Dig Dis Sci (2006) 51:1538–1545DOI 10.1007/s10620-005-9047-3

ORIGINAL PAPER

The Effect of Melatonin on TNBS-Induced ColitisAhmet Necefli · Burcu Tulumoglu · Murat Giris ·Umut Barbaros · Mucteba Gunduz · Vakur Olgac ·Recep Guloglu · Gulcin Toker

Received: 30 August 2005 / Accepted: 9 September 2005 / Published online: 22 August 2006C© Springer Science+Business Media, Inc. 2006

Abstract Ulcerative colitis is a multifactorial inflammatorydisease of the colon and rectum with an unknown etiology.The present study was undertaken to investigate the effect ofmelatonin administration on oxidative damage and apoptosisin 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced coli-tis. Rats were divided into four groups as follows: Group 1(n = 8)—TNBS colitis; Group 2 (n = 8)—melatonin,10 mg/kg/day ip, for 15 days in addition to TNBS; Group3 (n = 8)—melatonin alone, 10 mg/kg/day ip, for 15 days;and Group 4 (n = 8)—isotonic saline solution, 1ml/rat ip,for 15 days (sham control group). Colonic myeloperoxidase(MPO) activities, malondialdehyde (MDA) levels, and glu-tathione (GSH) levels are indicators of oxidative damage,while caspase-3 activities reveal the degree of apoptosis ofthe colonic tissue. In all TNBS-treated rats, colonic MPOactivity and MDA levels were found to be increased signif-icantly compared to those in the sham group. Colonic MPO

A. Necefli · B. Tulumoglu · U. Barbaros · M. Gunduz · R. GulogluDepartments of General Surgery, Istanbul Medical Faculty,Istanbul University,Istanbul, Turkey

M. Giris · G. TokerDepartments of Biochemistry, Istanbul Medical Faculty, IstanbulUniversity,Istanbul, Turkey

V. OlgacDepartments of Oncologic Pathology, Istanbul Medical Faculty,Istanbul University,Istanbul, Turkey

B. Tulumoglu (�)Department of General Surgery, Istanbul Medical Faculty,Istanbul University, 34340 Capa,Istanbul, Turkeye-mail: burcutulumoglu@yahoo.com.tr

activity and MDA levels were significantly lower in the mela-tonin treatment group compared to TNBS-treated rats. GSHlevels of colonic tissues were found to be significantly lowerin TNBS-treated rats compared to the sham group. Treatmentwith melatonin significantly increased GSH levels comparedto those in TNBS-treated rats. Caspas-3 activity of colonictissues was found to be significantly higher in TNBS-treatedrats compared to the sham group. Treatment with melatoninsignificantly decreased caspase-3 activity compared to thatin TNBS-treated rats. These results imply a reduction in mu-cosal damage due to anti-inflammatory and anti-apoptoticeffects of melatonin.

Keywords Melatonin . 2,4,6-trinitrobenzene sulfonicacid-induced colitis . Apoptosis . NFκ-B

Ulcerative colitis is a chronic infammatory disease of thecolon and rectum that is characterized by a set of clinical,endoscopic, and histological features [1–5]. Although theprecise etiology of ulcerative colitis remains unknown, it isbelieved to involve an abnormal host response to endoge-nous or environmental antigens, genetic factors, or oxidativedamage. Histologically, ulcerative colitis is characterized bymassive infiltration of inflammatory cells in the mucosa andsubmucosa. Activation of these infiltrating cells results in therelease of inflammatory mediators [3, 4, 6]. These mediatorsinclude various pro-inflammatory cytokines, chemokines, re-active oxygen species, nitric oxide, and the eicosanoids [7–10]. Since increased lipid peroxidation by products and mu-cosal production of reactive oxygen metabolites have beendescribed in colorectal biopsy specimens of patients with ul-cerative colitis, increasing attention has been focused on therole of free radicals in the pathogenesis of ulcerative colitis[10–15].

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The aim of the present study was to evaluate the effectsof melatonin on tissue injury and oxidative stress in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ulcerative col-itis to set up a possible basis for treatment of ulcerative colitis.

Materials and methods

Study design Thirty-two male Wistar-albino rats weighing250–300 g (Institute of Experimental Medicine and Re-search, Istanbul University) were used in the study. All an-imals were housed in wire mesh-bottomed cages under a12/12-hr light/dark cycle. Rats were kept in a room at aconstant temperature 22 ± 2◦C. The rats were fed a stan-dard chow diet and water until the experiment. The ethicscommittee of Istanbul University, Istanbul Medical School,approved the study.

The rats were divided into four groups as follows: Group 1(n = 8)—TNBS colitis; Group 2 (n = 8)—melatonin (SigmaChemical Co.,USA), 10 mg/kg/day ip, for 15 days in additionto TNBS; Group 3 (n = 8)—melatonin alone, 10 mg/kg/dayip, for 15 days; and Group 4 (n = 8)—isotonic saline solu-tion, 1 ml/rat ip, for 15 days (sham control group).

Induction of colitis Rats were fasted overnight before induc-tion of colitis and then anesthetized by ether inhalation. Toinduce colitis a 5-French polypropylene canula was insertedinto the rectum until the tip was 10 cm. above the anus, and asolution of 80 mg/kg TNBS (Sigma Chemical Co.) dissolvedin 0.25 ml of 50% ethanol was instilled. The rats were thenmaintained in the supine Trendelenburg position for 15 min.

Macroscopic and microscopic colitis score All animals weresacrificed 15 days after TNBS treatment. The distal 12 cmof the colon was excised freed of adherent adipose tissue,rinsed with ice-cold saline, and opened longitudinally. Thecolon was immediately examined visually and damage wasscored on a scale of 0–5 as previously described [16]. Scor-ing of macroscopic colon damage in TNBS-induced colitiswas as follows: (0) no colonic damage, (1) hyperemia andno ulcer, (2) linear ulcer and no colonic wall thickening,(3) linear ulcer and colonic wall thickening in one area,(4) colonic ulcer in multiple areas, and (5) major ul-cer and perforation. Rat colons were fixed and paraffin-embedded tissue sections were stained with hemotoxylinand eosin (HE). Colonic pathological changes were ob-served and evaluated by two independent researchers usinga modified histopathological score formula [17] as follows:(1) infiltration of acute inflammatory cells—0 = without;1 = mild, 2 = severe; (2) infiltration of chronic inflamma-tory cells: 0 = without, 1 = mild, 2 = severe; (3) fib-rin deposition—0 = negative, 1 = positive; (4) submucosaledema—0 = without, 1 = focal, 2 = diffuse; (5) cecrosis of

epithelial cells—0 = without, 1 = focal; 2 = diffuse; and(6) mucosal ulcer—0 = negative, 1 = positive.

NF-κB expression Colon specimens were fixed in 10%buffered formalin. Paraffin blocks prepared from routinelyprocessed specimens were cut into 5-µm slices and deparaf-finized. Antigen retrieval was performed after this process.After microwave incubation of the peroxyblock, followedby the ultra V block procedure, primary antibodies (NF-κBneomarker, anti-rabbit P50Ab-2) were applied. Followingthis process, biotinylated secondary antibody, streptovidinperoxidase, and substrate-cromogen (AEC) solution was ap-plied, respectively. Nuclear staining was done with hema-toxylin. Staining intensity was defined as percentages andwas given scores ranging from 1 to 4: >75%, 4 + ; 50–75%,3 + ; 25–50%, 2 + ; and <25%, 1+ .

Myeloperoxidase activity Colonic myeloperoxidase activitywas assayed by the o-dianisidine method [18]. Tissue sam-ples were suspended in 50 mM, pH 6.0, potassium phos-phate buffer containing 0.5% hexadecyltrimethylammoniumbromide and homogenized. A sample of homogenate wassonicated for 20 sec, freeze-thawed three times, sonicatedagain for 10 sec, and centrifuged at 15,000g for 10 min at4◦C. The reaction was started by mixing and incubating 100µl of the supernatant at 25◦C with a solution composed of2.810 µl of 50 mM potassium phosphate buffer, pH 6.0,30 µl of 20 mg/ml o-dianisidine dihydrochloride, and 30µl of 20 mM H2O2. After 10 min the reaction was termi-nated by the addition of 30 µl of 2% sodium azide. Thechange in absorbance was read at 460 nm and results werecalculated using the molar absorptivity coefficient of oxi-dized o-dianisidine (1.0062 × 104 M−1 cm−1). MPO activity(1 unit) was expressed as the amount of enzyme necessaryfor the degradation of 1 µmol H2O2/min/100 mg tissue at25◦C.

Malondialdehyde assays The levels of MDA in the colonictissues were measured in order to assess lipid peroxidation.Samples of small intestine, liver, and pancreas tissues werehomogenized with ice-cold 150 nmol/L potassium chlo-ride for determination of MDA levels. MDA levels weremeasured spectrophotometrically. Results are expressed asnanomoles of MDA per gram of tissue [19].

Glutathione Glutathione level in the colonic homogenateswas determined by the method of Sedlak and Lindsay [20].After precipitation with metaphosphoric acid, supernatantwas reacted with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB).Absorbance was read spectrophotometrically at 412 nm.

Caspase-3 activity Caspase-3 activity was measuredby quantifying the cleavage of acetyl-Asp-Glu-Val-Asp

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Fig. 1 Mean ± SD colonicmacroscopic score in the groups.Values with different letters aresignificant according to ANOVA

p-nitroaniline (Ac-DEVD-pNA) with a colorimetric caspase-3 assay kit (Sigma). p-itroaniline released by the enzy-matic hydrolysis of Ac-DEVD-pNA was calculated froma p-nitroaniline standard curve and the molar absorptiv-ity coefficient of p-NA at 405 nm (10,500 M−1 cm−1).The supernatant obtained by the centrifugation of 10% tis-sue homogenate at 15,000g for 5 min at 4◦C was usedas an enzyme source and incubated with and without thecaspase-3 inhibitor Ac-DEVCHO, according to the assayprotocol for the kit. Caspase activity was calculated by sub-tracting the absorbance measured in the presence of sub-strate plus inhibitor from the absorbance observed on in-cubation with substrate alone for 20 min at 37◦C. Enzymeactivity was expressed as p-nitroaniline liberated per mil-ligram of protein per minute. The protein concentrationof the enzyme source was measured using bicinchoninicacid [21].

Statistical analysis All data are expressed as mean ± SD.ANOVA was used for statistical analysis and P < 0.05 wasaccepted as significant (SPSS 11.0 for Windows).

Results

All TNBS-treated rats developed diarrhea. None of the ratsdied.

Macroscopic and microscopic colitis scores

In TNBS-treated rats, macroscopic and microscopic patho-logical scores were found to be significantly higher comparedto those of the sham group (P < 0.001). Treatment with mela-tonin significantly decreased the pathological scores com-pared to those of TNBS-treated rats (P < 0.001) (Figures 1–3). NF-κB positivity was strongest in the colon of animals inGroup 1. Expression was decreased in the melatonin treat-ment group (Figures 4 and 5).

Myeloperoxidase activity

In all TNBS-treated rats, colonic MPO activity was foundto be increased significantly compared to that in the shamgroup. Colonic MPO activity was significantly lower in the

Fig. 2 Mean ± SD colonicmicroscopic score in the groups.Values with different letters aresignificant according toANOVA

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Fig. 3 (A) Colonic mucosafrom the TNBS group. Largeulceration. (B) Colonic mucosafrom melatonin + TNBS-treatedgroup. Marked inflammatorycell infiltration. (C) Normalcolonic mucasa frommelatonin-treated group. (D)Normal colonic mucosa fromsham control group. (HE;original magnifications, ×100)

melatonin treatment group compared to the TNBS-treatedrats (P < 0.001) (Figure 6).

Malondialdehyde levels

MDA levels in colonic tissues were found to be significantlyhigher in TNBS-treated rats compared to the sham group(P < 0.001). Treatment with melatonin significantly de-

creased MDA levels compared to those in TNBS-treatedrats (p < 0.001) (Figure 7).

Glutathione levels

GSH levels in colonic tissues were found to be significantlylower in TNBS-treated rats compared to the sham group(P < 0.001). Treatment with melatonin significantly

Fig. 4 Mean ± SD ratio ofNF-κB expression in the rats’colon in the various groups.Values with different letters aresignificant according to ANOVA

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Fig. 5 (A) NF-κBoverexpression in the colon inthe TNBS group. (NFB; originalmagnification, ×40.) (B) Colonfrom the melatonin +TNBS-treated group. NF-κBexpression is not seen. (NF;original magnification, ×100)

Fig. 6 Colonic MPO activityof all groups. Values withdifferent letters are significantaccording to ANOVA

increased GSH levels compared to those in TNBS-treatedrats (P < 0.001) (Figure 8).

Caspase-3 activity

The caspase-3 activity in colonic tissues was found to besignificantly higher in TNBS-treated rats compared to thesham group (P < 0.001). Treatment with melatonin signifi-

cantly decreased the caspase-3 activity compared to that inTNBS-treated rats (P < 0.001) (Figure 9).

Discussion

The results of the present study suggest that oxidative dam-age and apoptosis contribute to the development of colonicdamage following TNBS treatment, as assessed by increased

Fig. 7 Colonic MDA levels ofall groups. Values with differentletters are significant accordingto ANOVA

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Fig. 8 Colonic GSH levels ofall groups. Values with differentletters are significant accordingto ANOVA

lipid peroxidation, MPO activity, and caspase-3 levels anddecreased GSH levels in the colonic tissue. Our results alsodemonstrate that melatonin protects against TNBS-inducedcolonic damage. The protective effect of melatonin is as-sociated with a reduction of tissue inflammation and thesuppression of apoptosis.

Ulcerative colitis is a multifactorial inflammatory dis-ease of the colon and rectum with an unknown etiol-ogy. Many factors have been implicated in the pathogene-sis of ulcerative colitis, such as neutrophil infiltration andoverproduction of pro-inflammatory mediators includingcytokines, arachidonate metabolites, and reactive oxygenmetabolites. The depletion of tissue antioxidants throughconsumption by increased free radical production remainsa major pathophysiological question in inflammatory boweldiseases [2, 3, 6–9].

In our study, in all TNBS-treated rats, colonic MPO activ-ity was found to be significantly increased compared to thatin the sham control group. MDA levels in the colonic tissueswere found to be significantly higher in TNBS-treated ratscompared to the sham group. GSH levels in colonic tissueswere found to be significantly decreased compared to those

in the sham control group. In TNBS-treated rats, macro-scopic and microscopic pathological scores were foundto be significantly higher compared to those of the shamgroup.

During the inflammatory process, various cytokines aresecreted to the circulation. NF-κB is activated by a widevariety of agents, including hydrogen peroxide, ozone, reac-tive oxygen intermediates, IL-1, TNF-α, bacteria, and viraltranscriptions. Once activated, NF-κB transcriptionally reg-ulates many cellular genes implicated in early immune, acutephase, and inflammatory responses. The activation of NF-κBdepends on the cellular redox potential and a reduced intra-cellular GSH/oxidized GSH ratio. The amount of activatedNF-κB correlates with the degree of mucosal inflammation[22–24]. In the present study, NF-κB positivity was strongestin the colonic tissue of TNBS-treated rats. Expression wasdecreased in the melatonin treatment group.

Apoptosis is an essential physiological process requiredfor maintenance of tissue homeostasis. Insufficient or exces-sive cell death can contribute to diseases [25–27]. Gastroin-testinal diseases in which apoptosis has been implicated areinflammatory bowel disease, colon cancer, pancreas cancer,

Fig. 9 Intestinal Caspase-3activity of all groups. Valueswith different letters aresignificant according toANOVA

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acute pancreatitis, hepatitis, and radiation enteritis [28, 29].Disregulation or inhibition of apoptosis appears to be impor-tant in cell proliferation, tissue hyperplasia, and malignanttransformation of gastrointestinal epithelia. Although manyfactors are involved in the apoptotic program, caspases havebeen shown to play a major role in the transduction of apop-totic signals [25–27]. In the present study, we determinedthat TNBS-induced colitis caused an elevation of caspase-3levels in colonic tissue.

Melatonin, the main secretory product of the pineal gland,was recently found to be a potent free radical scavenger andantioxidant. Melatonin receptors are found both in neuraltissues and in the gastrointestinal tract. Melatonin is presentin all body fluids. It is thought to play a protective role in theinitial and advanced stages of diseases whose pathogenesisinvolves damage by reactive oxygen metabolites [30, 31].Recent studies suggested that administration of melatonindiminished oxidative damage in pathologic conditions suchas ischemia reperfusion, thermal injury, radiation injury, andchemotherapy [32–37].

In the present study, treatment with melatonin signifi-cantly decreased the elevation of MDA levels in colonic tis-sue. Treatment with melatonin significantly increased GSHlevels in colonic tissue. In all TNBS-treated rats, colonicMPO activity was found to be increased significantly com-pared to that in the sham group, while melatonin treatmentresulted in a decrease in this elevation. Melatonin treatmentprotected the colonic mucosal structure. The decrease in theexpression of NF-κB in the melatonin-treated rats supportedthese findings. These results imply a reduction in mucosaldamage due to anti-inflammatory and anti-apoptotic effectsof melatonin.

In conclusion, melatonin reduced mucosal damage inTNBS-induced colitis. The mechanism of the protectionassociated with melatonin was due to anti-oxidant, anti-apoptotic, and anti-inflamatory effects of melatonin whichwere documented by the decrease in NF-κB expression,MPO activity, and MDA and caspase-3 levels and the con-current increase in GSH levels in colonic mucosa.

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