Melatonin modulates adiponectin expression on murine colitis with sleep deprivation

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1 Submit Mnuscript: Help Desk: DOI: /wjg.v22.i World J Gstroenterol 216 September 7; 22(33): ISSN (print) ISSN (online) 216 Bishideng Publishing Group Inc. All rights reserved. ORIGINAL ARTICLE Bsic Study Meltonin modultes diponectin expression on murine colitis with sleep deprivtion Te Kyun Kim, Young Sook Prk, Hing-Woon Bik, Jin Hyun Jun, Eun Kyung Kim, Je Woong Sull, Ho Joong Sung, Jin Woo Choi, Sook Hee Chung, Myung Chn Gye, Ju Yeon Lim, Jun Bong Kim, Seong Hwn Kim Te Kyun Kim, Young Sook Prk, Jun Bong Kim, Seong Hwn Kim, Deprtment of Gstroenterology, Eulji University School of Medicine, Eulji Hospitl, Seoul , South Kore Hing-Woon Bik, Deprtment of Biochemistry nd Moleculr Biology, Eulji University School of Medicine, Dejeon 31-7, South Kore Jin Hyun Jun, Je Woong Sull, Ho Joong Sung, Jin Woo Choi, Deprtemnt of Biomedicl Lbortory Science, Grdute School of Helth Science, Eulji University, Seongnm-si 13135, South Kore Jin Hyun Jun, Deprtemnt of Senior Helthcre, BK21 plus Progrm, Grdute School, Eulji University, Dejeon 34824, South Kore Eun Kyung Kim, Deprtment of Pthology, Eulji University School of Medicine, Seoul , South Kore Sook Hee Chung, Deprtment of Gstroenterology, Ajou University School of Medicine, Suwon , South Kore Myung Chn Gye, Ju Yeon Lim, Deprtment of Life Science nd Institute of Nturl Sciences, Hnyng University, Seoul 4763, South Kore Author contributions: All the uthors contributed to this mnuscript. Supported by 212 reserch fund from Eulji University. Institutionl review bord sttement: The Institutionl Review Bord of Eulji Hospitl t Eulji University College of Medicine pproved the study protocol (Approvl number: EUIACUC12-11). Institutionl niml cre nd use committee sttement: All procedures in experiments were conducted ccording to the Animl Cre Guidelines of the Ntionl Institutes of Helth nd Koren Acdemy of Medicl Sciences. Conflict-of-interest sttement: There is no conflict of interest in this study. Dt shring sttement: No dditionl dt re vilble. Open-Access: This rticle is n open-ccess rticle which ws selected by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ccordnce with the Cretive Commons Attribution Non Commercil (CC BY-NC 4.) license, which permits others to distribute, remix, dpt, build upon this work non-commercilly, nd license their derivtive works on different terms, provided the originl work is properly cited nd the use is non-commercil. See: licenses/by-nc/4./ Mnuscript source: Invited mnuscript Correspondence to: Young Sook Prk, MD, PhD, Deprtment of Gstroenterology, Eulji University School of Medicine, Eulji Hospitl, 68 Hngeulbiseok-Ro, Nowon-Gu, Seoul , South Kore. pys119@eulji.c.kr Telephone: Fx: Received: My 5, 216 Peer-review strted: My 6, 216 First decision: June 2, 216 Revised: July 11, 216 Accepted: July 31, 216 Article in press: July 31, 216 Published online: September 7, 216 Abstrct AIM To determine diponectin expression in colonic tissue of murine colitis nd systemic cytokine expression fter meltonin tretments nd sleep deprivtion September 7, 216 Volume 22 Issue 33

2 METHODS The following five groups of C57BL/6 mice were used in this study: (1) group Ⅰ, control; (2) group Ⅱ, 2% DSS induced colitis for 7 d; (3) group Ⅲ, 2% DSS induced colitis nd meltonin tretment; (4) group Ⅳ, 2% DSS induced colitis with sleep deprivtion (SD) using specilly designed nd modified multiple pltform wter bths; nd (5) group V, 2% DSS induced colitis with SD nd meltonin tretment. Meltonin (1 mg/kg) or sline ws intrperitonelly injected dily to mice for 4 d. The body weight ws monitored dily. The degree of colitis ws evluted histologiclly fter scrificing the mice. Immunohistochemicl stining nd Western blot nlysis ws performed using nti-diponectin ntibody. After smpling by intrcrdic punctures, levels of serum cytokines were mesured by ELISA. RESULTS Sleep deprivtion in wter bth excerbted DSS induced colitis nd worsened weight loss. Meltonin injection not only llevited the severity of mucosl injury, but lso helped survivl during stressful condition. The expression level of diponectin in mucos ws decresed in colitis, with the lowest level observed in colitis combined with sleep deprivtion. Meltonin injection significntly (P <.5) recovered the expression of diponectin. The expression levels of IL-6 nd IL-17 were incresed in the serum of mice with DSS colitis but decresed fter meltonin injection. CONCLUSION This study suggested tht meltonin modulted diponectin expression in colonic tissue nd meltonin nd diponectin synergisticlly potentited ntiinflmmtory effects on colitis with sleep deprivtion. Key words: Colitis; Meltonin; Adiponectin; Sleep deprivtion The Author(s) 216. Published by Bishideng Publishing Group Inc. All rights reserved. Core tip: We report this first study tht meltonin nd sleep deprivtion re relted to diponectin expression in the colonic mucos of murine colitis. C57BL/6 mice were feeding with 2% DSS for inducing colitis nd using specilly designed multiple pltform wter bths for sleep deprivtion. Immuno-histochemicl stining nd Western blot nlysis ws performed using nti-diponectin ntibody. The expression level of diponectin in mucos ws decresed in colitis, with the lowest level observed in colitis combined with sleep deprivtion. Meltonin injection significntly (P <.5) recovered the expression of diponectin. This study suggests tht meltonin nd diponectin synergisticlly potentite the nti-inflmmtory effects in murine colitis. Kim TK, Prk YS, Bik HW, Jun JH, Kim EK, Sull JW, Sung HJ, Choi JW, Chung SH, Gye MC, Lim JY, Kim JB, Kim SH. Meltonin modultes diponectin expression on murine colitis with sleep deprivtion. World J Gstroenterol 216; 22(33): Avilble from: URL: com/ /full/v22/i33/7559.htm DOI: org/1.3748/wjg.v22.i INTRODUCTION Inflmmtory bowel disese (IBD) is cused by multiple genetic, environmentl, nd host fctors [1]. Environmentl fctors such s stress nd sleep disturbnce cn ffect the progression nd relpse of ptients with IBD [2,3]. Stress frequently influences sleep qulity. IBD ptients hve significnt sleep disturbnce even during inctive stte. Sleep disturbnce might ffect the qulity of life nd gstrointestinl symptoms. In ddition, it cn increse the risk of flre-up of IBD [4]. Inflmmtory cytokines such s tumor necrosis fctor, interleukin-1 (IL-1), nd IL-6 re known s significnt contributors to sleep disturbnces. On the other hnd, sleep disturbnces cn upregulte these inflmmtory cytokines [5]. Meltonin is secreted by pinel glnd. It functions s regultor of circdin rhythms nd n ntioxidnt [6]. Meltonin levels in the gut re independent of pinel production. Pinelectomy hs no influence on gut meltonin concentrtions in rts [7]. At ny time of the dy or night, the gut contins t lest 4 times more meltonin thn tht of the pinel glnd, emphsizing the functionl importnce of meltonin in the gut [8]. The meltonin in GI trct hs nti-inflmmtory effect in experimentl models of colitis in mny previous reports [9-13]. Our previous study lso shows tht mrna level of diponectin is down regulted by sleep deprivtion but up-regulted by meltonin bsed on microrrys nd rel-time PCR nlysis of mice colon tissues [14]. Currently, the reltionship between meltonin nd diponectin on colitis with sleep deprivtion remins unknown. It is meningful to evlute the level of diponectin expressed in colon tissue of mice by immunohistochemicl stining nd western blotting to understnd the pthogenesis of inflmmtory bowel disese. Therefore, the objective of this study ws to investigte tissue expression of diponectin in colitis with sleep deprivtion nd meltonin injection. The expression levels of cytokines in mouse during sleep deprivtion with meltonin injection were lso nlyzed in this study. MATERIALS AND METHODS Animls A totl of 3 eight-week-old C57BL mle mice with body weight of 2-25 g were purchsed from Smtko Inc. (Gyunggido, Kore). They were rndomly ssigned 756 September 7, 216 Volume 22 Issue 33

3 Group Ⅰ (control) Group Ⅱ (DSS) Group Ⅲ (DSS + M) 2% DSS Sleep deprivtion Meltonin Scrifice d d d wter bth specificlly designed for sleep deprivtion. In wter tnk, 4 pltforms nd 2 mice were plced. Every mouse in the wter bth could move from one pltform to nother by jumping. The wter bth ws filled with wter for 4 cm from the bse (enough to drown). When mice strted to sleep, muscle toni led mice to fll down into the wter, wking up the mice. They would try to climb up the pltform to void being drowned. Through ll experiments, wter ws chnged every dy to clen wter. Prtil sleep deprivtion of mice begn from 2 PM to 1 AM (2 h) for 4 d. Mice hd 4 h of sleep dily from 1 AM to 2 PM during the 4 d. After finishing the 4 d of prtil sleep deprivtion, mice were scrificed for nlysis. Group Ⅳ (DSS + SD) Group Ⅴ (DSS + SD + M) d d Figure 1 Experimentl procedure. Experimentl colitis of mice ws induced by feeding mice with 2% DSS in group Ⅱ. Meltonin ws dministered to mice for 4 d with 2% DSS in group Ⅲ. Sleep deprivtion ws induced prtilly with 2% DSS in group Ⅳ. 2% DSS with prtil sleep deprivtion nd meltonin ws dministered to mice in group Ⅴ. M: Meltonin; SD: Sleep deprivtion. to the following five groups (6 mice per group): (1) Group Ⅰ, control; (2) Group Ⅱ, 2% Dextrose Sodium Sulfte(DSS) induced colitis for 7 d; (3) Group Ⅲ, 2% DSS induced colitis nd meltonin tretment; (4) Group Ⅳ, 2% DSS induced colitis with sleep deprivtion (SD) (2 h wkening/d) using specilly designed nd modified multiple pltform wter bths; nd (5) Group V, 2% DSS induced colitis with SD nd meltonin (Figure 1). Temperture of nd humidity of 55%-6% were mintined with 12-h light/drk cycle (lights on t 8:). During the experiment period, mice hd free ccess to food nd wter. Their body weights were mesured dily. All procedures in experiments were conducted ccording to the Animl Cre Guidelines of the Ntionl Institutes of Helth nd Koren Acdemy of Medicl Sciences. The Institutionl Review Bord of Eulji Hospitl t Eulji University College of Medicine pproved the study protocol (Approvl number: EUIACUC12-11). Experimentl procedures 2% DSS induced colitis model: For 7 d, mice were fed with 2% DSS (Sigm-Aldrich, Inc., United Sttes) solution to induce colitis. Prtil sleep deprivtion: On the 3 rd dy, ll mice were moved to wter bth. Wter bth hd either wide pltforms for norml sleep or nrrow pltforms for sleep deprivtion. Prtil sleep deprivtion of mice ws chieved by using modified multiple pltform Administrtion of meltonin: After induction of colitis with 2% DSS, meltonin ws dministered to mice intrperitonelly t dose of 1 mg/kg for 4 d. Norml sline ws dministered to mice intrperitonelly s control. Assessment of the severity of colitis: The severity of colitis ws ssessed by mesuring weight loss nd histologicl nlysis. Colon tissues were fixed with 1% formldehyde solution. Fixed tissues were dehydrted with lcohol nd embedded with prffin. Tissues were sliced into 4-μm thick sections. These sections were stined with hemtoxylin nd eosin to evlute inflmmtory chnge. The severity of inflmmtion in colon ws determined, including loss of mucosl structure (score -3), crypt bscess (score -1), thickened muscle (score -3), cellulr infiltrtion (score -3), nd depletion of goblet cell (score -1). The finl score for the severity of inflmmtion rnged from to 11 [15]. Immunohistochemicl stining of diponectin: Immunohistochemistry procedures were crried out using DAKO Autostiner plus (DAKO Cytomtion, Crpinteri, CA, United Sttes). Briefly, 4-μm sections of formlin-fixed nd prffin-embedded tissues were positioned onto poly-l-lysine slides. After deprffiniztion nd rehydrtion, ntigen retrievl ws performed using citrte buffer solution (ph 6.) t 121 for 1 min. Endogenous peroxidse ws blocked with 3% hydrogen peroxide for 5 min. The sections were then incubted with specific ntibodies ginst Adiponectin (Abcm, Cmbridge, United Kingdom, 1:5). Slides were then stined with 3,3 -diminobenzidine nd counterstined with hemtoxylin. Immunorectivity ws determined bsed on the percentge of stined cells: 1 + for less thn 1%, 2 + for 1%-5%, nd 3 + for over 5%. Quntifiction of diponectin by western blot nlysis: Tissues were homogenized in 1 volumes of extrction buffer (2 mmol/l Tris-HCl, ph 7.5, 1% Triton X-1) contining protese inhibitor cocktil (Complete TM, Roche, Germny) on ice September 7, 216 Volume 22 Issue 33

4 Group Ⅰ (control) Group Ⅱ (DSS) Group Ⅲ (DSS + M) Group Ⅳ (DSS + SD) Group Ⅴ (DSS + SD + M) Move to wter bth Group Ⅰ (control) Group Ⅱ (DSS) Group Ⅲ (DSS + M) Group Ⅳ (DSS + SD) Group Ⅴ (DSS + SD + M) t (d) t (d) Figure 2 Chnges in body weight of ech group. Group Ⅰ: control, Group Ⅱ: 2% DSS induced colitis, Group Ⅲ: 2% DSS induced colitis with meltonin tretment, Group Ⅳ: 2% DSS induced colitis with sleep deprivtion, Group Ⅴ: 2% DSS induced colitis with sleep deprivtion nd meltonin tretment). Dt were presented s men ± SD. M: Meltonin; SD: Sleep deprivtion. Figure 3 Survivl rte of ech group. Group Ⅰ: control, Group Ⅱ: 2% DSS induced colitis, Group Ⅲ: 2 % DSS induced colitis with meltonin tretment, Group Ⅳ: 2 % DSS induced colitis with sleep deprivtion, Group Ⅴ: 2% DSS induced colitis with sleep deprivtion nd meltonin tretment). M: Meltonin; SD: Sleep deprivtion. Homogentes were centrifuged t 12g for 1 h t 4. The superntnt ws collected nd its protein concentrtion ws determined using commercil protein ssy kit (Bio-Rd, CA). Protein smples were mixed with 2X smple buffer (Lemmli, 197), boiled for 5 min, nd cooled t room temperture. After briefly spinning, cler superntnts were resolved on 8% SDS-polycrylmide gels. Proteins were electrophoreticlly trnsferred to nitrocellulose membrnes (GE Helthcre, United Kingdom) nd blocked with Tris-buffered sline (TBS) contining 1% bovine serum lbumin overnight t 4. After rinsing three times with TBS/.1% Tween 2 (TBST) for 1 min ech, protein blots were incubted with rbbit nti-diponectin polyclonl ntibody (b62551, bcm, United Kingdom) diluted 1:1 in TBS for 2 h t room temperture. After rinsing with TBST three times for 1 min ech, the blots were incubted with peroxidse-lbeled got nti-rbbit IgG (Invitrogen, OR, United Sttes) diluted 1:1 in TBST for 1 h. The blots were wshed with TBST for 1 min followed by wshing with TBS for 1 min. Signls of protein bnds were detected using n ECL kit (GE Helthcre). Mesurement of serum cytokines: Blood were collected by intrcrdic smpling from the experimentl mice just before scrificing. Serum ws prepred to mesure cytokine levels. Levels of IL-17 (Ry Bio ELISA Kit Mouse IL-17; RyBiotech, Norcross, GA, United Sttes), IL-6 (Ry Bio ELISA Kit Mouse IL-6; RyBiotech), nd TNF-α (Ry Bio ELISA KitMouse TNF-lph; RyBiotech) were evluted using commercilly vilble kits following the mnufcturer s instructions. Sttisticl nlysis All dt were represented s men ± SD. Sttisticl Pckge for Socil Sciences softwre (SPSS; Koren version 18.) ws used for ll sttisticl nlyses. One wy nlysis of vrince (ANOVA) ws used to clculte the sttisticl significnce. Significnce ws considered when P vlue ws less thn.5. RESULTS Effect of sleep deprivtion nd meltonin tretment on body weight nd survivl On the 3 rd d, mice were moved to wter bth. From tht time, the body weight of ll mice strted to decrese due to stressful wter bth condition. The body weight of 2% DSS induced colitis group ws significntly (P =.5) lower thn tht of the control group (Figure 2). Sleep deprivtion induced severe weight loss. Hlf of mice were ded during sleep deprivtion (Figure 3). Meltonin filed to significntly recover the weight loss, but it could help ll mice survive during sleep deprivtion. Effect of sleep deprivtion with meltonin tretment on colonic inflmmtion In histologicl nlysis, the colon of 2% DSS induced colitis mice showed edem nd infiltrtion of inflmmtory cells into the mucos compred to tht of the control group. Meltonin tretment reduced inflmmtion in the colon of 2% DSS induced colitis in group Ⅲ. The number of infiltrting cells nd mucosl injury such s ulcer or necrosis were incresed by sleep deprivtion in the colon of 2% DSS induced colitis mice. Meltonin tretment lso diminished the inflmmtion nd erosion in the colon of 2% DSS induced colitis mice with sleep deprivtion. The microscopic inflmmtory score of the colitis group (Group Ⅱ) ws 6.5 ±.6. It ws significntly (P =.3) decresed to 4.4 ±.8 by meltonin tretment (Group Ⅲ). However, it ws significntly (P =.18) incresed to 8.5 ±.5 by sleep deprivtion (Group Ⅳ). The histologic score of 8.5 ±.5 of 2%DSS induced colitis mice with sleep 7562 September 7, 216 Volume 22 Issue 33

5 A B C D E Figure 4 Immunohistochemicl stining of diponectin (A: 1, B-E: 2). A: control; B: DSS: 2% DSS induced colitis; C: DSS + M: 2% DSS induced colitis with meltonin tretment; D: DSS + SD: 2% DSS induced colitis with sleep deprivtion; E: DSS + SD + M: 2% DSS induced colitis with sleep deprivtion nd meltonin tretment. Immunorectivity DSS Adiponectin DSS + M DSS + SD Figure 5 Adiponectin immunorectivity bsed on immunohistochemicl stining. Meltonin injection significntly recovered diponectin expression, P <.5 vs control. M: Meltonin; SD: Sleep deprivtion. DSS + SD + M deprivtion (group Ⅳ) ws decresed (P =.67) by meltonin tretment (group V, score of 7. ± 1.). Chnges of inflmmtory score in ll groups were sttisticlly significnt (P <.1). Immunohistochemicl stining of diponectin expression by meltonin tretment Adiponectin expression in colonic mucos ws determined by immunohistochemicl stining (Figure 4). Colitis mice nd colitis mice with sleep deprivtion showed decresed diponectin immunorectivity compred to mice of the control group. In meltonin injection group, mice showed significnt recovery in diponectin immunorectivity (Figure 5). Western blotting of diponectin expression fter meltonin tretment Bsed on western blot nlysis, the expression levels 7563 September 7, 216 Volume 22 Issue 33

6 Tble 1 Effect of meltonin nd sleep deprivtion on inflmmtory chnge of 2% DSS induced colitis mice bsed on histology DSS DSS+M DSS+SD DSS+SD+M P vlue Microscopic scores ± 6.5 ± ± ± ± 1. <.1 M: Meltonin; SD: Sleep deprivtion. Adiponectin/GAPDH DSS DSS + M DSS + SD DSS + SD + M DSS DSS + M DSS + SD Adiponectin (32 kd) GAPDH (37 kd) Figure 6 Western blot nlysis of diponectin. The expression of diponectin ws reduced in DSS colitis nd DSS colitis with sleep deprivtion. Meltonin injection significntly recovered diponectin expression, P <.5 vs control. M: Meltonin; SD: Sleep deprivtion. of diponectin were decresed in the colitis group nd the colitis with sleep deprivtion group compred to tht in the control group (Figure 6). In the group of colitis with sleep deprivtion, diponectin expression ws significntly (P <.5) recovered by meltonin injection (Figure 6). These results re consistent with results from immunohistochemicl stining. Estimtion of inflmmtory cytokines in the serum of mice The levels of pro-inflmmtory cytokines such s IL-6, IL-17, nd TNF-α in the serum of mice were incresed in DSS colitis group nd colitis with sleep deprivtion group compred to those of the control group. Their levels were decresed fter meltonin injection. Even in the control group, the levels of these proinflmmtory cytokines were reltively higher level thn tht in the meltonin tretment group. This could be due to the fct tht ll mice were moved to wter bth t the 3 rd experimentl dy when control mice got stressed out due to their fer of wter. The expression levels of IL-6, IL-17, nd TNF-α were significntly (P <.5) decresed fter meltonin tretment (Figure 7). The level of nti-inflmmtory cytokine IL-1 ws DSS + SD + M lower in colitis. However, it hd tendency of increse fter meltonin injection, indicting tht meltonin tretment might hve systemic nti-inflmmtory properties. DISCUSSION Meltonin plys vrious importnt roles in the GI trct. As physiologicl ntgonist to serotonin, it decreses peristlsis nd stimultes the secretion of mucosl bicrbonte medited by MT2 receptor. An importnt receptor independent ction of meltonin in the GI trct is tht it is free rdicl scvenger. The preventive role of meltonin ginst ulcer formtion for heling hs been well estblished [16]. Previous studies hve suggested tht meltonin dministrtion is beneficil for IBD vi its ntioxidnt, nti-poptotic, nd nti-inflmmtory properties. In this study, pthologic exmintion showed tht meltonin could ttenute inflmmtion nd decrese the severity of inflmmtion under sleep deprivtion (Tble 1). During our experiment, moving to wter bth ws stressful to mouse. mice lso lost weight in the wter bth. The sleep deprivtion group showed ggrvted weight loss nd hlf of this group could not survive until the end of experiments. However, meltonin injection could help the survivl of mice under sleep deprivtion. In our previous study, we found the genetic expression of diponectin fter meltonin injection with sleep deprivtion using microrry nd PCR [14]. Here, we tried to confirm the tissue expression of diponectin fter meltonin injection through immunohistochemicl stining (Figure 4) nd Western blotting (Figure 6). Our results showed tht diponectin expression ws decresed in both colitis nd colitis with sleep deprivtion condition. However, meltonin injection significntly recovered the diponectin expression in colon tissues, in consistent with genetic chnge tht we found erlier. Adiponectin is minly expressed by mture dipocytes. It circultes t high levels in the blood strem [17]. There re some reports bout the ntiinflmmtory effect of diponectin. Adiponectin could inhibit the expression of NF-kB, TNF-α, IL-6, nd IL- 1b but induce the expression of nti-inflmmtory cytokines such s IL-1 nd IL-1 receptor ntgonists [18]. Adiponectin cn lso induce the production of IL-1 nd IL-1R in humn PBMC, mcrophges, nd DC while impiring the production of IFNγ in mcrophges [19-21]. Overexpressed diponectin in mice cn decrese 7564 September 7, 216 Volume 22 Issue 33

7 A IL-6 B IL-17A pg/ml 2 15 pg/ml Colitis Colitis + M Colitis + SD Colitis + SD + M Colitis Colitis + M Colitis + SD Colitis + SD + M C TNF- D IL pg/ml 15 1 pg/ml Colitis Colitis + M Colitis + SD Colitis + SD + M Colitis Colitis + M Colitis + SD Colitis + SD + M Figure 7 Cytokine levels in serum of mice. Pro-inflmmtory cytokines such s IL-6 (A), IL-17 (B), nd TNFα (C) were reduced by meltonin injection; IL-1 (D) expression ws not significntly chnged. P <.5 vs control. M: Meltonin; SD: Sleep deprivtion. pro-inflmmtory cytokines including cellulr stress mrkers, TNF, IL-6, nd IL-1b in mice [22,23]. Our study showed tht mucosl expression of diponectin ws significntly decresed in DSS-induced colitis compred to tht in the control. This result ws consistent with other reports. Adiponectin mrna level is reported to be decresed in inflmed colonic mucos of DSS induced murine colitis [24]. Adiponectin cn inhibit chemokine production in intestinl epithelil cells. It hs been reported tht diponectin KO mice hs much more severe colitis compred to wild type mice [25]. Recently, diponectin expression in humn tissue hs been identified. In ptients with ulcertive colitis, the mrna expression of diponectin in colonic mucos is decresed [24]. Adiponectin concentrtion in mesenteric dipose tissue of CD ptients hs been found to be significntly lower in ptients with internl fistul compred to tht in ptients without fistul [26]. Interction of meltonin with diponectin ws first reported by Ríos-Lugo et l [18]. They showed tht meltonin ttenuted body weight increse nd hyperglycemi, but incresed men plsm diponectin in high ft-fed rts. The high-ft diet disrupted the norml 24-h pttern of diponectin, which ws countercted by meltonin [18]. Direct effect of meltonin on diponectin hs not been clrified yet. An in vitro study using pre-dipocytes hs shown n inhibitory effect of meltonin on diponectin synthesis [18,27]. Another study hs demonstrted tht chronic meltonin dministrtion to rts minly ffects the 24-h rhythm of diponectin secretion [28]. A recent study hs shown significnt increse in medin plsm diponectin levels nd insulin sensitivity s erly s 4 weeks fter meltonin dministrtion in cohort of ptients with non-lcoholic stetoheptitis [29] September 7, 216 Volume 22 Issue 33

8 In this study, the expression level of diponectin ws lower in DSS-induced colitis thn tht in the control, with the lowest level in the sleep deprivtion group. After meltonin injection, the expression level of diponectin ws significntly incresed, even higher thn tht in the control. Bsed on these results, diponectin expression in colonic tissue might be modulted by meltonin injection. These results re comprble to genetic expression of diponectin fter meltonin tretment in our previous study [14]. To understnd the mechnism of inflmmtion, the levels of serum cytokines of mice were determined. Pro-inflmmtory cytokines such s IL-6, IL-17A, nd TNF-α were incresed in colitis. As one of mechnisms to control inflmmtion, meltonin cn modulte vriety of moleculr trgets, including NF-kB, cyclooxygense-2, interleukin 17, mtrix metlloproteinse-9, nd connective tissue growth fctor [3]. We lso found tht the levels of proinflmmtory cytokines such s IL-6, IL-17A, nd TNF-α were decresed while the level of IL-1 ws incresed fter meltonin tretment (Figure 7). Therefore, diponectin decresed levels of proinflmmtory cytokines but incresed level of IL-1 in mcrophge nd PBMC. Meltonin nd diponectin might hve synergistic effect in these cytokine profiles. However, there ws no significnt cytokine chnge fter sleep deprivtion. This could be due to the fct tht we could only check serum cytokine levels in survived mice. Ded mice might hve higher proinflmmtory cytokine levels thn survived mice. Our results showed tht sleep deprivtion group mice hd more severe pthologic inflmmtion with worse survivl rte, indicting tht sleep is importnt for survivl. Under the sme condition, ll meltonin injected mice survived during the experimentl period (Figure 3). Although the precise mechnism on how meltonin helped the survivl of mice is currently unknown, meltonin might be beneficil to overcome stressful conditions such s sleep deprivtion for humn. Th17 cell linege is importnt for pthogenesis of IBD. It produces importnt inflmmtory cytokine IL-17. Our results lso demonstrted tht meltonin injection decresed IL-17 level. Th17 linegespecific trnscription fctor RORα is nturl trget of meltonin in T cells [31]. RORα, long with RORγ, cn regulte Th17 cell differentition [32], wheres down regultion of RORα expression is prt of the typicl Treg trnscriptionl signture [33]. A preliminry study hs highlighted the in vivo inhibitory ctions of meltonin on Treg cell genertion in cncer ptients [34]. It hs been reported tht in vivo dministrtion of meltonin to mice subjected to experimentl cncer hs down regultion of CD4+CD25+ Treg cells nd Foxp3 expression in tumor tissues [35]. These results suggest tht meltonin my hve djuvnt effect in inhibiting colitis ssocited cncer development in ptients with IBD. The levels of plsm meltonin in ptients with ulcertive colitis (UC) hve been reported to be significntly lower thn those of helthy control [36]. They re higher in remission stte thn in ctive disese. Meltonin cn lso cuse the disppernce of clinicl symptoms of ptients with UC. These symptoms re reported to hve reppered when meltonin consumption is stopped [37]. Adjuvnt tretment with meltonin in UC ptients is reported to keep remission for 12 mo with norml C- rective protein rnges nd high hemoglobin levels, suggesting tht djuvnt meltonin therpy my help sustin remission of ptients with UC [38]. However, more clinicl trils should be performed to confirm the beneficil effect of meltonin on IBD, lthough bsic studies strongly suggest such beneficil effect. To the best of our knowledge, this is the first study to report tht meltonin nd sleep deprivtion re relted to diponectin expression in colonic mucos of DSS colitis. Results of this study were consistent with results of previous study bout the genetic expression of diponectin fter meltonin nd sleep deprivtion. In summry, we found tht sleep deprivtion ggrvted inflmmtion nd lowered survivl rte. However, meltonin hd protective effect on inflmmtory chnge nd helped the survivl of DSS-induced colitis of mice. In ddition, meltonin modulted diponectin expression in colitis mice with sleep deprivtion. This study suggests tht meltonin nd diponectin synergisticlly potentite the ntiinflmmtory effects in the colitis. Becuse severe colitis with sleep deprivtion is frequent condition of ctive IBD ptients, meltonin is expected to be used for control of inflmmtion nd sleep deprivtion. COMMENTS Bckground Meltonin is secreted by pinel glnd. It functions s regultor of circdin rhythms nd n ntioxidnt. Meltonin levels in the gut re independent of pinel production. The gut contins t lest 4 times more meltonin thn the pinel glnd, emphsizing the functionl importnce of meltonin in the gut. The meltonin in GI trct hs nti-inflmmtory effect in experimentl models of colitis in mny previous reports. Reserch frontiers This previous study lso shows tht mrna level of diponectin is down regulted by sleep deprivtion but up-regulted by meltonin bsed on microrrys nd rel-time PCR nlysis of mice colon tissues Innovtions nd brekthroughs This is the first study tht meltonin nd sleep deprivtion re relted to diponectin expression in the colonic mucos of murine colitis being performed by immuno-histochemicl stining nd Western blot nlysis. So, the uthors confirmed previous genetic chnge of diponectin on microrrys nd rel-time PCR nlysis. Applictions The expression level of diponectin in mucos ws decresed in colitis, with the lowest level observed in colitis combined with sleep deprivtion. Meltonin injection significntly recovered the expression of diponectin. This study 7566 September 7, 216 Volume 22 Issue 33

9 suggests tht meltonin nd diponectin synergisticlly potentite the ntiinflmmtory effects in murine colitis. Terminology No specific terminology re used in this pper Peer-review Using the DSS-induced colitis mice model, the uthors studied the diponectin expression in colonic tissue of murine colitis nd cytokine expression in response to meltonin tretments nd sleep deprivtion. The results showed tht sleep deprivtion excerbted DSS colitis, which cn be llevited by meltonin injection. The expression level of diponectin in mucos ws decresed in colitis, with the lowest level observed in colitis combined with sleep deprivtion. REFERENCES 1 Bmis G, Cominelli F. Immunopthogenesis of inflmmtory bowel disese: current concepts. Curr Opin Gstroenterol 27; 23: [ PMID: DOI: / MOG.b13e3281c55eb2] 2 Frrell RJ, Peppercorn MA. Ulcertive colitis. Lncet 22; 359: [PMID: ] 3 Niess JH, Mönnikes H, Dignss AU, Klpp BF, Arck PC. Review on the influence of stress on immune meditors, neuropeptides nd hormones with relevnce for inflmmtory bowel disese. Digestion 22; 65: [PMID: ] 4 Rnjbrn Z, Keefer L, Frhdi A, Stepnski E, Sedghi S, Keshvrzin A. Impct of sleep disturbnces in inflmmtory bowel disese. J Gstroenterol Heptol 27; 22: [PMID: DOI: /j x] 5 Ali T, Choe J, Awb A, Wgener TL, Orr WC. Sleep, immunity nd inflmmtion in gstrointestinl disorders. World J Gstroenterol 213; 19: [PMID: DOI: /wjg.v19. i ] 6 Mrquez E, Sánchez-Fidlgo S, Clvo JR, l de Lstr CA, Motilv V. Acutely dministered meltonin is beneficil while chronic meltonin tretment ggrvtes the evolution of TNBSinduced colitis. J Pinel Res 26; 4: [PMID: DOI: /j.16-79X x] 7 Bubenik GA, Brown GM. Pinelectomy reduces meltonin levels in the serum but not in the gstrointestinl trct of rts. Biol Signls 1997; 6: 4-44 [PMID: DOI: /1917] 8 Chen CQ, Fichn J, Bshshti M, Li YY, Storr M. Distribution, function nd physiologicl role of meltonin in the lower gut. World J Gstroenterol 211; 17: [PMID: DOI: /wjg.v17.i ] 9 Necefli A, Tulumoğlu B, Giriş M, Brbros U, Gündüz M, Olgç V, Güloğlu R, Toker G. The effect of meltonin on TNBS-induced colitis. Dig Dis Sci 26; 51: [PMID: DOI: 1.17/s ] 1 Li JH, Yu JP, Yu HG, Xu XM, Yu LL, Liu J, Luo HS. Meltonin reduces inflmmtory injury through inhibiting NF-kppB ctivtion in rts with colitis. Meditors Inflmm 25; 25: [PMID: DOI: /MI ] 11 Akcn A, Kucuk C, Sozuer E, Esel D, Akyildiz H, Akgun H, Muhtroglu S, Arits Y. Meltonin reduces bcteril trnsloction nd poptosis in trinitrobenzene sulphonic cid-induced colitis of rts. World J Gstroenterol 28; 14: [PMID: DOI: /wjg ] 12 Cuzzocre S, Mzzon E, Serrino I, Lepore V, Terrnov ML, Ciccolo A, Cputi AP. Meltonin reduces dinitrobenzene sulfonic cid-induced colitis. J Pinel Res 21; 3: 1-12 [PMID: DOI: 1.134/j.16-79X x] 13 Dong WG, Mei Q, Yu JP, Xu JM, Xing L, Xu Y. Effects of meltonin on the expression of inos nd COX-2 in rt models of colitis. World J Gstroenterol 23; 9: [PMID: DOI: /wjg.v9.i6.137] 14 Chung SH, Prk YS, Kim OS, Kim JH, Bik HW, Hong YO, Kim SS, Shin JH, Jun JH, Jo Y, Ahn SB, Jo YK, Son BK, Kim SH. Meltonin ttenutes dextrn sodium sulfte induced colitis with sleep deprivtion: possible mechnism by microrry nlysis. Dig Dis Sci 214; 59: [PMID: DOI: 1.17/ s ] 15 Thn G, Grmignoli R, Mrongiu F, Aktolg S, Cetinky A, Thn V, Dorko K. Meltonin expresses powerful ntiinflmmtory nd ntioxidnt ctivities resulting in complete improvement of cetic-cid-induced colitis in rts. Dig Dis Sci 211; 56: [PMID: DOI: 1.17/ s ] 16 Slominski RM, Reiter RJ, Schlbritz-Loutsevitch N, Ostrom RS, Slominski AT. Meltonin membrne receptors in peripherl tissues: distribution nd functions. Mol Cell Endocrinol 212; 351: [PMID: DOI: 1.116/j.mce ] 17 Fjs L, Lndsberg RL, Huss-Grci Y, Srdet C, Lees JA, Auwerx J. E2Fs regulte dipocyte differentition. Dev Cell 22; 3: [PMID: DOI: 1.116/S (2)19-9] 18 Ríos-Lugo MJ, Cno P, Jiménez-Orteg V, Fernández-Mteos MP, Sccchi PA, Crdinli DP, Esquifino AI. Meltonin effect on plsm diponectin, leptin, insulin, glucose, triglycerides nd cholesterol in norml nd high ft-fed rts. J Pinel Res 21; 49: [PMID: DOI: /j.16-79X x] 19 Kdowki T, Ymuchi T. Adiponectin nd diponectin receptors. Endocr Rev 25; 26: [PMID: DOI: 1.121/ er.25-5] 2 Kern PA, Di Gregorio GB, Lu T, Rssouli N, Rngnthn G. Adiponectin expression from humn dipose tissue: reltion to obesity, insulin resistnce, nd tumor necrosis fctor-lph expression. Dibetes 23; 52: [PMID: DOI: /dibetes ] 21 Bruun JM, Lihn AS, Verdich C, Pedersen SB, Toubro S, Astrup A, Richelsen B. Regultion of diponectin by dipose tissuederived cytokines: in vivo nd in vitro investigtions in humns. Am J Physiol Endocrinol Metb 23; 285: E527-E533 [PMID: DOI: /jpendo.11.23] 22 Hung H, Prk PH, McMullen MR, Ngy LE. Mechnisms for the nti-inflmmtory effects of diponectin in mcrophges. J Gstroenterol Heptol 28; 23 Suppl 1: S5-S53 [PMID: DOI: /j x] 23 Thkur V, Pritchrd MT, McMullen MR, Ngy LE. Adiponectin normlizes LPS-stimulted TNF-lph production by rt Kupffer cells fter chronic ethnol feeding. Am J Physiol Gstrointest Liver Physiol 26; 29: G [PMID: DOI: / jpgi ] 24 Mtsung H, Hokri R, Kurihr C, Okd Y, Tkebyshi K, Okudir K, Wtnbe C, Komoto S, Nkmur M, Tsuzuki Y, Kwguchi A, Ngo S, Itoh K, Miur S. Omeg-3 ftty cids excerbte DSS-induced colitis through decresed diponectin in colonic subepithelil myofibroblsts. Inflmm Bowel Dis 28; 14: [PMID: DOI: 1.12/ibd.2491] 25 Nishihr T, Mtsud M, Arki H, Oshim K, Kihr S, Funhshi T, Shimomur I. Effect of diponectin on murine colitis induced by dextrn sulfte sodium. Gstroenterology 26; 131: [PMID: DOI: 1.153/ j.gstro ] 26 Ymmoto K, Kiyohr T, Murym Y, Kihr S, Okmoto Y, Funhshi T, Ito T, Nezu R, Tsutsui S, Miygw JI, Tmur S, Mtsuzw Y, Shimomur I, Shinomur Y. Production of diponectin, n nti-inflmmtory protein, in mesenteric dipose tissue in Crohn s disese. Gut 25; 54: [PMID: DOI: /gut ] 27 Alonso-Vle MI, Andreotti S, Peres SB, Anhê GF, ds Neves Borges-Silv C, Neto JC, Lim FB. Meltonin enhnces leptin expression by rt dipocytes in the presence of insulin. Am J Physiol Endocrinol Metb 25; 288: E85-E812 [PMID: DOI: /jpendo ] 28 Lee JH, Chn JL, Yinnkouris N, Kontoginni M, Estrd E, Seip R, Orlov C, Mntzoros CS. Circulting resistin levels re 7567 September 7, 216 Volume 22 Issue 33

10 not ssocited with obesity or insulin resistnce in humns nd re not regulted by fsting or leptin dministrtion: cross-sectionl nd interventionl studies in norml, insulin-resistnt, nd dibetic subjects. J Clin Endocrinol Metb 23; 88: [PMID: DOI: 1.121/jc ] 29 Goncirz M, Bielński W, Prtyk R, Brzozowski T, Konturek PC, Eszyk J, Celiński K, Reiter RJ, Konturek SJ. Plsm insulin, leptin, diponectin, resistin, ghrelin, nd meltonin in nonlcoholic stetoheptitis ptients treted with meltonin. J Pinel Res 213; 54: [PMID: DOI: /j.16-79X x] 3 Trivedi PP, Jen GB. Meltonin reduces ulcertive colitisssocited locl nd systemic dmge in mice: investigtion on possible mechnisms. Dig Dis Sci 213; 58: [PMID: DOI: 1.17/s ] 31 Lrdone PJ, Guerrero JM, Fernández-Sntos JM, Rubio A, Mrtín-Lcve I, Crrillo-Vico A. Meltonin synthesized by T lymphocytes s lignd of the retinoic cid-relted orphn receptor. J Pinel Res 211; 51: [PMID: DOI: /j.16-79X x] 32 Yng XO, Pppu BP, Nuriev R, Akimzhnov A, Kng HS, Chung Y, M L, Shh B, Pnopoulos AD, Schluns KS, Wtowich SS, Tin Q, Jetten AM, Dong C. T helper 17 linege differentition is progrmmed by orphn nucler receptors ROR lph nd ROR gmm. Immunity 28; 28: [PMID: DOI: 1.116/j.immuni ] 33 Du J, Hung C, Zhou B, Ziegler SF. Isoform-specific inhibition of ROR lph-medited trnscriptionl ctivtion by humn FOXP3. J Immunol 28; 18: [PMID: DOI: 1.449/jimmunol ] 34 Vigoré L, Messin G, Brivio F, Fumglli L, Rovelli F, DI Fede G, Lissoni P. Psychoneuroendocrine modultion of regultory T lymphocyte system: in vivo nd in vitro effects of the pinel immunomodulting hormone meltonin. In Vivo 21; 24: [PMID: ] 35 Liu H, Xu L, Wei JE, Xie MR, Wng SE, Zhou RX. Role of CD4+ CD25+ regultory T cells in meltonin-medited inhibition of murine gstric cncer cell growth in vivo nd in vitro. Ant Rec (Hoboken) 211; 294: [PMID: DOI: 1.12/ r.21361] 36 Chen M, Mei Q, Xu J, Lu C, Fng H, Liu X. Detection of meltonin nd homocysteine simultneously in ulcertive colitis. Clin Chim Act 212; 413: 3-33 [PMID: DOI: 1.116/ j.cc ] 37 Mnn S. Meltonin for ulcertive colitis? Am J Gstroenterol 23; 98: [PMID: DOI: /j x] 38 Chojncki C, Wisniewsk-Jrosinsk M, Wleck-Kpic E, Klupinsk G, Jworek J, Chojncki J. Evlution of meltonin effectiveness in the djuvnt tretment of ulcertive colitis. J Physiol Phrmcol 211; 62: [PMID: ] P- Reviewer: Chen JF S- Editor: Qi Y L- Editor: A E- Editor: Wng CH 7568 September 7, 216 Volume 22 Issue 33

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