Dig Dis Sci DOI 10.1007/s10620-013-3013-2
ORIGINAL ARTICLE
Melatonin Attenuates Dextran Sodium Sulfate Induced Colitis with Sleep Deprivation: Possible Mechanism by Microarray Analysis Sook Hee Chung • Young Sook Park • Ok Soon Kim • Ja Hyun Kim • Haing Woon Baik • Young Ok Hong • Sang Su Kim • Jae-Ho Shin • Jin-Hyun Jun • Yunju Jo • Sang Bong Ahn • Young Kwan Jo • Byoung Kwan Son Seong Hwan Kim
•
Received: 14 August 2013 / Accepted: 20 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Background Inflammatory bowel disease is a chronic inflammatory condition of the gastrointestinal tract. It can be aggravated by stress, like sleep deprivation, and improved by anti-inflammatory agents, like melatonin. We aimed to investigate the effects of sleep deprivation and melatonin on inflammation. We also investigated genes regulated by sleep deprivation and melatonin. Methods In the 2 % DSS induced colitis mice model, sleep deprivation was induced using modified multiple platform water bath. Melatonin was injected after induction of colitis and colitis with sleep deprivation. Also mRNA
S. H. Chung Department of Gastroenterology, Ajou University School of Medicine, Suwon, Korea Y. S. Park (&) Y. Jo S. B. Ahn Y. K. Jo B. K. Son S. H. Kim Department of Internal Medicine, Eulji Hospital, Eulji University School of Medicine, 280-1 H«agye 1-dong, Nowon-gu, Seoul 139-711, Korea e-mail: [email protected] O. S. Kim J. H. Kim H. W. Baik Department of Biochemistry, Eulji University School of Medicine, Seoul, Korea Y. O. Hong Department of Pathology, Eulji University School of Medicine, Seoul, Korea S. S. Kim Eulji Bio-Medical Research Institute, Eulji University School of Medicine, Seoul, Korea J.-H. Shin J.-H. Jun Eulji University School of Medicine, Seoul, Korea
was isolated from the colon of mice and analyzed via microarray and real-time PCR. Results Sleep deprivation induced reduction of body weight, and it was difficult for half of the mice to survive. Sleep deprivation aggravated, and melatonin attenuated the severity of colitis. In microarrays and real-time PCR of mice colon tissues, mRNA of adiponectin and aquaporin 8 were downregulated by sleep deprivation and upregulated by melatonin. However, mRNA of E2F transcription factor (E2F2) and histocompatibility class II antigen A, beta 1 (H2-Ab1) were upregulated by sleep deprivation and downregulated by melatonin. Conclusion Melatonin improves and sleep deprivation aggravates inflammation of colitis in mice. Adiponectin, aquaporin 8, E2F2 and H2-Ab1 may be involved in the inflammatory change aggravated by sleep deprivation and attenuated by melatonin. Keywords Inflammatory bowel disease Melatonin Sleep deprivation
Introduction Inflammatory bowel disease is caused by multiple genetic, environmental and host factors [1]. Environmental factors such as a stress increase the progression and relapse of patients with inflammatory bowel disease [2, 3]. Therefore, decreased immunity by stress might make people more susceptible to inflammatory bowel disease [2]. For example, the stress, such as devotion, bereavement, and sleep deprivation can aggravate the activity of inflammatory bowel disease. Sleep deprivation such as a stress could be a risk factor for more frequent inflammatory bowel disease in night-shift workers than day-time workers [4].
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Experimental Procedures 2 % DSS Induced Colitis Model All the mice were fed with 2 % DSS solution for 7 days to induce colitis. Partial Sleep Deprivation Fig. 1 Experimental procedure (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment). M melatonin, SD sleep deprivation
Melatonin is secreted by a pineal gland and functions as a regulator of circadian rhythms and an antioxidant [5]. The melatonin has been found to have anti-inflammatory effects in experimental models of colitis in many previous reports [6–10]. However, the exact mechanisms through which sleep deprivation and melatonin act on inflammatory bowel disease have not been clarified yet. Thus, the aim of this study was to investigate the effect of sleep deprivation and melatonin on inflammatory change in the colitis mouse model. Additionally, we also tried to investigate genetic expression altered by sleep deprivation and melatonin using DNA microarray analysis and RT-PCR.
Materials and Methods Animals For the experiment, a total of 30 C57BL mice, 8 weeks old and weighing 20–25 g, were purchased from Samtako, Inc. (Kyunggido, Korea). Five groups of six mice each were used as follows: group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment (Fig. 1). A temperature of 22–24 °C and humidity of 55–60 % were maintained with a 12-h light/dark cycle (lights on at 08:00). During the experimental period, all mice took food and water freely. Every day, body weights of all the mice were measured. All procedures in experiments were conducted according to the animal care guidelines of the national institutes of health and Korean Academy of Medical Sciences. The institutional review board of Eulji Hospital at the Eulji University College of Medicine approved the study protocol (Approval number: EMBRI2010-SN-05).
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The mice had partial sleep deprivation induced via a modified multiple platform water bath. Eighteen platforms were placed in a water tank. Five mice were placed in the water bath. Every mouse in the water bath could move from one platform to another by jumping. The water filled the water bath 4 cm from the base. When the mice had rapid eye movement stage of sleep, which is the paradoxical phase of sleep, muscle atonia could cause the mouse to fall into the water. Then the mice awakened and would try to climb up the platform to avoid being drowned. Through all experiments the water was changed with clean water in the tank. Partial sleep deprivation of the mice began from 2 PM to 10 AM, 20 h for 4 days using a modified multiple platform water bath. The mice had 4 h of sleep from 10 AM to 2 PM over 4 days. The mice were sacrificed after finishing 4 days of partial sleep deprivation. Administration of Melatonin Melatonin was administered to the mice intraperitoneally at a dose of 10 mg/kg for 4 days after induction of colitis with 2 % DSS. Normal saline was administered to control mice intraperitoneally. The mice were sacrificed after the administration of melatonin for 4 days. Assessment of Severity of Colitis The severity of colitis was assessed by measuring weight loss and histological analysis. Colon of mice was stained with hematoxylin and eosin in order to evaluate inflammatory change. For 24 h, 10 % formaldehyde solution fixed the tissue. The fixed tissue was placed in a 70 % ethanol solution and embedded by paraffin. The tissue was sliced into 4-lm thick sections. The severity of the inflammation in the colon was measured by various findings including loss of mucosal structure (score 0–3), crypt abscess (score 0–1), thickened muscle (score 0–3), cellular infiltration (score 0–3), and depletion of goblet cell (score 0–1). It was finally graded from 0 to 11 [11]. RNA Isolation and Microarray Analysis Colon tissues from the mice were removed. Total RNAs of colon tissue for microarray were extracted via Conventional
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TRIzolTM reagent (Invitrogen, Carlsbad, CA, USA). Agilent’s Low RNA Input Linear Amplification kit PLUS was used for synthesizing target cDNA probes and hybridization. T7 promoter primer mix and total RNA (1 lg) were incubated at 65 °C for 10 min. cDNA master mix solution (59 first strand buffer, 0.1 M DTT, 10 mM dNTP mix, RNaseOut, and MMLV-RT) was mixed with the primer mixture and RNA. Reverse-transcription and synthesis of doublestrand cDNA (dsDNA) were finished by incubating the mixtures at 40 °C for 2 h. After incubating the mixture at 65 °C for 15 min, all the reaction was terminated. The transcription master mixture (49 transcription buffer, 0.1 M DTT, NTP mix, 50 % PEG, RNase-Out, Inorganic pyrophosphatase, T7-RNA polymerase, and Cyanine 3/5-CTP) was added to the dsDNA reaction mixture. For transcription of dsDNA, the mixture was incubated at 40 °C for 2 h. The control and test cRNAs were labeled with Cy3-CTP and Cy5-CTP. For fragmentation of cRNA, the mixture was incubated at 60 °C for 30 min with 109 blocking agent and 259 fragmentation buffer. For microarray analysis, a 44 K Mouse Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA, USA) kit was used. It contained 41,174 transcripts and variants. Scanned images in all groups were analyzed using Feature Extraction software (Agilent Technologies). Fold-changed genes were selected using GeneSpring 7.3.1 software (Agilent Technologies, Inc., Palo Alto, CA, USA).
Fig. 2 Changes of body weight in all groups (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment). Data represent mean ± SD. M melatonin, SD sleep deprivation
statistical significance. Significance was determined if the P value was less than 0.05.
cDNA Synthesis and Real-Time PCR to Validate Gene
Results
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from the colons of mice. ImProm-IITM reverse transcriptase (Promega Corp., Madison, WI, USA) and oligo(dT) primers15 for aquaporin, adiponectin, E2F2 and H2-Ab were used in synthesis of reversely transcribed cDNA. A 20-lL reaction mixture containing 10 lL of SYBR Green qPCR Premix (Finnzymes Oy, Espoo, Finland), 10 lmol of forward primer, 10 lmol of reverse primer and 1 lg of cDNA was used for real-time polymerase chain reaction via the DNA Engine Opticon System (MJ Research Inc., CA, USA). Initial denaturation was performed at 95 °C for 5 min. Forty cycles of PCR were composed under the following conditions: denaturation at 95 °C for 1 min, annealing at 50–59 °C for 30 min, and extension at 72 °C for 1 min). The value of the threshold cycle (Ct) normalized to the mRNA value of b-actin was used to assess mRNA expression.
Effect of Sleep Deprivation and Melatonin Treatment on Body Weight of Mouse
Statistical Analysis All data were represented as the mean ± standard deviation. The Statistical Package for the Social Sciences software (SPSS; Korean version 18.0) was used in all statistical analyses. One-way ANOVA was used to calculate
From 7 days after 2 % DSS treatment, the body weights of the 2 % DSS induced colitis groups had decreased to much lower levels than that of the control group with statistical significance (Fig. 2, P = 0.005). Sleep deprivation induced weight loss in the 2 % DSS induced colitis groups even if there was no statistical significance. Half of the mice died during the sleep deprivation period. The mice in melatonin treatment group (group III and V) did not significantly recover the weight loss by sleep deprivation. Effect of Sleep Deprivation and Melatonin Treatment on Histological Findings of 2 % DSS Induced Colitis Mouse In histological analysis, the colons of the 2 % DSS induced colitis mice (Fig. 3b) showed edema and infiltration of inflammatory cells into the mucosa compared to those of the control group (Fig. 3a). Melatonin treatment reduced inflammation in the colon of the 2 % DSS induced colitis mice (Fig. 3c). An increased number of infiltrating cells and mucosal injury, such as ulcer or necrosis, were observed
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Fig. 3 Histological analyses of inflammatory change by sleep deprivation and melatonin in 2 % DSS induced colitis mice model (H&E stain 9400) (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment;
group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment)
increased by sleep deprivation (group IV, 8.7 ± 0.6) with P = 0.018. The histologic score of the 2 % DSS-induced colitis with sleep deprivation (group IV, 8.7 ± 0.6) decreased by melatonin treatment (group V, 7.0 ± 1.0) with P = 0.067. The changes of inflammatory score in all groups were statistically significant (P \ 0.001). DNA Microarray Analyses and Change of Genetic Expression Induced by 2 % DSS, Sleep Deprivation, and Melatonin Treatment
Fig. 4 Effects of melatonin and sleep deprivation on inflammatory change of 2 % DSS induced colitis model in histology (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment). *P \ 0.05 by analysis of variance. #P \ 0.10 by analysis of variance. M melatonin, SD sleep deprivation
in the colon of 2 % DSS induced colitis mice in the sleep deprivation group (Fig. 3d). Melatonin treatment also diminished inflammation and erosion in the colon of the 2 % DSS induced colitis mice with sleep deprivation (Fig. 3e). In Fig. 4, the microscopic inflammatory score of the colitis group (group II, 6.3 ± 0.6) decreased by melatonin treatment (group III, 4.6 ± 0.6) with P = 0.003, but
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Under sleep loss, 692 genes were upregulated more than twofold, 14,938 genes were upregulated 1–2 fold, 895 genes were downregulated more than twofold and 14,773 genes were downregulated 1–2 fold. Under melatonin treatment, 676 genes were upregulated more than twofold, 14,219 genes were upregulated 1–2 fold, 891 genes were downregulated more than twofold and 15,770 genes were downregulated 1–2 fold. We selected four genes (adiponectin, aquaporin 8, H2-Ab1 and E2F2) for real-time PCR based on fold changes of expression in microarray and biological plausibility which were affected significantly by melatonin and sleep deprivation (Table 1). Real Time PCR Validation of Microarray Data In real-time PCR adiponectin, aquaporin 8, E2F2 and H2Ab1 showed results similar to the microarray data (Fig. 5).
Dig Dis Sci Table 1 Significant genes changed by sleep deprivation and melatonin Gene symbol
Gene name
Gene bank
Fold change by melatonin
Fold change by sleep deprivation
E2F2
E2F transcription factor
NM_177733
0.96
1.78
H2-Ab1
Histocompatibility class II antigen A, beta 1
NM_207105
0.94
1.79
Adipoq
Adiponectin
NM_009605
5.89
0.28
Aqp8
Aquaporin 8
NM_007474
1.75
0.45
Fig. 5 RT-PCR. a Adiponectin. b Aquaporin 8. c E2F2. d H2-Ab1 (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced
colitis with sleep deprivation and melatonin treatment). M melatonin, SD sleep deprivation. *P \ 0.05 and **P \ 0.001 by analysis of variance
mRNA of adiponectin and aquaporin 8 were upregulated by melatonin and downregulated by sleep deprivation in real time PCR. Contrarily, mRNA of E2F2 and H2-Ab1 were upregulated by melatonin and downregulated by sleep deprivation in real-time PCR.
disease [2, 3]. Sleep deprivation as a stress could affect the inflammatory bowel disease by inducing inflammatory cytokines [12]. In the colitis induced animal model, disrupted sleep induced more severe inflammation and immune activity [13]. Sleep deprivation in shift workers could increase the risk of inflammatory bowel disease (IBD) [4, 12, 14]. Melatonin is secreted by the pineal gland and functions as a regulator of circadian rhythms and an antioxidant [5]. Also melatonin has been shown to have anti-inflammatory effects in experimental models of colitis in many previous
Discussion Environmental factors such as stress could be risk factors for the progression and relapse of inflammatory bowel
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reports [6–10]. Melatonin was shown to have anti-inflammatory and anti-apoptotic effects by decreasing TNF-a, IL1b, Fas ligand, phosphorylation of c-Jun, and Bax in DNBS treated rats [15]. The results of our study demonstrated that sleep deprivation increased the severity of inflammation and was compatible with other reports [6–10]. In this study melatonin treatment decreased the inflammation with and without sleep deprivation in colitis induced mice model histopathologically and clinically. In our results, the survival rate of mice in the sleep deprivation group was only 50 %. However, there were no dead mice in the melatonintreated group. In this study we tried to investigate the mechanisms of the regulatory process of inflammation by sleep deprivation and melatonin. In both microarray and real-time PCR analysis, mRNA of adiponectin, aquaporin 8, E2F2 and H2-Ab1 were changed by sleep deprivation and melatonin. Adiponectin had anti-inflammatory effects by inhibiting nuclear factor-kappa B, TNF-a and IL-6 and by inducing anti-inflammatory cytokines such as IL-10 and IL-1 receptor antagonists [16–21]. Overexpressed adiponectin in mice decreased proinflammatory cytokines including the cellular stress markers, TNF, IL-6 and IL-1 [22, 23]. mRNA of adiponectin decreased in inflamed colonic mucosa of murine colitis and patients with IBD [24]. On the other hand, another study showed that adiponectin had proinflammatory effects by producing TNF-r, IL-6 and IL-10 [25–27]. The regulatory effects of melatonin on adiponectin were not consistent in several studies [28–32]. Therefore, the regulatory effects of adiponectin on inflammation were also not completely discovered yet. Aquaporin 8 is located in the apical side of the superficial cells and known to be involved in fluid transport in the colon [33, 34]. Aquaporin 8 was known to regulate osmoregulation and fluid fluxes [35–37]. Aquaporin 8 mRNA was downregulated in the murine model of colitis and in patients with active inflammatory bowel disease [37]. The degree of colonic injury was related to downregulated aquaporin 8 expression [37]. But in another report aquaporin 8 mRNA and protein levels increased in the colon of ulcerative colitis patients [38]. The regulatory effect of aquaporin 8 on inflammation was also not completely discovered yet. E2F2 is a member of the E2F which is a family of transcription factors. E2F was known to regulate metabolic and proliferative genes [39]. The E2F family might be involved in the development, G1/S cell-cycle transition apoptosis, differentiation and DNA repair [39–41]. H2-Ab1 is a family of molecules found in antigen-presenting cells and lymphocytes. Extracellular proteins as antigens were presented by class II peptides in the immune response [42].
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The effects of adiponectin, aquaporin 8, E2F2 and H2Ab1 on the inflammatory change were inconsistent and not completely known in previous studies. Even if we do not know the exact regulatory mechanism of adiponectin, aquaporin 8, E2F2 and H2-Ab1 on inflammation, they might be involved in the inflammatory process by sleep deprivation and melatonin. There were three strong points in this study. First, this study is the first to investigate the effects of sleep deprivation and melatonin on the inflammation in the DSSinduced colitis model simultaneously. Second, we used a specialized sleep deprivation model using a modified multiple platform water bath that was independently developed in our department. There was a study which had used a different method to induce sleep deprivation in the mouse model like a rotating wheel [43]. In this study, we decided to use a sleep deprivation model using a modified multiple platform water bath because this model was used in other partial sleep deprivation studies [44–47]. Third, we found that sleep deprivation and melatonin might be related to adiponectin, aquaporin, E2F2 and H2-Ab1 in the regulation of inflammatory processes of the colon. However, there were also weak points in this study. First, we only know the effect of melatonin and sleep deprivation on the inflammatory change through in vitro test. We should confirm this result in in vivo testing. Second, we decided to choose the dose of melatonin as 10 mg/kg because many other studies used 10 mg/kg as a dose of melatonin showing an anti-inflammatory effect [6, 8, 48]. The doses of melatonin used in many experiments were different ranging from 0.5 to 15 mg/kg [49]. We should perform the experiment with various melatonin doses to know the dose effect of melatonin. In conclusion, sleep deprivation aggravates and melatonin protects inflammatory change in 2 % DSS-induced colitis of mice. In the regulatory mechanism of inflammation by sleep deprivation and melatonin, adiponectin, aquaporin 8, E2F2 and H2-Ab1 might be involved in the inflammatory process. Acknowledgments DNA data.
We thank E biogen for help in analyzing micro
Conflict of interest This research was supported by EMBRI Grants 2010-SN-05 from the Eulji University.
References 1. Bamias G, Cominelli F. Immunopathogenesis of inflammatory bowel disease: current concepts. Curr Opin Gastroenterol. 2007; 23:365–369. 2. Farrell RJ, Peppercorn MA. Ulcerative colitis. Lancet. 2002;359: 331–340. 3. Niess JH, Monnikes H, Dignass AU, et al. Review on the influence of stress on immune mediators, neuropeptides and hormones
Dig Dis Sci
4. 5.
6. 7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
with relevance for inflammatory bowel disease. Digestion. 2002; 65:131–140. Sonnenberg A. Occupational distribution of inflammatory bowel disease among German employees. Gut. 1990;31:1037–1040. Marquez E, Sanchez-Fidalgo S, Calvo JR, et al. Acutely administered melatonin is beneficial while chronic melatonin treatment aggravates the evolution of TNBS-induced colitis. J Pineal Res. 2006;40:48–55. Necefli A, Tulumoglu B, Giris M, et al. The effect of melatonin on TNBS-induced colitis. Dig Dis Sci. 2006;51:1538–1545. Li JH, Yu JP, Yu HG, et al. Melatonin reduces inflammatory injury through inhibiting NF-kappaB activation in rats with colitis. Mediators Inflamm. 2005;2005:185–193. Akcan A, Kucuk C, Sozuer E, et al. Melatonin reduces bacterial translocation and apoptosis in trinitrobenzene sulphonic acidinduced colitis of rats. World J Gastroenterol. 2008;14:918–924. Cuzzocrea S, Mazzon E, Serraino I, et al. Melatonin reduces dinitrobenzene sulfonic acid-induced colitis. J Pineal Res. 2001; 30:1–12. Dong WG, Mei Q, Yu JP, et al. Effects of melatonin on the expression of iNOS and COX-2 in rat models of colitis. World J Gastroenterol. 2003;9:1307–1311. Tahan G, Gramignoli R, Marongiu F, et al. Melatonin expresses powerful anti-inflammatory and antioxidant activities resulting in complete improvement of acetic-acid-induced colitis in rats. Dig Dis Sci. 2011;56:715–720. Swanson GR, Burgess HJ, Keshavarzian A. Sleep disturbances and inflammatory bowel disease: a potential trigger for disease flare? Expert Rev Clin Immunol. 2011;7:29–36. Preuss F, Tang Y, Laposky AD, et al. Adverse effects of chronic circadian desynchronization in animals in a ‘‘challenging’’ environment. Am J Physiol Regul Integr Comp Physiol. 2008;295: R2034–R2040. Jean-Louis G, Kripke DF, Ancoli-Israel S, et al. Sleep duration, illumination, and activity patterns in a population sample: effects of gender and ethnicity. Biol Psychiatry. 2000;47:921–927. Mazzon E, Esposito E, Crisafulli C, et al. Melatonin modulates signal transduction pathways and apoptosis in experimental colitis. J Pineal Res. 2006;41:363–373. Yokota T, Oritani K, Takahashi I, et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood. 2000;96:1723–1732. Wulster-Radcliffe MC, Ajuwon KM, Wang J, et al. Adiponectin differentially regulates cytokines in porcine macrophages. Biochem Biophys Res Commun. 2004;316:924–929. Nishihara T, Matsuda M, Araki H, et al. Effect of adiponectin on murine colitis induced by dextran sulfate sodium. Gastroenterology. 2006;131:853–861. Masaki T, Chiba S, Tatsukawa H, et al. Adiponectin protects LPS-induced liver injury through modulation of TNF-alpha in KK-Ay obese mice. Hepatology. 2004;40:177–184. Wolf AM, Wolf D, Rumpold H, et al. Adiponectin induces the anti-inflammatory cytokines IL-10 and IL-1RA in human leukocytes. Biochem Biophys Res Commun. 2004;323:630–635. Kumada M, Kihara S, Ouchi N, et al. Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages. Circulation. 2004;109: 2046–2049. Huang H, Park PH, McMullen MR, et al. Mechanisms for the anti-inflammatory effects of adiponectin in macrophages. J Gastroenterol Hepatol. 2008;23:S50–S53. Thakur V, Pritchard MT, McMullen MR, et al. Adiponectin normalizes LPS-stimulated TNF-alpha production by rat Kupffer cells after chronic ethanol feeding. Am J Physiol Gastrointest Liver Physiol. 2006;290:G998–G1007.
24. Matsunaga H, Hokari R, Kurihara C, et al. Omega-3 fatty acids exacerbate DSS-induced colitis through decreased adiponectin in colonic subepithelial myofibroblasts. Inflamm Bowel Dis. 2008;14: 1348–1357. 25. Liao W, Yu C, Wen J, et al. Adiponectin induces interleukin-6 production and activates STAT3 in adult mouse cardiac fibroblasts. Biol Cell. 2009;101:263–272. 26. Tang CH, Chiu YC, Tan TW, et al. Adiponectin enhances IL-6 production in human synovial fibroblast via an AdipoR1 receptor, AMPK, p38, and NF-kappa B pathway. J Immunol. 2007;179: 5483–5492. 27. Ehling A, Schaffler A, Herfarth H, et al. The potential of adiponectin in driving arthritis. J Immunol. 2006;176:4468–4478. 28. Zaouali MA, Reiter RJ, Padrissa-Altes S, et al. Melatonin protects steatotic and nonsteatotic liver grafts against cold ischemia and reperfusion injury. J Pineal Res. 2011;50:213–221. 29. Alonso-Vale MI, Andreotti S, Peres SB, et al. Melatonin enhances leptin expression by rat adipocytes in the presence of insulin. Am J Physiol Endocrinol Metab. 2005;288:E805–E812. 30. Alonso-Vale MI, Peres SB, Vernochet C, et al. Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBPbeta transcriptional activity. J Pineal Res. 2009;47:221–227. 31. Gonciarz M, Bielanski W, Partyka R, et al. Plasma insulin, leptin, adiponectin, resistin, ghrelin, and melatonin in nonalcoholic steatohepatitis patients treated with melatonin. J Pineal Res. 2013;54:154–161. 32. Rios-Lugo MJ, Cano P, Jimenez-Ortega V, et al. Melatonin effect on plasma adiponectin, leptin, insulin, glucose, triglycerides and cholesterol in normal and high fat-fed rats. J Pineal Res. 2010;49:342–348. 33. Ma T, Yang B, Verkman AS. Cloning of a novel water and ureapermeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem Biophys Res Commun. 1997; 240:324–328. 34. Laforenza U, Cova E, Gastaldi G, et al. Aquaporin-8 is involved in water transport in isolated superficial colonocytes from rat proximal colon. J Nutr. 2005;135:2329–2336. 35. Calamita G, Mazzone A, Bizzoca A, et al. Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract. Eur J Cell Biol. 2001;80:711–719. 36. Liu K, Nagase H, Huang CG, et al. Purification and functional characterization of aquaporin-8. Biol Cell. 2006;98:153–161. 37. Hardin JA, Wallace LE, Wong JF, et al. Aquaporin expression is downregulated in a murine model of colitis and in patients with ulcerative colitis, Crohn’s disease and infectious colitis. Cell Tissue Res. 2004;318:313–323. 38. Zahn A, Moehle C, Langmann T, et al. Aquaporin-8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis. World J Gastroenterol. 2007;13:1687–1695. 39. Dimova DK, Dyson NJ. The E2F transcriptional network: old acquaintances with new faces. Oncogene. 2005;24:2810–2826. 40. Fajas L, Landsberg RL, Huss-Garcia Y, et al. E2Fs regulate adipocyte differentiation. Dev Cell. 2002;3:39–49. 41. Tsantoulis PK, Gorgoulis VG. Involvement of E2F transcription factor family in cancer. Eur J Cancer. 2005;41:2403–2414. 42. Blum JS, Wearsch PA, Cresswell P. Pathways of antigen processing. Annu Rev Immunol. 2013;31:443–473. 43. Tang Y, Preuss F, Turek FW, et al. Sleep deprivation worsens inflammation and delays recovery in a mouse model of colitis. Sleep Med. 2009;10:597–603. 44. Lee YD, Kim JY, Lee KH, et al. Melatonin attenuates lipopolysaccharide-induced acute lung inflammation in sleep-deprived mice. J Pineal Res. 2009;46:53–57. 45. Machado RB, Hipolide DC, Benedito-Silva AA, et al. Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery. Brain Res. 2004;1004: 45–51.
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Dig Dis Sci 46. Suchecki D, Tufik S. Social stability attenuates the stress in the modified multiple platform method for paradoxical sleep deprivation in the rat. Physiol Behav. 2000;68:309–316. 47. Silva RH, Kameda SR, Carvalho RC, et al. Anxiogenic effect of sleep deprivation in the elevated plus-maze test in mice. Psychopharmacology. 2004;176:115–122.
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48. Nosal’ova V, Zeman M, Cerna S, et al. Protective effect of melatonin in acetic acid induced colitis in rats. J Pineal Res. 2007;42:364–370. 49. Terry PD, Villinger F, Bubenik GA, et al. Melatonin and ulcerative colitis: evidence, biological mechanisms, and future research. Inflamm Bowel Dis. 2009;15:134–140.
ORIGINAL ARTICLE
Melatonin Attenuates Dextran Sodium Sulfate Induced Colitis with Sleep Deprivation: Possible Mechanism by Microarray Analysis Sook Hee Chung • Young Sook Park • Ok Soon Kim • Ja Hyun Kim • Haing Woon Baik • Young Ok Hong • Sang Su Kim • Jae-Ho Shin • Jin-Hyun Jun • Yunju Jo • Sang Bong Ahn • Young Kwan Jo • Byoung Kwan Son Seong Hwan Kim
•
Received: 14 August 2013 / Accepted: 20 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Background Inflammatory bowel disease is a chronic inflammatory condition of the gastrointestinal tract. It can be aggravated by stress, like sleep deprivation, and improved by anti-inflammatory agents, like melatonin. We aimed to investigate the effects of sleep deprivation and melatonin on inflammation. We also investigated genes regulated by sleep deprivation and melatonin. Methods In the 2 % DSS induced colitis mice model, sleep deprivation was induced using modified multiple platform water bath. Melatonin was injected after induction of colitis and colitis with sleep deprivation. Also mRNA
S. H. Chung Department of Gastroenterology, Ajou University School of Medicine, Suwon, Korea Y. S. Park (&) Y. Jo S. B. Ahn Y. K. Jo B. K. Son S. H. Kim Department of Internal Medicine, Eulji Hospital, Eulji University School of Medicine, 280-1 H«agye 1-dong, Nowon-gu, Seoul 139-711, Korea e-mail: [email protected] O. S. Kim J. H. Kim H. W. Baik Department of Biochemistry, Eulji University School of Medicine, Seoul, Korea Y. O. Hong Department of Pathology, Eulji University School of Medicine, Seoul, Korea S. S. Kim Eulji Bio-Medical Research Institute, Eulji University School of Medicine, Seoul, Korea J.-H. Shin J.-H. Jun Eulji University School of Medicine, Seoul, Korea
was isolated from the colon of mice and analyzed via microarray and real-time PCR. Results Sleep deprivation induced reduction of body weight, and it was difficult for half of the mice to survive. Sleep deprivation aggravated, and melatonin attenuated the severity of colitis. In microarrays and real-time PCR of mice colon tissues, mRNA of adiponectin and aquaporin 8 were downregulated by sleep deprivation and upregulated by melatonin. However, mRNA of E2F transcription factor (E2F2) and histocompatibility class II antigen A, beta 1 (H2-Ab1) were upregulated by sleep deprivation and downregulated by melatonin. Conclusion Melatonin improves and sleep deprivation aggravates inflammation of colitis in mice. Adiponectin, aquaporin 8, E2F2 and H2-Ab1 may be involved in the inflammatory change aggravated by sleep deprivation and attenuated by melatonin. Keywords Inflammatory bowel disease Melatonin Sleep deprivation
Introduction Inflammatory bowel disease is caused by multiple genetic, environmental and host factors [1]. Environmental factors such as a stress increase the progression and relapse of patients with inflammatory bowel disease [2, 3]. Therefore, decreased immunity by stress might make people more susceptible to inflammatory bowel disease [2]. For example, the stress, such as devotion, bereavement, and sleep deprivation can aggravate the activity of inflammatory bowel disease. Sleep deprivation such as a stress could be a risk factor for more frequent inflammatory bowel disease in night-shift workers than day-time workers [4].
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Experimental Procedures 2 % DSS Induced Colitis Model All the mice were fed with 2 % DSS solution for 7 days to induce colitis. Partial Sleep Deprivation Fig. 1 Experimental procedure (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment). M melatonin, SD sleep deprivation
Melatonin is secreted by a pineal gland and functions as a regulator of circadian rhythms and an antioxidant [5]. The melatonin has been found to have anti-inflammatory effects in experimental models of colitis in many previous reports [6–10]. However, the exact mechanisms through which sleep deprivation and melatonin act on inflammatory bowel disease have not been clarified yet. Thus, the aim of this study was to investigate the effect of sleep deprivation and melatonin on inflammatory change in the colitis mouse model. Additionally, we also tried to investigate genetic expression altered by sleep deprivation and melatonin using DNA microarray analysis and RT-PCR.
Materials and Methods Animals For the experiment, a total of 30 C57BL mice, 8 weeks old and weighing 20–25 g, were purchased from Samtako, Inc. (Kyunggido, Korea). Five groups of six mice each were used as follows: group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment (Fig. 1). A temperature of 22–24 °C and humidity of 55–60 % were maintained with a 12-h light/dark cycle (lights on at 08:00). During the experimental period, all mice took food and water freely. Every day, body weights of all the mice were measured. All procedures in experiments were conducted according to the animal care guidelines of the national institutes of health and Korean Academy of Medical Sciences. The institutional review board of Eulji Hospital at the Eulji University College of Medicine approved the study protocol (Approval number: EMBRI2010-SN-05).
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The mice had partial sleep deprivation induced via a modified multiple platform water bath. Eighteen platforms were placed in a water tank. Five mice were placed in the water bath. Every mouse in the water bath could move from one platform to another by jumping. The water filled the water bath 4 cm from the base. When the mice had rapid eye movement stage of sleep, which is the paradoxical phase of sleep, muscle atonia could cause the mouse to fall into the water. Then the mice awakened and would try to climb up the platform to avoid being drowned. Through all experiments the water was changed with clean water in the tank. Partial sleep deprivation of the mice began from 2 PM to 10 AM, 20 h for 4 days using a modified multiple platform water bath. The mice had 4 h of sleep from 10 AM to 2 PM over 4 days. The mice were sacrificed after finishing 4 days of partial sleep deprivation. Administration of Melatonin Melatonin was administered to the mice intraperitoneally at a dose of 10 mg/kg for 4 days after induction of colitis with 2 % DSS. Normal saline was administered to control mice intraperitoneally. The mice were sacrificed after the administration of melatonin for 4 days. Assessment of Severity of Colitis The severity of colitis was assessed by measuring weight loss and histological analysis. Colon of mice was stained with hematoxylin and eosin in order to evaluate inflammatory change. For 24 h, 10 % formaldehyde solution fixed the tissue. The fixed tissue was placed in a 70 % ethanol solution and embedded by paraffin. The tissue was sliced into 4-lm thick sections. The severity of the inflammation in the colon was measured by various findings including loss of mucosal structure (score 0–3), crypt abscess (score 0–1), thickened muscle (score 0–3), cellular infiltration (score 0–3), and depletion of goblet cell (score 0–1). It was finally graded from 0 to 11 [11]. RNA Isolation and Microarray Analysis Colon tissues from the mice were removed. Total RNAs of colon tissue for microarray were extracted via Conventional
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TRIzolTM reagent (Invitrogen, Carlsbad, CA, USA). Agilent’s Low RNA Input Linear Amplification kit PLUS was used for synthesizing target cDNA probes and hybridization. T7 promoter primer mix and total RNA (1 lg) were incubated at 65 °C for 10 min. cDNA master mix solution (59 first strand buffer, 0.1 M DTT, 10 mM dNTP mix, RNaseOut, and MMLV-RT) was mixed with the primer mixture and RNA. Reverse-transcription and synthesis of doublestrand cDNA (dsDNA) were finished by incubating the mixtures at 40 °C for 2 h. After incubating the mixture at 65 °C for 15 min, all the reaction was terminated. The transcription master mixture (49 transcription buffer, 0.1 M DTT, NTP mix, 50 % PEG, RNase-Out, Inorganic pyrophosphatase, T7-RNA polymerase, and Cyanine 3/5-CTP) was added to the dsDNA reaction mixture. For transcription of dsDNA, the mixture was incubated at 40 °C for 2 h. The control and test cRNAs were labeled with Cy3-CTP and Cy5-CTP. For fragmentation of cRNA, the mixture was incubated at 60 °C for 30 min with 109 blocking agent and 259 fragmentation buffer. For microarray analysis, a 44 K Mouse Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA, USA) kit was used. It contained 41,174 transcripts and variants. Scanned images in all groups were analyzed using Feature Extraction software (Agilent Technologies). Fold-changed genes were selected using GeneSpring 7.3.1 software (Agilent Technologies, Inc., Palo Alto, CA, USA).
Fig. 2 Changes of body weight in all groups (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment). Data represent mean ± SD. M melatonin, SD sleep deprivation
statistical significance. Significance was determined if the P value was less than 0.05.
cDNA Synthesis and Real-Time PCR to Validate Gene
Results
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from the colons of mice. ImProm-IITM reverse transcriptase (Promega Corp., Madison, WI, USA) and oligo(dT) primers15 for aquaporin, adiponectin, E2F2 and H2-Ab were used in synthesis of reversely transcribed cDNA. A 20-lL reaction mixture containing 10 lL of SYBR Green qPCR Premix (Finnzymes Oy, Espoo, Finland), 10 lmol of forward primer, 10 lmol of reverse primer and 1 lg of cDNA was used for real-time polymerase chain reaction via the DNA Engine Opticon System (MJ Research Inc., CA, USA). Initial denaturation was performed at 95 °C for 5 min. Forty cycles of PCR were composed under the following conditions: denaturation at 95 °C for 1 min, annealing at 50–59 °C for 30 min, and extension at 72 °C for 1 min). The value of the threshold cycle (Ct) normalized to the mRNA value of b-actin was used to assess mRNA expression.
Effect of Sleep Deprivation and Melatonin Treatment on Body Weight of Mouse
Statistical Analysis All data were represented as the mean ± standard deviation. The Statistical Package for the Social Sciences software (SPSS; Korean version 18.0) was used in all statistical analyses. One-way ANOVA was used to calculate
From 7 days after 2 % DSS treatment, the body weights of the 2 % DSS induced colitis groups had decreased to much lower levels than that of the control group with statistical significance (Fig. 2, P = 0.005). Sleep deprivation induced weight loss in the 2 % DSS induced colitis groups even if there was no statistical significance. Half of the mice died during the sleep deprivation period. The mice in melatonin treatment group (group III and V) did not significantly recover the weight loss by sleep deprivation. Effect of Sleep Deprivation and Melatonin Treatment on Histological Findings of 2 % DSS Induced Colitis Mouse In histological analysis, the colons of the 2 % DSS induced colitis mice (Fig. 3b) showed edema and infiltration of inflammatory cells into the mucosa compared to those of the control group (Fig. 3a). Melatonin treatment reduced inflammation in the colon of the 2 % DSS induced colitis mice (Fig. 3c). An increased number of infiltrating cells and mucosal injury, such as ulcer or necrosis, were observed
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Fig. 3 Histological analyses of inflammatory change by sleep deprivation and melatonin in 2 % DSS induced colitis mice model (H&E stain 9400) (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment;
group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment)
increased by sleep deprivation (group IV, 8.7 ± 0.6) with P = 0.018. The histologic score of the 2 % DSS-induced colitis with sleep deprivation (group IV, 8.7 ± 0.6) decreased by melatonin treatment (group V, 7.0 ± 1.0) with P = 0.067. The changes of inflammatory score in all groups were statistically significant (P \ 0.001). DNA Microarray Analyses and Change of Genetic Expression Induced by 2 % DSS, Sleep Deprivation, and Melatonin Treatment
Fig. 4 Effects of melatonin and sleep deprivation on inflammatory change of 2 % DSS induced colitis model in histology (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced colitis with sleep deprivation and melatonin treatment). *P \ 0.05 by analysis of variance. #P \ 0.10 by analysis of variance. M melatonin, SD sleep deprivation
in the colon of 2 % DSS induced colitis mice in the sleep deprivation group (Fig. 3d). Melatonin treatment also diminished inflammation and erosion in the colon of the 2 % DSS induced colitis mice with sleep deprivation (Fig. 3e). In Fig. 4, the microscopic inflammatory score of the colitis group (group II, 6.3 ± 0.6) decreased by melatonin treatment (group III, 4.6 ± 0.6) with P = 0.003, but
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Under sleep loss, 692 genes were upregulated more than twofold, 14,938 genes were upregulated 1–2 fold, 895 genes were downregulated more than twofold and 14,773 genes were downregulated 1–2 fold. Under melatonin treatment, 676 genes were upregulated more than twofold, 14,219 genes were upregulated 1–2 fold, 891 genes were downregulated more than twofold and 15,770 genes were downregulated 1–2 fold. We selected four genes (adiponectin, aquaporin 8, H2-Ab1 and E2F2) for real-time PCR based on fold changes of expression in microarray and biological plausibility which were affected significantly by melatonin and sleep deprivation (Table 1). Real Time PCR Validation of Microarray Data In real-time PCR adiponectin, aquaporin 8, E2F2 and H2Ab1 showed results similar to the microarray data (Fig. 5).
Dig Dis Sci Table 1 Significant genes changed by sleep deprivation and melatonin Gene symbol
Gene name
Gene bank
Fold change by melatonin
Fold change by sleep deprivation
E2F2
E2F transcription factor
NM_177733
0.96
1.78
H2-Ab1
Histocompatibility class II antigen A, beta 1
NM_207105
0.94
1.79
Adipoq
Adiponectin
NM_009605
5.89
0.28
Aqp8
Aquaporin 8
NM_007474
1.75
0.45
Fig. 5 RT-PCR. a Adiponectin. b Aquaporin 8. c E2F2. d H2-Ab1 (group I, control; group II, 2 % DSS induced colitis; group III, 2 % DSS induced colitis with melatonin treatment; group IV, 2 % DSS induced colitis with sleep deprivation; group V, 2 % DSS induced
colitis with sleep deprivation and melatonin treatment). M melatonin, SD sleep deprivation. *P \ 0.05 and **P \ 0.001 by analysis of variance
mRNA of adiponectin and aquaporin 8 were upregulated by melatonin and downregulated by sleep deprivation in real time PCR. Contrarily, mRNA of E2F2 and H2-Ab1 were upregulated by melatonin and downregulated by sleep deprivation in real-time PCR.
disease [2, 3]. Sleep deprivation as a stress could affect the inflammatory bowel disease by inducing inflammatory cytokines [12]. In the colitis induced animal model, disrupted sleep induced more severe inflammation and immune activity [13]. Sleep deprivation in shift workers could increase the risk of inflammatory bowel disease (IBD) [4, 12, 14]. Melatonin is secreted by the pineal gland and functions as a regulator of circadian rhythms and an antioxidant [5]. Also melatonin has been shown to have anti-inflammatory effects in experimental models of colitis in many previous
Discussion Environmental factors such as stress could be risk factors for the progression and relapse of inflammatory bowel
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reports [6–10]. Melatonin was shown to have anti-inflammatory and anti-apoptotic effects by decreasing TNF-a, IL1b, Fas ligand, phosphorylation of c-Jun, and Bax in DNBS treated rats [15]. The results of our study demonstrated that sleep deprivation increased the severity of inflammation and was compatible with other reports [6–10]. In this study melatonin treatment decreased the inflammation with and without sleep deprivation in colitis induced mice model histopathologically and clinically. In our results, the survival rate of mice in the sleep deprivation group was only 50 %. However, there were no dead mice in the melatonintreated group. In this study we tried to investigate the mechanisms of the regulatory process of inflammation by sleep deprivation and melatonin. In both microarray and real-time PCR analysis, mRNA of adiponectin, aquaporin 8, E2F2 and H2-Ab1 were changed by sleep deprivation and melatonin. Adiponectin had anti-inflammatory effects by inhibiting nuclear factor-kappa B, TNF-a and IL-6 and by inducing anti-inflammatory cytokines such as IL-10 and IL-1 receptor antagonists [16–21]. Overexpressed adiponectin in mice decreased proinflammatory cytokines including the cellular stress markers, TNF, IL-6 and IL-1 [22, 23]. mRNA of adiponectin decreased in inflamed colonic mucosa of murine colitis and patients with IBD [24]. On the other hand, another study showed that adiponectin had proinflammatory effects by producing TNF-r, IL-6 and IL-10 [25–27]. The regulatory effects of melatonin on adiponectin were not consistent in several studies [28–32]. Therefore, the regulatory effects of adiponectin on inflammation were also not completely discovered yet. Aquaporin 8 is located in the apical side of the superficial cells and known to be involved in fluid transport in the colon [33, 34]. Aquaporin 8 was known to regulate osmoregulation and fluid fluxes [35–37]. Aquaporin 8 mRNA was downregulated in the murine model of colitis and in patients with active inflammatory bowel disease [37]. The degree of colonic injury was related to downregulated aquaporin 8 expression [37]. But in another report aquaporin 8 mRNA and protein levels increased in the colon of ulcerative colitis patients [38]. The regulatory effect of aquaporin 8 on inflammation was also not completely discovered yet. E2F2 is a member of the E2F which is a family of transcription factors. E2F was known to regulate metabolic and proliferative genes [39]. The E2F family might be involved in the development, G1/S cell-cycle transition apoptosis, differentiation and DNA repair [39–41]. H2-Ab1 is a family of molecules found in antigen-presenting cells and lymphocytes. Extracellular proteins as antigens were presented by class II peptides in the immune response [42].
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The effects of adiponectin, aquaporin 8, E2F2 and H2Ab1 on the inflammatory change were inconsistent and not completely known in previous studies. Even if we do not know the exact regulatory mechanism of adiponectin, aquaporin 8, E2F2 and H2-Ab1 on inflammation, they might be involved in the inflammatory process by sleep deprivation and melatonin. There were three strong points in this study. First, this study is the first to investigate the effects of sleep deprivation and melatonin on the inflammation in the DSSinduced colitis model simultaneously. Second, we used a specialized sleep deprivation model using a modified multiple platform water bath that was independently developed in our department. There was a study which had used a different method to induce sleep deprivation in the mouse model like a rotating wheel [43]. In this study, we decided to use a sleep deprivation model using a modified multiple platform water bath because this model was used in other partial sleep deprivation studies [44–47]. Third, we found that sleep deprivation and melatonin might be related to adiponectin, aquaporin, E2F2 and H2-Ab1 in the regulation of inflammatory processes of the colon. However, there were also weak points in this study. First, we only know the effect of melatonin and sleep deprivation on the inflammatory change through in vitro test. We should confirm this result in in vivo testing. Second, we decided to choose the dose of melatonin as 10 mg/kg because many other studies used 10 mg/kg as a dose of melatonin showing an anti-inflammatory effect [6, 8, 48]. The doses of melatonin used in many experiments were different ranging from 0.5 to 15 mg/kg [49]. We should perform the experiment with various melatonin doses to know the dose effect of melatonin. In conclusion, sleep deprivation aggravates and melatonin protects inflammatory change in 2 % DSS-induced colitis of mice. In the regulatory mechanism of inflammation by sleep deprivation and melatonin, adiponectin, aquaporin 8, E2F2 and H2-Ab1 might be involved in the inflammatory process. Acknowledgments DNA data.
We thank E biogen for help in analyzing micro
Conflict of interest This research was supported by EMBRI Grants 2010-SN-05 from the Eulji University.
References 1. Bamias G, Cominelli F. Immunopathogenesis of inflammatory bowel disease: current concepts. Curr Opin Gastroenterol. 2007; 23:365–369. 2. Farrell RJ, Peppercorn MA. Ulcerative colitis. Lancet. 2002;359: 331–340. 3. Niess JH, Monnikes H, Dignass AU, et al. Review on the influence of stress on immune mediators, neuropeptides and hormones
Dig Dis Sci
4. 5.
6. 7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
with relevance for inflammatory bowel disease. Digestion. 2002; 65:131–140. Sonnenberg A. Occupational distribution of inflammatory bowel disease among German employees. Gut. 1990;31:1037–1040. Marquez E, Sanchez-Fidalgo S, Calvo JR, et al. Acutely administered melatonin is beneficial while chronic melatonin treatment aggravates the evolution of TNBS-induced colitis. J Pineal Res. 2006;40:48–55. Necefli A, Tulumoglu B, Giris M, et al. The effect of melatonin on TNBS-induced colitis. Dig Dis Sci. 2006;51:1538–1545. Li JH, Yu JP, Yu HG, et al. Melatonin reduces inflammatory injury through inhibiting NF-kappaB activation in rats with colitis. Mediators Inflamm. 2005;2005:185–193. Akcan A, Kucuk C, Sozuer E, et al. Melatonin reduces bacterial translocation and apoptosis in trinitrobenzene sulphonic acidinduced colitis of rats. World J Gastroenterol. 2008;14:918–924. Cuzzocrea S, Mazzon E, Serraino I, et al. Melatonin reduces dinitrobenzene sulfonic acid-induced colitis. J Pineal Res. 2001; 30:1–12. Dong WG, Mei Q, Yu JP, et al. Effects of melatonin on the expression of iNOS and COX-2 in rat models of colitis. World J Gastroenterol. 2003;9:1307–1311. Tahan G, Gramignoli R, Marongiu F, et al. Melatonin expresses powerful anti-inflammatory and antioxidant activities resulting in complete improvement of acetic-acid-induced colitis in rats. Dig Dis Sci. 2011;56:715–720. Swanson GR, Burgess HJ, Keshavarzian A. Sleep disturbances and inflammatory bowel disease: a potential trigger for disease flare? Expert Rev Clin Immunol. 2011;7:29–36. Preuss F, Tang Y, Laposky AD, et al. Adverse effects of chronic circadian desynchronization in animals in a ‘‘challenging’’ environment. Am J Physiol Regul Integr Comp Physiol. 2008;295: R2034–R2040. Jean-Louis G, Kripke DF, Ancoli-Israel S, et al. Sleep duration, illumination, and activity patterns in a population sample: effects of gender and ethnicity. Biol Psychiatry. 2000;47:921–927. Mazzon E, Esposito E, Crisafulli C, et al. Melatonin modulates signal transduction pathways and apoptosis in experimental colitis. J Pineal Res. 2006;41:363–373. Yokota T, Oritani K, Takahashi I, et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood. 2000;96:1723–1732. Wulster-Radcliffe MC, Ajuwon KM, Wang J, et al. Adiponectin differentially regulates cytokines in porcine macrophages. Biochem Biophys Res Commun. 2004;316:924–929. Nishihara T, Matsuda M, Araki H, et al. Effect of adiponectin on murine colitis induced by dextran sulfate sodium. Gastroenterology. 2006;131:853–861. Masaki T, Chiba S, Tatsukawa H, et al. Adiponectin protects LPS-induced liver injury through modulation of TNF-alpha in KK-Ay obese mice. Hepatology. 2004;40:177–184. Wolf AM, Wolf D, Rumpold H, et al. Adiponectin induces the anti-inflammatory cytokines IL-10 and IL-1RA in human leukocytes. Biochem Biophys Res Commun. 2004;323:630–635. Kumada M, Kihara S, Ouchi N, et al. Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages. Circulation. 2004;109: 2046–2049. Huang H, Park PH, McMullen MR, et al. Mechanisms for the anti-inflammatory effects of adiponectin in macrophages. J Gastroenterol Hepatol. 2008;23:S50–S53. Thakur V, Pritchard MT, McMullen MR, et al. Adiponectin normalizes LPS-stimulated TNF-alpha production by rat Kupffer cells after chronic ethanol feeding. Am J Physiol Gastrointest Liver Physiol. 2006;290:G998–G1007.
24. Matsunaga H, Hokari R, Kurihara C, et al. Omega-3 fatty acids exacerbate DSS-induced colitis through decreased adiponectin in colonic subepithelial myofibroblasts. Inflamm Bowel Dis. 2008;14: 1348–1357. 25. Liao W, Yu C, Wen J, et al. Adiponectin induces interleukin-6 production and activates STAT3 in adult mouse cardiac fibroblasts. Biol Cell. 2009;101:263–272. 26. Tang CH, Chiu YC, Tan TW, et al. Adiponectin enhances IL-6 production in human synovial fibroblast via an AdipoR1 receptor, AMPK, p38, and NF-kappa B pathway. J Immunol. 2007;179: 5483–5492. 27. Ehling A, Schaffler A, Herfarth H, et al. The potential of adiponectin in driving arthritis. J Immunol. 2006;176:4468–4478. 28. Zaouali MA, Reiter RJ, Padrissa-Altes S, et al. Melatonin protects steatotic and nonsteatotic liver grafts against cold ischemia and reperfusion injury. J Pineal Res. 2011;50:213–221. 29. Alonso-Vale MI, Andreotti S, Peres SB, et al. Melatonin enhances leptin expression by rat adipocytes in the presence of insulin. Am J Physiol Endocrinol Metab. 2005;288:E805–E812. 30. Alonso-Vale MI, Peres SB, Vernochet C, et al. Adipocyte differentiation is inhibited by melatonin through the regulation of C/EBPbeta transcriptional activity. J Pineal Res. 2009;47:221–227. 31. Gonciarz M, Bielanski W, Partyka R, et al. Plasma insulin, leptin, adiponectin, resistin, ghrelin, and melatonin in nonalcoholic steatohepatitis patients treated with melatonin. J Pineal Res. 2013;54:154–161. 32. Rios-Lugo MJ, Cano P, Jimenez-Ortega V, et al. Melatonin effect on plasma adiponectin, leptin, insulin, glucose, triglycerides and cholesterol in normal and high fat-fed rats. J Pineal Res. 2010;49:342–348. 33. Ma T, Yang B, Verkman AS. Cloning of a novel water and ureapermeable aquaporin from mouse expressed strongly in colon, placenta, liver, and heart. Biochem Biophys Res Commun. 1997; 240:324–328. 34. Laforenza U, Cova E, Gastaldi G, et al. Aquaporin-8 is involved in water transport in isolated superficial colonocytes from rat proximal colon. J Nutr. 2005;135:2329–2336. 35. Calamita G, Mazzone A, Bizzoca A, et al. Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract. Eur J Cell Biol. 2001;80:711–719. 36. Liu K, Nagase H, Huang CG, et al. Purification and functional characterization of aquaporin-8. Biol Cell. 2006;98:153–161. 37. Hardin JA, Wallace LE, Wong JF, et al. Aquaporin expression is downregulated in a murine model of colitis and in patients with ulcerative colitis, Crohn’s disease and infectious colitis. Cell Tissue Res. 2004;318:313–323. 38. Zahn A, Moehle C, Langmann T, et al. Aquaporin-8 expression is reduced in ileum and induced in colon of patients with ulcerative colitis. World J Gastroenterol. 2007;13:1687–1695. 39. Dimova DK, Dyson NJ. The E2F transcriptional network: old acquaintances with new faces. Oncogene. 2005;24:2810–2826. 40. Fajas L, Landsberg RL, Huss-Garcia Y, et al. E2Fs regulate adipocyte differentiation. Dev Cell. 2002;3:39–49. 41. Tsantoulis PK, Gorgoulis VG. Involvement of E2F transcription factor family in cancer. Eur J Cancer. 2005;41:2403–2414. 42. Blum JS, Wearsch PA, Cresswell P. Pathways of antigen processing. Annu Rev Immunol. 2013;31:443–473. 43. Tang Y, Preuss F, Turek FW, et al. Sleep deprivation worsens inflammation and delays recovery in a mouse model of colitis. Sleep Med. 2009;10:597–603. 44. Lee YD, Kim JY, Lee KH, et al. Melatonin attenuates lipopolysaccharide-induced acute lung inflammation in sleep-deprived mice. J Pineal Res. 2009;46:53–57. 45. Machado RB, Hipolide DC, Benedito-Silva AA, et al. Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery. Brain Res. 2004;1004: 45–51.
123
Dig Dis Sci 46. Suchecki D, Tufik S. Social stability attenuates the stress in the modified multiple platform method for paradoxical sleep deprivation in the rat. Physiol Behav. 2000;68:309–316. 47. Silva RH, Kameda SR, Carvalho RC, et al. Anxiogenic effect of sleep deprivation in the elevated plus-maze test in mice. Psychopharmacology. 2004;176:115–122.
123
48. Nosal’ova V, Zeman M, Cerna S, et al. Protective effect of melatonin in acetic acid induced colitis in rats. J Pineal Res. 2007;42:364–370. 49. Terry PD, Villinger F, Bubenik GA, et al. Melatonin and ulcerative colitis: evidence, biological mechanisms, and future research. Inflamm Bowel Dis. 2009;15:134–140.
