THE INFLUENCE OF PRETREATMENT WITH GHRELIN ON THE DEVELOPMENT OF ACETIC-ACID-INDUCED COLITIS IN RATS

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1 JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 5, 66, 6, D. MADUZIA,, A. MATUSZYK,, D. CERANOWICZ,3, Z. WARZECHA, P. CERANOWICZ, K. FYDEREK 3, K. GALAZKA 4, A. DEMBINSKI THE INFLUENCE OF PRETREATMENT WITH GHRELIN ON THE DEVELOPMENT OF ACETIC-ACID-INDUCED IN RATS Deprtment of Physiology, Jgiellonin University Medicl College, Crcow, Polnd; Deprtment of Antomy, Jgiellonin University Medicl College, Crcow, Polnd; 3 Deprtment of Peditrics, Gstroenterology nd Nutrition, Children s University, Jgiellonin University Medicl College, Crcow, Polnd; 4 Deprtment of Pthology, Jgiellonin University Medicl College, Crcow, Polnd Ghrelin hs been primrily shown to exhibit protective nd therpeutic effect in the gut. Pretretment with ghrelin inhibits the development of cute pncretitis nd ccelertes pncretic recovery in the course of this disese. In the stomch, ghrelin reduces gstric mucosl dmge induced by ethnol, stress or lendronte, s well s ccelertes the heling of cetic cid-induced gstric nd duodenl ulcer. The im of present studies ws to investigte the effect of pretretment with ghrelin on the development of cetic cid-induced colitis. Studies hve been performed on mle Wistr rts. Animls were treted intrperitonelly with sline (control) or ghrelin (4, 8 or 6 nmol/kg/dose). Sline or ghrelin ws given twice: 8 nd h before induction of colitis. Colitis ws induced by rectl enem with ml of 4% solution of cetic cid nd the severity of colitis ws ssessed or 4 hours fter induction of inflmmtion. Rectl dministrtion of cetic cid induced colitis in ll nimls. Dmge of colonic wll ws seen t the mcroscopic nd microscopic level. This effect ws ccompnied by reduction in colonic blood flow nd mucosl DNA synthesis. Moreover, induction of colitis significntly incresed mucosl concentrtion of pro-inflmmtory interleukin-β (IL- β), ctivity of myeloperoxidse nd concentrtion of mlondildehyde (MDA). Mucosl ctivity of superoxide dismutse (SOD) ws reduced. Pretretment with ghrelin reduced the re nd grde of mucosl dmge. This effect ws ccompnied by n improvement of blood flow, DNA synthesis nd SOD ctivity in colonic mucos. Moreover, ghrelin dministrtion reduced mucosl concentrtion of IL-β nd MDA, s well s decresed mucosl ctivity of myeloperoxidse. Administrtion of ghrelin protects the lrge bowel ginst the development of the cetic cid-induced colitis nd this effect seems to be relted to the ghrelin-evoked nti-inflmmtory nd nti-oxidtive effects. Key words: colitis, ghrelin, oxidtive stress, DNA synthesis, interleukin-β, colonic blood flow, mlondildehyde, myeloperoxidse INTRODUCTION Inflmmtory bowel disese (IBD) is chronic relpsing inflmmtion of the digestive trct with lternte periods of excerbtion nd remission. The min forms of IBD re Crohn s disese nd ulcertive colitis (). Epidemiologicl studies indicte tht the frequency of IBD incidence is rising globlly (-5). The etiology of IBD is still uncler, but reserch in bsic science, experimentl niml models, genetics nd humn clinicl trils hs provided new dt concerning the pthogenesis of bowel inflmmtion. It is currently suggested tht IBD results from n bnorml immunologicl response to micro flor present in the digestive system nd their pthogenesis is complex, nd requires co-existence of fctors of environmentl nd genetic nture (, 5, 6). There re numerous methods to tret IBD, but mediction tht would ensure permnent therpeutic effect hs not been found so fr (, 6, 7). Therefore, it is vitl to serch for the new therpeutic strtegies. Ghrelin, 8-mino cid peptide, discovered in humn nd rt stomchs (8-), is n endogenous lignd for receptor ctivted by fctors which stimulte growth hormone secretion (growth hormone secretgogue receptor (GHS-R)). The receptor is present minly in the pituitry glnd nd hypothlmus, but lso in other tissues of the body (). Besides inducing body mss growth through food intke stimultion nd decrese in utiliztion of ft tissue (), ghrelin demonstrtes ntiinflmmtory properties. Receptors for ghrelin re present, mong others, on the immunologicl system cells, suggesting tht ghrelin my modulte inflmmtory response (3). Previous studies hve shown ghrelin exhibits protective nd therpeutic on the gut. Pretretment with ghrelin inhibits the development of experimentl gstric ulcers induced by ethnol (4), stress (5) or lendronte (6). Moreover, dministrtion of ghrelin ccelertes the heling of gstric (7, 8), duodenl (7, 9) nd orl () ulcers evoked by different noxious gents. Protective nd therpeutic effect of ghrelin hs been lso found in the pncres. Pretretment with ghrelin inhibits the

2 876 development of cerulein- nd ischemi/reperfusion-induced pncretitis (, ) nd ccelertes its heling (3-5). The role of ghrelin in IBD is not cler. Clinicl studies hve shown tht expression of mrna of ghrelin in colonic mucos is significntly upregulted in ptients with IBD nd correlted with the grde of inflmmtion (6, 7). Moreover, ptients with ctive phse of IBD hve higher serum ghrelin concentrtion thn ptients in remission or helthy individuls nd the level of circulting ghrelin correltes with the severity of IBD (8-3). There re niml experimentl studies concerning the role of ghrelin in the development nd heling of colitis. Some studies hve shown tht dministrtion of exogenous ghrelin significntly ccelertes the heling of colitis evoked by trinitrobenzene sulfonic cid (TNBS) in mice (3) nd rts (6). Therpeutic effect of ghrelin dministrtion hs lso been found in dextrn sulfte sodium (DSS)-induced colitis in rts (3). In contrst to those dt re results obtined by De Smet et l. (33). They hve reported tht endogenous nd exogenous ghrelin enhnces the colonic mnifesttion of DSS-induced colitis in mice. Pro-inflmmtory properties of ghrelin in colitis hve been lso postulted by Zho et l. (34). They hve found tht ghrelin stimultes interleukin-8 gene expression through protein kinse C-medited NF-kppB pthwy in humn colonic epithelil cells. The objective of present study ws determine the influence of pretretment with ghrelin on the development of cetic cidinduced colitis in rts. The possible protective effect of ghrelin in the lrge intestine could be useful in clinicl condition in mintining the IBD ptients in remission. Animls nd tretment MATERIALS AND METHODS The reserch ws performed on Wistr mle rts weighing 5 7 g nd conducted following the experimentl protocol pproved by the First Locl Commission of Ethics for the Cre nd Use of Lbortory Animls in Crcow. During study, the nimls were kept in cges plced in temperture nd -hour light-drkness cycle ws mintined. The nimls, prior to colitis induction with cetic cid solution, were strved for 8 hours with free ccess to wter. Erlier nd fter this period food nd tp wter were vilble d libitum. One hundred sixty rts were rndomly divided in eight equl experimentl groups: [] control rts treted intrperitonelly (i.p.) with sline without induction of colitis; [-4] rts treted i.p. with ghrelin given t the dose of 4, 8 or 8 nmol/kg/dose without induction of colitis; [5] rts treted i.p. with sline before induction of colitis; [6-8] rts treted i.p. with ghrelin given t the dose of 4, 8 or 6 nmol/kg/dose before induction of colitis. Animls from ech experimentl group were divided into two equl sub-groups. In the first sub-groups, the severity of colitis ws ssessed fter h from cetic cid enem. In the second sub-groups, the severity of colitis ws ssessed fter 4 h from cetic cid enem. Experiments were repeted to obtin nimls in ech experimentl group nd ech time of observtion. According to group of nimls, sline or rt ghrelin (Ynihr Institute, Shizuok, Jpn) were dministered intrperitonelly twice, 8 nd h before rectl dministrtion of sline or cetic cid solution. Ghrelin ws dissolved in sline nd then dministered in n mount which did not exceed.3 ml/dose. Before induction of colitis, nimls were nesthetized with ketmine (5 mg/kg i.p., Bioketn, Vetoquinol Biowet, Gorzow Wielkopolski, Polnd). Colitis ws induced by intrrectl dministrtion of ml of 4% cetic cid queous solution through polyethylene ctheter, which the end ws inserted into the bowel by 4.5 cm from the nus. Animls without induction of colitis were treted with rectl enem of sline (shmopertion). Mesurement of colonic blood flood nd mucosl lesions One or 4 h fter rectl enem, rts were nesthetized gin with ketmine. After opening the bdominl cvity nd exposing the colon, the mesurement of colonic blood flow volume ws performed using lser Doppler flowmeter (PeriFlux 4 Mster monitor, Perimed AB, Jrfll, Sweden), in ccordnce with the methodology described before (35). The blood flow mesurement ws performed every time in five vrious prts of the descending nd sigmoid colon nd men vlue of five records ws expressed s the percentge of vlue obtined in nimls from the control group. After mesurement of colonic blood flow, the re of mucosl dmge ws mesured, using computerized plnimeter (Morphomt, Crl Zeiss, Berlin, Germny), in ccordnce to the method described erlier (9). Biochemicl nlysis After mesurement of colonic blood flow nd lesions re frgment of colonic wll for histologicl exmintion ws cut out by scissors. After tht mucos from remining prts of the colon ws scrpped using glss microscope slides over glss plte. Then smples of mucos from the colon were divided for determintion of mucosl DNA synthesis (n index of mucosl cell prolifertion), concentrtion of pro-inflmmtory interleukin-β, ctivity of myeloperoxidse, concentrtion of mlondildehyde (MDA) (s n index of lipid peroxidtion) nd ctivity of superoxide dismutse (SOD). Determintion of DNA synthesis in colonic mucos DNA synthesis ws determined by mesurement of [ 3 H]thymidine incorportion ([6-3H]-thymidine, 3 Ci/mmol, Institute for Reserch, Production nd Appliction of Rdioisotopes, Prgue, Czech Republic) into mucosl DNA s described previously (36). The incorportion of lbeled thymidine into DNA ws determined by counting.5 ml DNAcontining superntnt in liquid scintilltion system. The rte of DNA synthesis ws expressed s tritium disintegrtions per minute per µg of DNA (dpm/µg DNA). Determintion of interleukin-β concentrtion in colonic mucos Smples of colonic mucos were homogenized in ice-cold phosphte buffered sline (PBS, mm, ph 7.4). Homogente ws centrifuged t 5 g for min t 4 C. Content of interleukin-β in the superntnt ws mesured using the BioSource Cytoscreen rt IL-β kit (BioSource Interntionl, Cmrillo, Cliforni, USA) bsed on ELISA. Concentrtion of interleukin-β in colonic mucos ws expressed s ng per g of tissue. Determintion of myeloperoxidse ctivity in colonic mucos Smples of colonic mucos were frozen in liquid nitrogen, nd, until the mrking ws done, stored t the temperture of 6 C. Myeloperoxidse ctivity ws ssessed using modifiction of the method described by Brdley et l. (37). Mucos ws homogenized in ml of 5 mm potssium phosphte buffer (ph 6.) contining.5% of

3 877 hexdecyltrimethyl mmonium bromide. Then the homogente ws freeze-thwed three times, subjected to soniction in ice bth for s nd centrifuged t 3 g for 5 min t the temperture of 4 C. µl of the superntnt ws tken nd.9 ml of 5 mm phosphte buffer ws dded, which contined 57 µg/ml of o-dinisidine dihydrochloride nd.5% of hydrogen peroxide. Hydrogen peroxide reduction through myeloperoxidse cuses the oxidtion of o-dinisidine nd produces stined finl product. The intensity of stining ws mesured spectrophotometriclly with light wve of the length of 46 nm. The obtined results were clculted in units per grm of tissue nd finlly expressed s the percentge of the vlue observed in the control group. Determintion of mlondildehyde concentrtion in colonic mucos Peroxidtion of lipids in colonic mucos ws tested by mesurement of MDA concentrtion using commercil kit Bioxytech LPO-586 TM (OxisReserch TM, OXIS Helth Products, Inc., Portlnd, OR, USA), s described previously (38). Prior to homogeniztion of tissue smples, µl.5 M butylted hydroxytoluene in cetonitrile ws dded to prevent smple oxidtion during homogeniztion. Mucos ws homogenized in ice-cold Tris buffer ( mm, ph 7.4), centrifuged (3 g t 4 C for min) nd superntnt ws used for the ssy. Results were expressed in nnomoles per grm of colonic mucos. Determintion of superoxide dismutse ctivity in colonic mucos To determine the ctivity of SOD in colonic mucos, tissue ws homogenized in mm HEPES buffer, ph 7., contining mm EGTA, mm mnnitol, nd sucrose. Homogente ws centrifuged t 5 g for 5 min t 4 C. Activity of SOD in the superntnt ws mesured using Superoxide Dismutse Assy Kit (Cymn Chemicl Compny, Ann Arbor, MI, USA). Results hve been expressed in units per g of colonic mucos. Histologicl exmintion of the colon Smples of the colon were fixed in % buffered formldehyde nd embedded in prffin. Prffin sections were stined with hemtoxylin nd eosin. Slides were exmined by two experienced pthologists without knowledge of the tretment given. The histologicl grding of colonic dmge ws determined using scle previously presented by Vilsec (39). The histologicl grding of lesions ws mde using scle rnging from to ( = no lesions; = smll lesions < 3mm; = lrge lesions > 3 mm). Inflmmtory infiltrtion ws grded from to 3 ( = none; smll; = moderte; 3 = hevy), depth of the lesions ws grded from to 3 ( = no lesions; = lesions reching submucos; = lesions reching musculris propri; 3 = lesions reching seros). The presence of fibrosis ws grding from to ( = none; = mild; = severe). Moreover, the presence of rteril vessels inflmmtion ws ssessed. Sttisticl nlysis Results were presented s men vlue ± stndrd error (S.E.M.). Sttisticl ssessment ws done through one wy nlysis of vrince followed by Tukey s multiple comprison test using GrphPdPrism (GrphPd Softwre, Sn Diego, CA, USA). Differences were considered to be sttisticlly significnt if P ws less thn.5. RESULTS Mcroscopic nd microscopic morphologicl fetures obtined from rts treted with sline (control) or ghrelin without induction of colitis, did not show ny dmge of the colon (Fig., Tble ). Tht lck of dmge ws found in both times of observtion, nd 4 hours fter enem with sline. Rectl dministrtion of 4% solution of cetic cid cused the development of colitis in ll rts. One hour fter dministrtion of cetic cid, the verge re of colonic lesions ws 5. ±.3 mm ; wheres 3 h lter the re of colonic lesions enlrged to 8.6 ±.4 mm (Fig. ). In nimls with colitis, histologicl exmintion performed hour fter cetic cid enem, showed the presence of lrge lesions reching the level of musculr membrne, with moderte inflmmtory cell infiltrtion (Tble ). Twenty three hour lter, histologicl exmintion showed n increse in colonic dmge. There were lrge ulcers, reching the musculr membrne or, in hlf of cses, even serous membrne of the colonic wll. These chnges were ccompnied by hevy inflmmtory leukocytic infiltrtion nd severe fibrosis together with rteril vessels inflmmtion (Tble, Fig. AREA OF COLONIC LESIONS (mm ) C G 4 G 8 G 6 NCl G 4 G 8 G 6,b,b,b h 4 h,b Fig.. Influence of pretretment with ghrelin given intrperitonelly t the dose of 4, 8 or 6 nmol/kg/dose (G4, G8 or G6) nd colitis evoked by rectl enem with cetic cid on the re of colonic lesions observed or 4 h fter induction of colitis. Men ± stndrd error. N = nimls in ech group. P<.5 compred to control (C) t the sme time of observtion; b P<.5 compred to NCl + colitis t the sme time of observtion.

4 878 Tble. Influence of pretretment with ghrelin (given intrperitonelly t the dose of 4, 8 or 6 µg/kg/dose) nd colitis evoked by rectl enem with cetic cid on morphologicl signs of colonic dmge observed h or 4 hours fter rectl dministrtion of sline or induction of colitis. MORPHOLOGICAL CHANGES GRADING OF COLONIC DAMAGE (-) INFLAMMATORY INFILTRATION DEPTH OF DAMAGE FIBROSIS (-3) (-3) (-3) HOUR CONTROL GHRELIN 4 GHRELIN 8 GHRELIN 6 GHRELIN GHRELIN 8 + GHRELIN HOURS CONTROL GHRELIN 4 PRESENCE OF INFLAMMATION IN ARTERIAL BLOOD VESSELS (+) GHRELIN 8 GHRELIN (+) GHRELIN (+) GHRELIN 8 + GHRELIN 6 + Numbers represent the predominnt histologicl grding in ech group. Fig.. Morphologicl signs of colonic dmge observed 4 hours fter rectl enem with cetic cid. Hemtoxylineosin stin, originl mgnifiction. ). Pretretment with ghrelin reduced the development of colitis. One hour fter cetic cid enem, the re of colonic dmge ws reduced by, 56 or 6% in rts pretreted ghrelin given t the dose of 4, 8 or 6 nmol/kg/dose, respectively. Twenty three hours lter in rts pretreted with ghrelin given t the dose of 4, 8 or 6 nmol/kg/dose, the re of colonic lesion ws reduced by 8, 5 or 56% (Fig. ). The effect cused by ghrelin given t the dose of 8 nd 6 nmol/kg/dose ws sttisticlly significnt in both times of observtion, nd 4 hours fter enem with cetic cid solution.

5 879 Fig. 3. Influence of pretretment with ghrelin given t the dose of 8 nmol/kg/dose on morphologicl signs of colonic dmge observed 4 hours fter rectl enem with cetic cid. Hemtoxylin-eosin stin, originl mgnifiction. h 4 h 45 DNA SYNTHESIS (dpm/µg DNA) 4 35,b 3 5,b,b,b 5 5 C G4 G8 G 6 NCl G4 G8 G 6 h 4 h BLOOD FLOW (% of control) Fig. 4. Influence of pretretment with ghrelin given intrperitonelly t the dose of 4, 8 or 6 nmol/kg/dose (G4, G8 or G6) nd colitis evoked by rectl enem with cetic cid on DNA synthesis in colonic mucos observed or 4 hours fter induction of colitis. Men ± stndrd error. N = nimls in ech group. P<.5 compred to control (C) t the sme time of observtion; bp<.5 compred to NCl + colitis t the sme time of observtion. 8,b,b,b,b 6 4 C G4 G8 G 6 NCl G4 G8 Pretretment with ghrelin reduced lso histologicl mnifesttion of colonic dmge evoked by cetic cid (Tble, Fig. 3). One hour fter enem with cetic cid, beneficil effect G 6 Fig. 5. Influence of pretretment with ghrelin given intrperitonelly t the dose of 4, 8 or 6 nmol/kg/dose (G4, G8 or G6) nd colitis evoked by rectl enem with cetic cid on mucosl blood flow in the colon observed or 4 hours fter induction of colitis. Men ± stndrd error. N = nimls in ech group. P<.5 compred to control (C) t the sme time of observtion; bp<.5 compred to NCl + colitis t the sme time of observtion. of pretretment with ghrelin ws found s decrese in grding nd depth of ulcers, nd reduction in inflmmtory infiltrtion of the colonic wll. Twenty three hours lter, the beneficil effect of

6 h 4 h INTERLEUKIN-β (ng/g tissue) 5 4 3,b,b,b,b C G 4 G 8 G 6 NCl G 4 G 8 G 6 Fig. 6. Influence of pretretment with ghrelin given intrperitonelly t the dose of 4, 8 or 6 nmol/kg/dose (G4, G8 or G6) nd colitis evoked by rectl enem with cetic cid on interleukin-β concentrtion in colonic mucos observed or 4 hours fter induction of colitis. Men ± stndrd error. N = nimls in ech group. P<.5 compred to control (C) t the sme time of observtion; b P<.5 compred to NCl + colitis t the sme time of observtion. pretretment with ghrelin ws mnifested, prt effects mentioned bove, by reduction in fibrosis nd prevention of inflmmtion in rteril blood vessels (Tble ). In the nimls from the control group, the rte of DNA synthesis in colonic mucos reched vlue of 35. ±.4 or 36. ±.3 dpm/µg DNA t the time of or 4 h fter sline enem, respectively (Fig. 4). Pretretment with ghrelin t the dosge used, tended to increse DNA synthesis in colonic mucos in rts without induction of colitis (Fig. 4), but this effect ws sttisticlly insignificnt (Fig. 4). Induction of colitis through rectl dministrtion of 4% solution of cetic cid inhibited DNA synthesis in the colon. One nd 4 h fter dministrtion of cetic cid, DNA synthesis ws significntly reduced by round 4 nd 6%, respectively. Pretretment with ghrelin before induction of colitis prtly reversed tht effect. In both times of observtion, ghrelin given t the dose of 4 nmol/kg/dose tended to increse mucosl DNA synthesis, but tht effect ws sttisticlly insignificnt (Fig. 4). In contrst to tht, ghrelin dministered t the dose of 8 or 6 nmol/kg/dose, led to sttisticlly significnt nd similr in cse of both doses, improvement of DNA synthesis in the colon mucos in nimls with induction of colitis. This effect ws observed fter nd 4 hours fter induction of colitis (Fig. 4). Double intrperitonel dministrtion of ghrelin, in doses used, hd no influence on mucosl blood flow in the colon in nimls without induction of colitis (Fig. 5). Induction of colitis led to sttisticlly significnt decrese in colonic mucosl blood flow. One hour fter induction of colitis mucosl blood flow in the colon ws reduced by lmost 68%, when compred to vlue observed in the nimls from the control group (Fig. 5). Twenty three hours lter, mucosl blood flow in the colon ws reduced by nerly 75%. Pretretment with ghrelin given t the dose of 8 or 6 nmol/kg/dose prtly, but significntly, reversed the colitis-evoked decrese in mucosl blood flow in the colon. This effect of ghrelin ws found in both times of observtion, nd 4 h fter induction of colitis. Effect of ghrelin given t the dose of 4 nmol/kg/dose ws wek nd sttisticlly insignificnt (Fig. 5). The concentrtion of pro-inflmmtory interleukin β (IL- β) in colonic mucos in control nimls ws round.5 ng/g of tissue (Fig. 6). Double dministrtion of ghrelin, t the dosge used, hd no influence on IL-β concentrtion in colonic mucos in nimls without induction of colitis. Induction of colitis through rectl dministrtion of 4% cetic cid led to sttisticlly significnt increse in the concentrtion of IL-β in colonic mucos. One hour fter induction of colitis, mucosl concentrtion of IL-β ws incresed by more thn %; wheres 3 hours lter lmost -fold increse in tht proinflmmtory cytokine ws observed (Fig. 6). Administrtion of ghrelin inhibited the colitis-induced increse in IL-β concentrtion in colon mucos. The influence of ghrelin, given t the dose 8 or 6 nmol/kg/dose, on this prmeter in rts with colitis ws sttisticlly significnt nd similr for both of those doses. This effect ws observed in both periods of observtion, h nd 4 hours fter induction of colitis. On the other hnd, pretretment with ghrelin dministered t the dose of 4 nmol/kg/dose filed to ffect the mucosl concentrtion of IL- β in the colon of rts with colitis (Fig. 6). In control sline-treted nimls, myeloperoxidse (MPO) ctivity in colonic mucos ws low nd reched vlue of.7 U/g of tissue (Fig. 7). Pretretment with ghrelin, t the doses used, filed to ffect MPO ctivity in colonic mucos in rts without induction of colitis. Induction of colitis resulted in significnt increse in MPO ctivity in colonic mucos. One h fter induction of colitis, MPO ctivity reched -fold increse; wheres 3 hours lter more thn 4-fold increse ws observed. Pretretment with ghrelin reduced the colitis-evoked increse in colonic ctivity of MPO. The effect of ghrelin, dministered t the dose of 4 nmol/kg/dose, ws sttisticlly insignificnt; wheres the two remining doses inhibited significntly the mucosl MPO ctivity in the colon of nimls with colitis. This inhibitory effect ws similr for both doses of ghrelin nd ws observed ether or hours fter induction of colitis (Fig. 7). A vlue of mlondildehyde (MDA) concentrtion in colonic mucos in control sline-treted rts ws between 4.6 nd 4.5 nmol/g of tissue (Fig. 8). Pretretment with ghrelin hd no significnt influence on MDA concentrtion in colonic mucos in nimls without induction of colitis. Induction of colitis by rectl dministrtion of 4% solution of cetic cid cused sttisticlly significnt increse in MDA concentrtion in mucos of the colon. More thn 3-fold nd 5-fold rise in this prmeter ws observed nd 4 h fter induction of colitis; respectively. Pretretment with ghrelin inhibited the colitisinduced increse in MDA concentrtion in colonic mucos. In both periods of observtion, tht effect ws sttisticlly significnt nd reched similr intensity fter ghrelin dministered t the dose of 8 or 6 nmol/kg/dose. Ghrelin given t the dose of 4 nmol/kg/dose filed to significntly ffect MDA concentrtion in colonic mucos in rts with colitis (Fig. 8).

7 88 MYELOPEROXIDASE (U/g tissue) 4 3 C G 4 G 8 G 6 NCl G 4 G 8 G 6,b,b,b h 4 h,b Fig. 7. Influence of pretretment with ghrelin given intrperitonelly t the dose of 4, 8 or 6 nmol/kg/dose (G4, G8 or G6) nd colitis evoked by rectl enem with cetic cid on myeloperoxidse ctivity in colonic mucos observed or 4 hours fter induction of colitis. Men ± stndrd error. N = nimls in ech group. P<.5 compred to control (C) t the sme time of observtion; b P<.5 compred to NCl + colitis t the sme time of observtion. 5 h 4 h MALONDIALDEHYDE (nmol/g tissue) 5 5,b,b,b,b C G 4 G 8 G 6 NCl G 4 G 8 G 6 Fig. 8. Influence of pretretment with ghrelin given intrperitonelly t the dose of 4, 8 or 6 nmol/kg/dose (G4, G8 or G6) nd colitis evoked by rectl enem with cetic cid on concentrtion of mlondildehyde in colonic mucos observed or 4 hours fter induction of colitis. Men ± stndrd error. N = nimls in ech group. P<.5 compred to control (C) t the sme time of observtion; b P<.5 compred to NCl + colitis t the sme time of observtion. SUPEROXIDE DISMUTASE (U/g tissue) C G 4 G 8 G 6 NCl G 4 G 8 G 6 b,b b h 4 h,b Fig. 9. Influence of pretretment with ghrelin given intrperitonelly t the dose of 4, 8 or 6 nmol/kg/dose (G4, G8 or G6) nd colitis evoked by rectl enem with cetic cid on superoxide dismutse ctivity in colonic mucos observed or 4 hours fter induction of colitis. Men ± stndrd error. N = nimls in ech group. P<.5 compred to control (C) t the sme time of observtion; b P<.5 compred to NCl + colitis t the sme time of observtion. Superoxide dismutse (SOD) ctivity in mucos in control sline-treted rts ws round 36 U/g of tissue (Fig. 9). Administrtion of ghrelin in doses used filed to ffect mucosl SOD ctivity in the colon of nimls without induction of colitis. Induction of colitis significntly reduced SOD ctivity nd this effect ws found in both periods of observtion, nd 4 hours fter induction of colitis, mucosl SOD ctivity ws decresed by 34 nd 46%, respectively. Pretretment with ghrelin prtly reversed the colitis-induced reduction in mucosl SOD ctivity. This effect ws sttisticlly significnt fter ghrelin dministered

8 88 t the dose of 8 or 6 nmol/kg/dose. Ghrelin given t the dose of 4 nmol/kg/dose filed to significntly ffect SOD ctivity in colonic mucos in rts with colitis (Fig. 9). DISCUSSION Our present study hs provided severl importnt observtions regrding the influence of exogenous ghrelin on the integrity of the colonic mucos. We hve found tht pretretment with ghrelin exhibits protective effect in the colon nd inhibits the development of colitis evoked by cetic cid enem. Protective effect of ghrelin ws mnifested s n improvement of colonic morphology nd mucosl blood flow, decrese in biochemicl indexes of inflmmtion s well s prtil reversion of the colitis-evoked reduction in mucosl cell vitlity. Our study hs demonstrted tht the re of colonic dmge increses more thn 5-fold between the st nd 4 th h fter cetic cid enem. Pretretment with ghrelin reduced colonic dmge nd this effect ws observed in both periods of observtion. The decrese in colonic dmge ws mnifested s reduction of the re of dmge nd n improvement in the colon morphology observed in the microscopic exmintion. A sttisticlly significnt protective effect of ghrelin ws observed if this peptide ws dministered t the dose of 8 or 6 nmol/kg/dose nd rte of this effect ws similr for of both bove doses. This observtion is in greement with previous studies showing tht those doses of ghrelin, 8 or 6 nmol/kg/dose, exhibit protective nd/or therpeutic effect in other orgns, such s the pncres (, 3), stomch (7), duodenum (7) nd orl cvity (). Cell prolifertion in mucos of the gut exhibits high dynmics nd colonic mucos is renewed every 3 8 dys (4). Insufficient cell prolifertion nd/or incresed cell loss my led to mucosl trophy nd the development of ulcertion (4, 4). On the other hnd, incresed cell prolifertion my result in hyperplsi, but it is lso ssocited with incresed protection of mucos ginst dmge evoked by noxious gents, s well s ccelertes mucosl regenertion fter dmge of the stomch nd intestine (43-46). In synthesis (S) phse of cell-division cycle, DNA is replicted nd this process is essentil for prepring of the cell to division (47). Our present study hs shown tht induction of colitis is ssocited with decrese in mucosl DNA synthesis in the colon. The rte of mucosl DNA synthesis ws inversely proportionl to the grde of colonic dmge, higher hour fter induction of colitis nd lower fter next 3 hours. In rts without induction of colitis, dministrtion of ghrelin ws without significnt influence on DNA synthesis in colonic mucos. On the other hnd, pretretment with ghrelin, prior to colitis induction with cetic cid, cused considerble reversl of the colitis-evoked decrese in DNA synthesis in the colon. These findings indicte tht protective effect of ghrelin in the colon is relted, t lest in prt, to n increse in vitlity of cells in colonic mucos. Previous studies hve shown tht exposure of gstric mucos to potentilly dmging fctors results in little or no dmge, s long s n dequte blood flow is sustined. On the other hnd, decrese in mucosl blood flow increses the severity nd extension of gstric lesions (48). The importnce of pproprite blood flow in mintining mucosl integrity nd heling of mucosl dmge hs been lso found in other prts of the digestive trct system such s the orl cvity (), esophgus (49), duodenum (5) nd colon (5). Findings of our present study re in hrmony with those dt. We hve found tht induction of colitis reduced mucosl blood flow in the colon nd the grde of this reduction ws well-correlted with the severity of colitis. Pretretment with ghrelin prior to colitis induction with cetic cid led to improvement of blood flood through colonic mucos nd tht effect ws ssocited with proportionl reduction in the re nd severity of colonic dmge. This observtion indictes tht ghrelin s protective effect on colonic mucos is connected with n improvement of mucosl blood flow. A question remins, however, whether the ghrelin-evoked improvement of blood flood through colonic mucos in rts with colitis is mechnism or result of ghrelin s protective effect in the colon. Previous studies hve documented tht the reltion between mucosl blood flow nd mucosl dmge is bidirectionl. Improvement of mucosl blood flow decreses mucosl injury, but simultneously decresing the severity of mucosl dmge leds to n improvement in the blood flow through mucosl membrne (36, 48, 5, 53). Another interesting discovery of our present study ws the observtion regrding the influence of intrperitonel dministrtion of ghrelin nd rectl dministrtion of cetic cid solution on mucosl concentrtion of interleukin-β (IL-β) nd myeloperoxidse ctivity in the colon. IL-β plys crucil role in in the induction of locl inflmmtion nd systemic cute phse response nd in the relese of subsequent proinflmmtory cytokines in the biochemicl cscde of inflmmtion (54, 55). Numerous experimentl nd clinicl studies confirm tht dministrtion of IL-β receptor ntgonists or ntibodies ginst IL-β prevents the increse in serum concentrtion of pro-inflmmtory interleukin-6 nd tumor necrosis fctor-α, s well s decreses the severity of systemic inflmmtion (56-6). Our present study hs shown tht induction of colitis by cetic cid enem leds to dmge nd inflmmtory infiltrtion of colonic mucos, nd increses mucosl concentrtion of IL-β. On the other hnd, pretretment with ghrelin hs reduced mucosl concentrtion of IL-β in the colon of rts with colitis. These dt indicte tht ghrelin reduces locl inflmmtory response in cetic cid-induced colitis nd this observtion seems to show the next mechnism involved in the ghrelin-evoked protective effect in the colon. Myeloperoxidse (MPO) is relesed from the grnules of neutrophils in inflmmtory rections nd for this reson ctivity of this enzyme reflects the degree of tissue infiltrtion by neutrophils (6-63). In our present study, induction of colitis incresed MPO ctivity, wheres pretretment with ghrelin prtly, but significntly, reversed this effect. This finding is the next evidence tht pretretment with ghrelin exhibits ntiinflmmtory effect in the colon. Oxidtive stress ws originlly defined s the imblnce between pro-oxidnts nd ntioxidnts in biologicl systems nd is result of incresed production of rective oxygen species (ROS) nd/or impired ntioxidnt cpcity (64). ROS consist of group of highly rective oxygen metbolites, including superoxide nion rdicls, hydrogen peroxide, hypochlorous cid nd the especilly rective hydroxyl rdicl (65). In physiologicl condition, the use of oxygen during norml metbolism produces low levels of ROS nd excess of ROS re sfely neutrlized by ntioxidnt mechnisms involving ntioxidnt enzymes nd scvengers. Superoxide dismutse (SOD), ctlse nd glutthione peroxidse re the min enzymes responsible for ROS neutrliztion (66). Oxidtive stress plys n importnt role in the pthogenesis of inflmmtion. Involvement of ROS hs been shown in the pthogenesis of the spirin- (67), stress- (68) or ischemi/reperfusion-induced gstric lesions (66). Also in the cse of inflmmtory bowel disese, the role of oxidtive stress is being emphsized (69). In pthology, when ROS production exceeds the cpcity of the ntioxidnt defense system, ROS cuses oxidtive dmge of ll cellulr elements (64, 7). Peroxidtion of cellulr lipids, clled lipid peroxidtion, is the first stge of ROS-medited cellulr dmge (65). Our present study hs shown tht the

9 883 development of cetic cid-induced colitis is ssocited with oxidtive stress. We hve found n increse in concentrtion of lipid peroxidtion product, MDA in colonic mucos nd this effect hs been ssocited with reduction in SOD ctivity in this mucos, leding to dditionl disruption of redox blnce. Pretretment with ghrelin, prior to rectl dministrtion of cetic cid resulted in reduction of redox imblnce. Mucosl level of MDA ws reduced, wheres mucosl ctivity of SOD ws prtly restored. These observtions indicte tht pretretment with ghrelin reduces the colitis-induced oxidtive stress in colonic mucos nd improves nti-oxidnt defense in this inflmmtion. Finlly, we conclude tht pretretment with ghrelin inhibits the development of the cetic cid-induced colitis nd this effect seems to be relted to the ghrelin evoked nti-inflmmtory nd nti-oxidtive effects. Conflict of interests: None declred. REFERENCES. Stenson WF, Hnuer SB, Cohen RD. Inflmmtory bowel disese. 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11 Evns MD, Cooke MS. Fctors contributing to the outcome of oxidtive dmge to nucleic cids. BioEssys 4; 6: R e c e i v e d: April, 5 A c c e p t e d: November 3, 5 Author s ddress: Assoc. Prof. Piotr Cernowicz, Deprtment of Physiology, Jgiellonin University Medicl College, 6 Grzegorzeck Street, 3-53 Crcow, Polnd. E-mil: mpcerno@cyf-kr.edu.pl