergy, ectoine (1) has been reported to interfere with signaling pathways, such as mitogen-activated protein (MAP) kinase and various cytokines, and to reduce neutrophilic infiltration, thereby reducing the inflammatory response.21,22 5α-Hydroxyectoine (2) was also shown to protect rat livers from cold ischemic reperfusion injury.23 A recent study showed that ectoines increase the hydration of a model biological membrane, resulting in higher membrane fluidity.19 The increased hydration and fluidization of the cell membrane may help to withstand membrane-damaging stressors and might also accelerate repair mechanisms. Recently, we have shown that ectoine (1), given for several days before the induction of colitis by 2,4,6-trinitrobenzene-
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RESULTS AND DISCUSSION Induction of colitis led to severe inhibition of the rate of weight gain of the rats during the experimental period as compared to normal untreated controls. Treatment with ectoine (1), 5αhydroxyectoine (2), or sulfasalazine tended to slightly ameliorate the rate of growth, albeit not to a statistically significant extent (Figure 1A). Intracolonic administration of TNBS also resulted in extensive inflammation and necrosis of the colon. Both 1 and 2 reduced the ulcerative area from 4.9 cm2 to 1.2−2.7 cm2 and 1.3−2.3 cm2, respectively, according to C
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Figure 3. Effect of treatment with ectoine (1) or 5α-hydroxyectoine (2) on TNF-α (A), IL-1ß (B), and ICAM-1 (C) levels in colonic tissue homogenates of rats with TNBS-induced colitis. Colitis was induced by rectal instillation of TNBS/ethanol. Forty-eight hours after induction, 1, 2, or sulfasalazine was administered orally once per day for 1 week in the indicated dose levels [mg/kg]. Each column represents the mean of at least 6 animals ± SEM. * denotes statistical significance at p < 0.05 vs TNBS control (black column); # denotes statistical significance at p < 0.05 vs untreated normal control (white column).
Previous studies on the prophylactic effect of the drug against the development of TNBS colitis had shown that doses lower than 50 mg/kg exerted less beneficial effects (unpublished observations). The same phenomenon was also observed for
the dose used. Ectoine (1) showed a peak activity at 100 mg/ kg. Smaller and higher doses had a lesser effect (Figure 1B). 5αHydroxyectoine (2), however, exhibited a maximum effect at a dose of 50 mg/kg. Higher doses were less active (Figure 1B). D
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was found to decrease nuclear translocation of NF-κB, to downregulate the expression of the pro-inflammatory mediators IL1α, IL-6, IL-8, TNF-α, and PGE2, and to inhibit MAP-kinase activation.20,22,24 These findings are in line with the present study, where TNF-α and IL-1β were markedly elevated after colitis induction, but effectively reduced to normal levels by ectoine (1) and 5α-hydroxyectoine (2). TNF-α is one of the most important mediators in inflammatory bowel disease, and TNFα antagonists (biologicals) are widely and successfully used to treat IBD patients and to induce remission. IL-1β was shown to promote Th17 cell differentiation, which seems to play an important role in IBD and other inflammatory diseases.35 Fecal calprotectin is one of the few established fecal disease markers for inflammatory bowel disease.36 Only a small amount of calprotectin could be detected in the feces of healthy control animals (Figure 4). The TNBS group that was not treated,
the colon mass index, an indicator of the degree of colonic edema,27 which was reduced by almost 50% in response to ectoine (1) (100 mg/kg) and 5α-hydroxyectoine (2) (50 mg/ kg) as compared to the TNBS control (from 7.9 mg/g to 4.3 mg/kg and 4.0 mg/kg, respectively; Figure 1C). The effect of these doses was comparable to that of sulfasalazine (300 mg/ kg). Colitis was also associated with massive neutrophilic infiltration, which was detected as a rise in colonic myeloperoxidase (MPO) activity (Figure 2A). The favorable effects of ectoine (1) and 5α-hydroxyectoine (2) on macroscopic changes were reflected in an improvement of MPO activity, showing again optimal effects at 100 mg/kg for 1 and 50 mg/kg for 2 (Figure 2A). On the other hand, the colonic GSH levels were hardly affected by either treatment or by sulfasalazine (Figure 2B). Only a slight, but statistically nonsignificant tendency to improve depleted GSH levels was observed with both agents. Treatment with ectoine (1) or 5α-hydroxyectoine (2) reduced the area of colonic lesions, colon mass index, and MPO activity to similar extents in a bell-shaped manner. A similar bell-shaped response was also reported by Buommino et al.,20 who showed that a prominent expression of the Hsp70B′ gene was observed in keratinocytes in vitro at a concentration of 100 μg/mL ectoine (1), whereas smaller and larger concentrations produced lesser effects. A similar bell-shaped effect was also shown for 1 in the prophylactic setting for TNBS-induced colitis.24 The current type of bell-shaped response 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 for ectoine (1) and 50 mg/kg for 5αhydroxyectoine (2). Several studies have shown that neutrophils play an important role in the pathogenesis of inflammatory bowel disease, as they release a variety of inflammatory mediators.28−30 In the present study, the elevated MPO activity was accompanied by a marked increase in colonic levels of the proinflammatory cytokines TNF-α and IL-1β (Figure 3A, B). TNF-α also acts as a potent chemoattractant31 and may account, in part, for neutrophil infiltration in the inflamed colonic tissue. Both ectoine (1) and 5α-hydroxyectoine (2) at all doses (except the highest dose of 2) were able to completely normalize the levels of TNF-α and IL-1β. The reduction in MPO activity, colon mass index, and TNFα and IL-1β levels points to an additional inflammationreducing effect that could be secondary to the intestinal barrier stabilizing activity. Earlier studies suggest that ectoine (1) might also suppress pro-inflammatory mediators, an effect that is probably mediated by its biophysical impact on membrane fluidity, leading to interference with membrane-coupled proinflammatory signaling.32 For instance, treatment with ectoine (1) was shown to protect against nanoparticle-induced neutrophilic lung inflammation in rats22 and to restore apoptosis rates in neutrophils obtained from healthy individuals and COPD patients.33 In vitro, pretreatment with ectoine (1) was also reported to reduce UVA-induced expression of the adhesion molecule ICAM-134 in keratinocytes. This effect on ICAM-1, however, was not observed in the current study, where neither TNBS, ectoine (1), nor 5α-hydroxyectoine (2) had any impact on ICAM-1 levels (Figure 3C). Furthermore, 1
Figure 4. Effect of treatment with ectoine (1) or 5α-hydroxyectoine (2) on fecal calprotectin levels in rats with TNBS-induced colitis. Colitis was induced by rectal instillation of TNBS/ethanol. Forty-eight hours after induction, 100 mg/kg ectoine (1) or 5α-hydroxyectoine (2) was administered orally once per day for 1 week. Fecal samples from 4 randomly chosen animals per group were harvested prior to sacrifice. All samples were run in duplicate, and the medians were calculated. The data points and the calculated medians are shown in a scatter plot.
either with ectoine (1) or 5α-hydroxyectoine (2), showed an erratic pattern. One animal showed only a small amount of calprotectin, but the other three samples contained significantly more calprotectin in comparison to the healthy control group. Ectoine (1) or 5α-hydroxyectoine (2) treatment reduced these levels nearly back to normal levels (Figure 4). Calprotectin is found in abundance in neutrophilic granulocytes, as well as in monocytes and macrophages. In active inflammatory bowel disease, an increased migration of polymorphonuclear neutrophils occurs from the circulation to the inflamed intestinal mucosa. Due to leukocyte shedding in the intestinal lumen, pro-inflammatory proteins such as calprotectin can be detected and measured in the stool. The concentration of calprotectin is directly proportional to the intensity of the neutrophilic infiltrate in the gut mucosa; hence it is used in the clinic as an indicator for disease activity as well as a predictor for relapse. It is also a useful and reliable surrogate for mucosal improvement and healing.36 In vitro studies indicated that ectoine (1) might affect Hsp70 expression.20 Hsp70 was shown to be elevated in the colonic mucosa of patients suffering from ulcerative colitis and to decrease upon treatment with conventional therapy (mesalazine or mesalazine and probiotics).37 It was also shown to stabilize and enhance the activity of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), an E
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Figure 5. Effect of treatment with ectoine (1) or 5α-hydroxyectoine (2) on heat shock protein (Hsp)70 levels in colonic tissue homogenates of rats with TNBS-induced colitis. Colitis was induced by rectal instillation of TNBS/ethanol. Forty-eight hours after induction, ectoine (1), 5αhydroxyectoine (2) (OH-ect.), or sulfasalazine was administered orally once per day for 1 week in the indicated dose levels (mg/kg). Each column represents the mean of at least 6 animals ± SEM. # denotes statistical significance at p < 0.05 vs untreated normal control (white column). ± 3 °C and a 12 h light/dark cycle as well as a constant relative humidity throughout the experimental period. The study was approved by the Ethical Committee for Animal Experimentation at the Faculty of Pharmacy, Cairo University (PCU09/27). Induction of Colitis. Colitis was induced by TNBS using essentially the method described by Morris et al.39 In brief, rats were deprived of food for 36 h prior to experimentation but had free access to water. Under light ether anesthesia, with the rats in a headdown position, 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. A 0.25 mL amount of 50% (v/v) ethanol containing 100 mg/kg TNBS was slowly instilled into the lumen of the colon. After 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. Experimental Design. Rats were randomly allocated to 11 groups of 8 animals each. Three groups served as normal controls. They were subjected to the same procedure described above, but instead of TNBS they had normal saline instilled in the colon. The control groups were treated orally with either water, ectoine (1) (100 mg/kg), or 5αhydroxyectoine (2) (100 mg/kg), respectively. Colitis was induced in the remaining 8 groups. Forty-eight hours later, animals were treated with water (colitis control), ectoine (1) (50, 100, or 300 mg/kg), 5αhydroxyectoine (2) (50, 100, or 300 mg/kg), or sulfasalazine (300 mg/kg) as a reference drug. All agents were administered orally once per day for 1 week after induction of colitis. Animals were weighed immediately before colitis induction and just before necropsy to determine the effect of colitis on body weight. Rats were sacrificed by cervical dislocation 24 h after the last compound administration, and the distal 8 cm portion of the colon was excised, opened longitudinally, and thoroughly rinsed in ice-cold normal saline. The colonic segments were then placed on an ice-cold dissecting surface, cleaned of fat and mesentery, blotted on filter paper, and weighed. Mucosal damage was assessed macroscopically by measuring planimetrically the ulcerative area (cm2) in the distal colon. The colon mass index (ratio of colon weight to total body weight) was calculated in terms of mg/g. This index was used as a measure of the degree of colonic edema and hence the severity of inflammation. Biochemical Parameters. The excised portion of the colon was then homogenized in ice-cold bidistilled water to obtain a 10% (w/v) homogenate, which was then divided into three aliquots. One aliquot was deproteinized with ice-cold 5% sulfosalicylic acid, then centrifuged at 3000 rpm for 20 min. The supernatant was used for spectrophotometric estimation of reduced glutathione (GSH) according to Beutler et al.40 The second aliquot was mixed with an equal volume of 100 mM phosphate buffer (pH 6) containing 1%
intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall in the intestine.38 NOD2 loses the ability to respond properly to bacterial cell wall fragments when it is mutated, and such mutations have been implicated in the pathogenesis of Crohn’s disease.38 Therefore, we measured Hsp70 concentration in our model. However, its concentration was only slightly affected by the current treatment regimens. Induction of colitis with TNBS tended to decrease its levels by only 15% (statistically nonsignificant). Treatment with the test compounds led to an additional decrease in its levels. This was especially true for 5αhydroxyectoine (2) and sulfasalazine (Figure 5). Treating normal animals with either ectoine (1) or 5αhydroxyectoine (2) did not affect any of the measured parameters to any statistically significant extent. In conclusion, the results of this study show that the natural compatible solutes ectoine (1) and 5α-hydroxyectoine (2) can ameliorate the colonic inflammatory changes in an established colitis in rats. 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-α and IL-1β. Although it is not always easy to extrapolate findings from animal models to humans, the present study suggests that ectoine (1) and 5α-hydroxyectoine (2), acting as naturally occurring intestinal barrier stabilizers, could represent a new class of therapeutic agents for the management of human inflammatory bowel disease.
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EXPERIMENTAL SECTION
Test Compounds. 2,4,6-Trinitrobenzenesulfonic acid was purchased from Sigma-Aldrich, Schnelldorf, Germany. Ectoine (1) and 5α-hydroxyectoine (2) were provided by Bitop AG (Witten, Germany) in a purity of ≥95% (HPLC) 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. The concentrations of all drugs used orally were adjusted so that the required dose per 200 g rat was made available in 1 mL of solution/suspension. 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 for 1 week before subjecting them to experimentation. They were provided with standard pellet diet and given water ad libitum. The animals were kept at a temperature of 22 F
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(13) Collins, K. D.; Washabaugh, M. W. Q. Rev. Biophys. 1985, 18, 323−422. (14) Yu, I.; Jindo, Y.; Nagaoka, M. J. Phys. Chem. B 2007, 111, 10231−10238. (15) Roychoudhury, A.; Haussinger, D.; Oesterhelt, F. Protein Pept. Lett. 2012, 19, 791−794. (16) Smiatek, J.; Harishchandra, R. K.; Rubner, O.; Galla, H.-J.; Heuer, A. Biophys. Chem. 2012, 160, 62−68. (17) Widderich, N.; Hoppner, A.; Pittelkow, M.; Heider, J.; Smits, S. H.; Bremer, E. PLoS One 2014, 9, e93809. (18) Kunte, H. J.; Lentzen, G.; Galinski, E. A. Curr. Biotechnol. 2014, 3, 10−25. (19) Harishchandra, R. K.; Sachan, A. K.; Kerth, A.; Lentzen, G.; Neuhaus, T.; Galla, H.-J. Biochim. Biophys. Acta: Biomembr. 2011, 1808, 2830−2840. (20) Buommino, E.; Schiraldi, C.; Baroni, A.; Paoletti, I.; Lamberti, M.; De Rosa, M.; Tufano, M. A. Cell Stress Chaperones 2005, 10, 197− 203. (21) Unfried, K.; Kroker, M.; Autengruber, A.; Gotic, M.; Sydlik, U. J. Allergy (Cairo) 2014, 2014, 708458. (22) Sydlik, U.; Gallitz, I.; Albrecht, C.; Abel, J.; Krutmann, J.; Unfried, K. Am. J. Respir. Crit. Care Med. 2009, 180, 29−35. (23) Kadaba Srinivasan, P.; Fet, N.; Bleilevens, C.; Afify, M.; Doorschodt, B.; Yagi, S.; van Echten-Deckert, G.; Tolba, R. H. Ann. Transplant. 2014, 19, 165−173. (24) Abdel-Aziz, H.; Wadie, W.; Abdallah, D. M.; Lentzen, G.; Khayyal, M. T. Phytomedicine 2013, 20, 585−591. (25) Wang, X.; Zhao, J.; Han, Z.; Tang, F. Int. J. Mol. Med. 2015, 35, 1699−1707. (26) Wu, Z.; Boersema, G. S.; Kroese, L. F.; Taha, D.; Vennix, S.; Bastiaansen-Jenniskens, Y. M.; Lam, K. H.; Kleinrensink, G. J.; Jeekel, J.; Peppelenbosch, M.; Lange, J. F. Inflamm. Bowel Dis. 2015, 21, 1038−1046. (27) Wang, W. P.; Guo, X.; Koo, M. W.; Wong, B. C.; Lam, S. K.; Ye, Y. N.; Cho, C. H. Am. J. Physiol. Gastrointest. Liver Physiol. 2001, 281, G586−G594. (28) Nielsen, O. H.; Rask-Madsen, J. Scand. J. Gastroenterol. Suppl. 1996, 216, 149−159. (29) Sartor, R. B. Am. J. Gastroenterol. 1997, 92, 5s−11s. (30) Kolios, G.; Petoumenos, C.; Nakos, A. Hepatogastroenterology 1998, 45, 1601−1609. (31) Stallmach, A.; Giese, T.; Schmidt, C.; Ludwig, B.; MuellerMolaian, I.; Meuer, S. C. Int. J. Colorectal Dis. 2004, 19, 308−315. (32) Peuschel, H.; Sydlik, U.; Haendeler, J.; Buchner, N.; Stockmann, D.; Kroker, M.; Wirth, R.; Brock, W.; Unfried, K. Biol. Chem. 2010, 391, 1327−1332. (33) Sydlik, U.; Peuschel, H.; Paunel-Gorgulu, A.; Keymel, S.; Kramer, U.; Weissenberg, A.; Kroker, M.; Seghrouchni, S.; Heiss, C.; Windolf, J.; Bilstein, A.; Kelm, M.; Krutmann, J.; Unfried, K. Eur. Respir. J. 2013, 41, 433−442. (34) Buenger, J.; Driller, H. Skin Pharmacol. Physiol. 2004, 17, 232− 237. (35) McGeachy, M. J.; Cua, D. J. Immunity 2008, 28, 445−453. (36) Lehmann, F. S.; Burri, E.; Beglinger, C. Ther. Adv. Gastroenterol. 2015, 8, 23−36. (37) Tomasello, G.; Sciume, C.; Rappa, F.; Rodolico, V.; Zerilli, M.; Martorana, A.; Cicero, G.; De Luca, R.; Damiani, P.; Accardo, F. M.; Romeo, M.; Farina, F.; Bonaventura, G.; Modica, G.; Zummo, G.; Conway de Macario, E.; Macario, A. J.; Cappello, F. Eur. J. Histochem. 2011, 55, e38. (38) Mohanan, V.; Grimes, C. L. J. Biol. Chem. 2014, 289, 18987− 18998. (39) Morris, G. P.; Beck, P. L.; Herridge, M. S.; Depew, W. T.; Szewczuk, M. R.; Wallace, J. L. Gastroenterology 1989, 96, 795−803. (40) Beutler, E.; Duron, O.; Kelly, B. M. J. Lab Clin. Med. 1963, 61, 882−888. (41) Krawisz, J. E.; Sharon, P.; Stenson, W. F. Gastroenterology 1984, 87, 1344−1350.
hexadecyltrimethylammonium bromide (HTAB) and sonicated for 10 s, then centrifuged at 10 000 rpm for 15 min at 4 °C. The supernatant was used for spectrophotometric estimation of myeloperoxidase activity according to Krawisz et al.41 The third aliquot was used to determine tumor necrosis factor alpha (TNF-α), interleukin-1β (IL1β), intercellular adhesion molecule-1 (ICAM-1), and heat shock protein 70 (Hsp70) using specific ELISA kits (Hölzel Diagnostika, Köln, Germany for Hsp70; R&D Systems, Wiesbaden, Germany, for the other kits). In addition, fecal samples from 4 randomly chosen animals from each of the following groups were collected prior to sacrifice: normal control, TNBS control, TNBS + 100 mg/kg ectoine (1), and TNBS + 100 mg/kg 5α-hydroxyectoine (2). They were analyzed for their calprotectin content using a commercial ELISA kit (S100A8/S100A9, Immundiagnostik AG, Bensheim, Germany). Determination was done according to the manufacturer’s instructions with slight modifications: the feces were weighed and suspended 1:10 in extraction buffer; the dilutions were incubated for 90 min at room temperature; the samples were incubated in the ELISA plates for 90 min at room temperature. Statistical Analysis. Values are presented as means ± SEM. Comparisons between means were carried out using one-way analysis of variance (ANOVA), followed by Student−Newman−Keuls multiple comparisons test. Differences were considered significant at p < 0.05. Instat software, version 2 (GraphPad Software, Inc., San Diego, CA, USA), was used to carry out the statistical tests.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +49 06151 3305 202. Fax: +49 06151 3305 471. E-mail:
[email protected]. Notes
The authors declare the following competing financial interest(s): Dr. Olaf Scherner is fully employed by Bitop AG, Witten, Germany.
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ACKNOWLEDGMENTS The authors wish to thank Bitop AG, Witten, Germany (currently the worldwide sole producer of ectoine and hydroxyectoine for commercial purposes), for financial support of this study.
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REFERENCES
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