MK-801 Verilen Ratlarm Cerebellumun'da Makromolekiiler Oksidasyon ve Cape'nin Koruyucu Etkisi
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1 Kocatepe TIp Dergisi The Medical Journal ofkocatepe 7: 63-67/ EylUl2006 Afyo n Kocatepe Universitesi MK-801 Verilen Ratlarm Cerebellumun'da Makromolekiiler Oksidasyon ve Cape'nin Koruyucu Etkisi The Macromoleculer Oxidation in Cerebellum ofrat Expos ed To MK-801 and The Protective Effect ofcape Birsen OZYURT 1, Huseyin OZYURT 2, Fatih EKicP, Hasan ERDOGAN 3, Mustafa SARSILMAZ 4 I Gaziosmanpasa Universitesi Tip Fakultesi, Anatomi AD. Tokat, 2 Gaziosmanpasa Uni versitesi Tip Fakiiltesi Biyokimya AD. Tokat, 3 Gaziosmanpasa Universi tesi Tip Fakiilt esi Psikoloj i AD. Tokat, 4 FIrat Universitesi Tip Fakiiltesi Anatomi AD., Elaztg. 6ZET: Bu cahs maru n arnaci MK- 80 I ile olusa n norotoksisitede oksidatif stresin etki sini incelemek, oksidatif stresi n onl enmesi ile pro gnozun duzelebileceginin gosterilmesi dir. MK- 801 en noro toksik NMDA rese p tor antagonistlerinden biridir. Wistar Albino ratlar 3 gruba ayn ldi : I.g rup: Kontrol, 2. grup: MK- 80:1, 3. grup: MK 80 1+CAPE. MK- 801 bes gun intraperitoneal verildi. Tedavi grubuna MK-801 'e maru zken CAPE verildi. Kontrol grubuna aym surede serum fizyolojik intraperitonea l verild i. 7 gun sonra ratlar dekapitasyonl a olduruldu, Serebellumlan biyokimyasal ana lizler icin cikan ldi. Oksidatif stres parametresi olan Nitrik oksit (NO) yam sira Malond iald ehit (MDA) ve Protein karb onil (PC) seviyeleri kont rol ile karsilastmldigmda MK- 801 grubu sere be llumunda anlarnh art rmsti. CA PE ile tedavi edilen grupta NO, MDA ve PC sev iyes i MK- 801 grubu ile karst Iastmldrgmda ise anlam h aza ldi, Bu ca hs marun sonuc lan serebellumda MK- 801 ile olusa n norotoksisiten in patogenesizinde oksid atif stresi n onemli rolu oldugunu ortaya koydu. Bu deneysel cahs rna ayrn zaman da MK- 801 ile olusa n norotoksisite uzerin e CA PE'nin koruyucu etkis i olabilecegi yonunde bazi delill er sag lar, Anahtar Kelimeler: MK- 80 I, CA PE, MDA, PC, NO ctnts Son yillarda, NMDA (N-metil D-aspartat) glutamat reseptor sisteminin sizofrenide potansiy el rolii olduguna yonelik artan cahsma lar mevcuttur 'li yrllarda disosiy atif ane stezik olarak kull am Ian fensi klidin' in (PC P) insanl arda sizofreni benzeri Yazisma ve ttpkt basim icin; Yrd. Doc. Dr. Birsen Ozyurt Gaziosmanpasa Oniversitesi, TIp Fakiiltesi Anatomi AD Tokat, Turkey Tel: +90 (356) /1232 Fax: +90 (356) ( birsenozyurt05@hotmail.com) ABSTRACT: The aims of this study were to investigate the contributio n effect of oxi dative stress in MK- 80 I induced neurotoxicity, and to show that preventi on of oxidative stress may improve prognosis. MK- 801 was shown to be one of the most neur otoxic NM DA receptor antago nists. Wistar Albino rats were divided into three gro ups: 1. group: control gro up, 2. gro up: MK -801, 3. gro up: MK 80 1+CAPE grou p. MK- 801 was given intra peritoneally for five days. CAPE was give n to the treatme nt group while exposed MK-801. In contro l grou p, saline was given intra peritoneally at the same time. Afte r 7 days, rats were killed by decapit ation. Ce rebe llums were removed for biochemica l analyses. Malondialdehyde, protein carbonyl, as well as nitric ox ide levels as oxidative param eters, levels was found to be increas ed sig nifica ntly in cere bellum of MK -80 I gro up compare d to control gro up. In CAPE treated rats, cerebe llum tissue mal ondi aldehyde, prot ein carbonyl, nitric oxide levels were significa ntly decreased when compared to MK-80 1 gro ups. The result s of this study revealed that oxidative stress in cerebellum may play an important role in the pathogenesis of MK -801 indu ced neuronal toxicity. This experim ental study also pro vides some ev idences for the protective effects of CA PE on MK-80 l-induced changes in cere bellum. Key Words: MK- 801, CA PE, MD A, PC, NO psikotik semptomlara neden oldugu gorulmustur. Klinikte PCP ve diger non-kompetitif reseptor antagoni stlerinin saglikh gonullulerde denenmesi, akut sizofrenid en ayn lamayan, negatif semptomlarla, dusunce bozuklugu ve hallusinasyonlann oldugu psikoz tablosunu olusturm ustur. Bu gozlernler sizofreni calisrnalan icin ilgin c bir ilac mod eli olarak PCP deneysel psikoz modelin in gelistirilmes ine yol acrmstrr ( I). NMDA blokaji yapan diger ilaclarla ardisik yapilan ca hsmalar akut psikotik reaksiyonlan tetikler ve bu nedenle NMDA glutamat reseptor fonksiyonundaki bozuklugun sizofreni patofizyolojisind e mod el olabil ecegi goruldt). NMDA rese ptor blokajirun sonucu se rebral kortekste eksitator
2 64 OZYURT ve ark. norotransmitterlerin fazla sahrnrm ve postsinaptik noronlann ardisik stimulusyonu NMDA reseptor hipofonksiyon (NMDARHipo) durumunu olusturacagi ve gorulen davrams degisikliklerini aciklayabilecegi belirtildi (2). NMDA antagonistlerinin uzun bir listesinin PCP psikozuna benzer sekilde insanda kusursuz psikotik reaksiyonu tetikledigi test edilmistir (I). Uzarms NMDARHipo durumu irreversibil dejenerasyona ve cogu kortikolimbik beyin noronlannm olumune sebep olur (3). Tahminen noronlardaki bu hiperstirnulasyon psikotik semptom olusmasma yardimcr olur. Boylelikle hayvan modelleri, NMDARHipo hiperstimulasyonu ve noron zedelenmesini onlernede farmakolojik yaklasimlar icin firsat vermektedir (I). NMDA reseptorleri, PCP ve MK-801 (dizocilpine) [(5R,IOS)-(+)-5-methyl-1O,II-dihydro 5H-dibenzo (a,d)-cyclohepten-5, lo-imine hydrogen maleate)] tarafmdan inhibe edilmektedir (4,5). PCP ya da MK-801 'in sistemik ya da lokal verilmesi, mezokortikal ve mezolimbik dopamin noronlannda ciddi bozukluga sebep olmus, davrams degerlendirmelerinde ise kacmma cevaplan, lokomotor aktivasyon, ataksi ve stereotipik davramslarda anlamh degisiklikler tespit edilmistir (6,7). Andine ve arkadaslannm yaptik Ian cahsmada, psikoz modeli olarak ratlarda MK-801 ile olusan davrams degisiklikleri, 1100 Sprague Dawley cinsi rat uzerinde doz, yas, cinsiyet farkhhklan ile gaz kromatografik metoduyla MK-801 ' in plazma ve beyin analizleri yapilarak tespit edilmistir. Vine MK 801 ile psikoz olusturulan gruplara verilen noroleptiklerin insandaki antipsikotik etkilerine korele olarak olusan davraruslan bloke ettigi gorulmustur (7). Boylelikle yeni ve daha iyi antipsikotik ajanlann denenmesi amaciyla psikoz modeli olabilecegine karar verilmistir, Davrarus cahsmalannda koordineli veya koordineli olmayan lokomotor aktivite artist, iki ayak uzerine kalkma, havayi koklama, bas sallama, kasmma, bir alandan obur alana gecis, kendi etrafmda donme, dusme, yuvarlanma, hayvanlann kendine ceki duzen vermelerinde eksiklik, ataksik davramslartespit edilmis ve sizofreni modeli olarak kullarnlrrusur (8,9,10). Sican serebellum ve bey in sapmda, PCP ve MK-80 I ile olusan Fos immunoreaktivitesine bakilan bir cahsmada bu ilaclann serebellar eksitasyon meydana getirdigi gorulmustur (II). Biz yapngirmz bu cahsmada, MK-801 verilen ratlarda gorulen ataksik davramsm temelinde oksidatif hasan tespit etmeyi ve antioksidan bir madde olan Kaffeik asit fenetil ester (CAPE)'nin oksidatif parametreler uzerine duzeltici rolu olup olmadrgmi saptamayi planladik. GERE<; VE YONTEM Bu calisrnada yerel etik kuru I onayi almarak, 21 Wistar Albino rat, rastgele secilen 3 gruba aynldi. I. grup: %0,9'luk serum fizyolojik intraperitoneal (lp) yolla gunde bir kez (I x I) 5 gun uygulandt. 2. grup: 0.5 rug/kg/gun dozunda MK- 801 Ixl kez IP olarak 5 gun uygulandi. 3. grup: 10 urnol/kg dozunda CAPE Ixl kez IP olarak 6 gun uygulandi. MK -801 'de uygulanan bu ratlara CAPE tedavisi I gun once uygulanmaya baslandi, Deneyin 7. gununde turn ratlar dekapite edilerek beyin dokulan crkartildr ve serebellumlan aynlarak biyokimyasal islernler icin donduruldu. Serebellum dokusunda NO, MDA ve PC duzeyleri spektrofotometrik yonternle olculdu. Nitrik oksit miktarmm tayini: Vucutta uretilen NO saniyeler icinde okside olarak once azot diokside (NOz) soma NOz' ve N0 3 ' donusur, Doku homojenatmdaki spesifik olmayan reaksiyonlar meydana gelmesini onlemek icin homojenatlan onc e deproteinize edip daha soma nitrit ve nitrat konsantrasyonlan Griess reaksiyonu ile belirlendi (12). Total nitrit (nitrit+nitrat) konsantrasyonu modifiye kadmiyum rediik siyon metodu ile degerlendirildi. ph 9.7 glisin tamponunda bakir (Cu) kaph kadmiyum graniilleri deproteinize ornek supernatanti ile 90 dakikahk inkiibasyon sonunda nitrat rediiksiyonu saglandi, Sulfanilamid ve buna bagh N naphthylethylene diamin (NNDA) diazotizasyonuyla reaksiyon sonu olusan pembe rengin 545 nm dalga boyunda spektro fotometrede okunmasi ile indirek NO olcumn yapildi. Sonuclar urnol /g yas doku olarak ifade edildi. Tiyobarbitiirik asit reaktif maddeleri (TBARS) miktarmm tayini: Esterbauer ve Cheeseman'nin metodu ile cahsildi (13). Asidik ortamdaki tiyobarbitiirik asit ile C'de reaksiyona giren malondialdehit (MDA) ve diger TBARS, pembe renkli kromojen meydana getirir. Onbes dakika soma hizla sogutulan omeklerin absorbanslan 532 nm 'de spektrofotometrik olarak okundu. Sonuclar nmol/g yas doku olarak ifade edildi. Protein karbonil (PC) miktan tayini: Metod; karbonil grubunun 2,4-dinitrofenil hidrazin ile reaksiyona girerek 2,4-dinitrofenilhidrazon olusturmasi prensibine dayamr (14,15). 2,4-dinitrofenilhidrazin oksidasyona maruz kalrms proteinlerde acrga cikan karbonil gruplarmm gosterilmesinde ku llaruian ajandir. 2M HCI icerisinde hazirlanan 2,4 dinitrofenilhidrazin soliisyonunun homojenattaki Kocatepe Tip Dergisi, c u. 7No: 3, Eyliil 2006.
3 MK-801 Veri/en Ratlartn Cerebellumun 'da Makromolekiiler Oksidasyon ve Cape 'nin Koruyu cu Etkisi / The Macromoleculer OXidation in Cerebellum ojrat Expos ed To MK-801 and The Protective Effect oj Cape 65 karbonil icerigi ile reaksiyona girmesi saglandi, etanolletil asetat kansmu ile yikanan cokelti bir sonraki asamada 100 mm NaOH cozeltisi icerisinde cozulmesinden soma 360 nm'de spektrofotometrik olarak oleum yapildi, Sonuclar; nmol/mg protein olarak ifade edildi. istatistik: Sonuclar Windows uyumlu SPSS programi ile degerlendirildi. Gruplann normal dagilim gosterip gostermedigi Kolmogorov Smimov testi ile incelendi. Tiim parametrelerde normal dagihrn gostermesinden dolayi parametrik testier kullamldi, Parametrik testlerden One-Way ANOVA testi ve Post Hoc testlerden LSD uygulandi. Sonuclar ortalama ± standart hata olarak verildi. p degeri 0.05 den kucuk olanlar anlamh olarak kabul edildi. SONUC;LAR MK-801 verilmesinden soma biyokimyasal olarak incelenen serebellumda, lipid peroksidasyonunun gostergesi olan MDA (p<o.ooi), protein oksidasyonunun gostergesi olan PC (0.045) ve NO (p< I) diizeyinin kontrol grubu ile karsilastmldigmda anlamh olarak artngi tespit edilrnistir. Cahsmamizda CAPE'nin bu parametreler uzerinde diizeltici etkisi oldugu (p< MDA icin, p< PC icin, p< NO icin MK-801 grubu ile karsilastmldigmda) ve kontrol grubu ile arasinda anlamh farkhhk olmadrgi tespit edilmistir. CAPE verilen grup ile kontrol grubu karsilastmldigmda CAPE'li grupta PC seviyesinin anlamh olarak dustiigii gorulrnusttlr (p< 0.003). TARTI~MA Reaktif oksijen iiriinlerinin (ROS) artmasi ve/veya antioksidan savunma sisteminin azalmasi ile olusan oksidatif stres, membran fosfolipidleri ozellikle esansiyel coklu doymarms yag asitlerinin esterlesmesine, membran fosfolipidlerinin peroksidasyonuna, protein ve DNA hasanna sebep olmaktadir (16). Sinir hiicreleri ve beyin dokusu oksidatif hasara viicudun diger dokulanndan daha fazla yatkmdir, Beyinde oksidatif stresin ROS atagimn sebep oldugu akut donemdeki en onemli olay lipit peroksidasyonudur. Lipit peroksidasyonu sonucu aciga cikan yikrm iiriinleri olan MDA, tiyobarbitiirik asit ile reaksiyona girebilen bilesikler aracihgi ile olculmektedir. Bu bilesiklerin ortamda bulunmasi oksidatif stres oldugu ongortilmektedir (17). Sizofrenide oksidatif stresin arastmldrgi klinik cahsmalarda, oksidatif hasan gostermede onemli bir parametre olarak MDA duzeyinin artrms oldugu saptanmisnr (16,18,19). Bizim bu cahsmarmzda, MK 80 I verilen grup kontrol grubu ile karsilastmldigmda istatistiksel olarak anlamh derecede artngi goriilmektedir. Yapngmuz literatiir taramalannda, deneysel hay van psikoz modelinde oksidatif hasann arasnnldigi az sayida makale vardir (20). Bu cahsma ile sonucta lipit peroksidasyonunun onemli bir bulgusu olan MDA diizeylerinin deneysel sizofreni modelinde arttigi, CAPE veri len grupta ise azaldigi ve bu azalrnanm istatistiksel olarak anlamh oldugu gorulmustur. Vine son yillarda lipitlerin peroksidatif son yikim iiriinii olan MDA dismda, proteinlerin oksidatif hasan sonucu aciga cikan protein karbonil ile ilgili cahsmalar yogunluk kazanrmsnr. Ancak oksidatif hasan saptamak icin kullamlan bu markirlar sizofreniye ozgu olmayip biitiin serbest radikal hasannm incelendigi cahsrnalarda parametre olarak kullamlmaktadir, Yapnus oldugumuz cahsmad a ise psikoz grubuna ait beyin dokusunda oksidatif hasann gostergesi olarak MDA diizeyindeki anlarnh degisiklikle beraber protein karbonil diizeyi ve nitrik oks it seviyesinin de kontrol grubuna gore anlamh olarak artrms oldugu tespit edilmistir. NO ise L-arginin'den NO sentaz enzimi aracihgryla sentezlenir. NO biyolojik noral messenger molekiilii olarak bilinmesi yanmda santral sinir sisteminde toksik reaktif serb est radikal olarak da bilinmektedir. Peroksinitrit ve onun ileri urunleri hucre hasanna sebep olmaktadir. Sizofreni hastalannda yapilan 9ahsmalarda NO seviyesinin yiiksek oldugu tespit edilmistir (21). CAPE flavonoid yapismda propolisin aktif bir komponentidir. CAPE'nin lipit peroksidasyonunu baskiladigi, antioksidan aktivite gosterdigi ve lipooksijenaz aktiviteyi inhibe ettigi rapor edilmistir (22). CAPE ile ilgili yapilan calismalarda noroprotektif etkisinden bahsedilmektedir (23). Bu cahsmada da gorulmustur ki CAPE, MK-801 ile olusan oksidatif hasan onlemis ve oksidatif stres parametrelerini kontrol grubu seviyelerine cekebilmistir. Bu cahsma ile MK-80 I ile olusturulan serebellum hasannda oksidatif stresin katkisi olabilecegi ortaya konmustur. Bu konuda ileri baska 9ahsmalara ihtiyac vardir. Ayru zamanda bir antioksidan olan CAPE'nin diizeltici rolu bu gorusu desteklemektedir. Kocatepe TIp Dergis i, CUt 7 No: 3. Eyliil 2006.
4 66 6 ZYURT ve ark. 1.2 Nitrik Oksit 1 * 0,8 0,6 0,4 0, _._ r., ~ G rafik I : Rat serebellumunda; Kontrol, MK -80 I ve MK 80 1+CA PE grubundaki NO seviyeleri. *P< MK 80 I grubu MK- 80 I+CAPE grubu ile karsila snnldiginda so _ TBARS * '--- Grafik 2: Rat serebellumunda; Kontrol, MK-80 1 ve MK 80 I+CA PE grubundaki TBARS sev iyeleri. *p< MK-80 1 grubu M K-80I+CAPE gru bu ile karsilas tm ldigmda 600 Protein Karbonil 500 * '-----_. G rafik 3: Rat serebellumunda; Kontrol, MK-801 ve MK 80 1+CAPE grubundaki PC seviyeleri. *p< MK 80 1 grubu MK-80 1+CAP E grubu ile karsil astmldrgmda KAYNAKLAR I. Olne y JW, Newcomer JW, Farber NB. NMDA receptor hypofun ction model of schizophrenia. J Psych iatr Res, 1999; 33: Javitt DC, Zukin SR. Recent adva nces in the phen cyclidine model of schizophrenia. Am J Psych iatry, 199 1; 148: Corso TD, Sesma MA, Tenkova TI, Der TC, Wozniak DF, Farber NB, Olney JW. Mu ltifocal bra in damage induc ed by phencyclidine is aug mented by pilocarpine. Brain Res, 1997; 752: Haring R, Pellegrini-G iampietro DE, Zu kin SR, Zukin RS, Scheideler MA. High efficien cy reconstitution of a phenc yclidine/mk-801 receptor binding site so lubilized from rat forebrain membranes. Mol Pharmacol. 1991; 40: Ferrer-Mont iel AV, Sun W, Montal M. Molecular design of the N-methyl-D-aspartate receptor binding site for phencyclidine and dizolcipine. Proc Natl Acad Sci U S A. 1995; 92: Ho ffman DC. Typical and atypic al neurolep tics antagonize MK-801-induced locomotion and stereotypy in rats, J Neural Transm Gen Sect ;89: Andine P, Widermark N, Axel sson R et al.. Characterization of MK- 801-induced behavior as a putative rat model of pschosis. JP ET. 1999;290: Mastropaolo J, Rosse RB, Deutsch Si. Anabasine, a selective nicotinic acetylcholine receptor ago nist, antagonizes MK- 80 I-elicited mouse poppi ng beha vior, an anima l model of schizophrenia. Behav Brain Res ;153: Deutsch SI, Rosse RB, Billingslea EN, Bellack AS, Mastropa olo J. Modulation of MK-801-elicited mouse poppin g behavior by galantamine is complex and dose-dependent. Life Sci : Rung JP, Carlsson A, Ryden Markinhuhta K, Carlsson ML. (+)-MK-80 1 induced socia l withdrawal in rats; a mode l for negat ive symptoms of schizophrenia. Prog Ne urops ychopharmacol Bioi Psyc hiatry : I I. Nakki R, Sharp F., Sagar SM. FOS expre ssion in the brain stem and cerebellum following phenc yclidine and MK 801. J Neurosci Res. 1996; 43 : Cortas, N. K., Wakid, N. W.:Determination of inorganic nitrate in serum and urine by a kineti c cadm ium-reduction method. Clin Chern 1990 ;36: Esterbauer H, Cheeseman KH. Determ ination of ald ehydic lipid peroxidation produ cts: Malonaldehyde and 4-hyd roxynonenal. In Methods in Kocatepe Tip Dergisi, Cilt 7 No: 3. Eylii12006.
5 MK-80 1 Veri/en Ratlann Cerebellumun 'da Makromolekiiler Oksidasyon ve Cape 'nin Koruyucu Etkisi / The Macromoleculer OXidation in Cerebellum ofrat Exposed To MK-801 and The Protective Effect ojcape 67 Enzymology, V 186, Oxygen radicals in biological systems edited by Packer L, Glazer AN. Academic Press, California, 1990, pp Packer L, Glazer NA. Determination of Carbonyl Content in Oxidatively Modified Protein s, Methods in Enzymology. California. 1990;186: Lowry 0, Rosenbraugh N, Farr L, Rondall R. Protein measurement with the folin-phenol reagent. J Bioi Chern 1951;183: Mahadik SP, Evans D, Lal H. Oxidative stress and role of antioxidant and omega-3 essential fatty acid supplementation in schizophrenia. Prog Neuropsychopharmacol Bioi Psychiatry. 200 I; 25: Mahad ik SP, Mukherjee S, Scheffer R, Correnti EE, Mahadik JS. Elevated plasma lipid peroxides at the onset of nonaffective psycho sis. Bioi Psychiatry. 1998; 43 : Mates JM, Perez-Gomez C, De Castro IN. Antioxidant enzymes and human diseases. Clin. Biocehmistry. 1999; 32: Akyol 0, Herken H, Uz E, Fadillioglu E, UnaI S, Sogut S, Ozyurt H, Savas HA. The indices of endogenous oxidative and antioxidative processes in plasma from schizophrenic patients. The possible role of oxidant/antioxidant imbalance. Prog Neurops ychopharmacol BioI Psychiatry ; 26: Ozyurt B, Sarsilmaz M, Akpolat N, Ozyurt H, Akyol 0, Herken H, Kus I. The protect ive effects of omega 3 fatty acids again st MK-801-induced neurotoxicity in prefrontal corte x of rat. Neurochem Int ; 50: Herken H, Uz E, Ozyurt H, Akyol O. Red blood cell nitric oxide levels in patients with schizophrenia. Schizophr Res. 200 I; 52: Ozyurt B, Gulec M, Ozyurt H, Ekici F, AtI ~ 0, Akbas A. The effect of antioxidant caffeic acid phen ethyl ester (CAP E) on some enzyme activities in cisplatininduced neurotoxicity in rats. Eur J Gen Med ; 3: Sud'ina GF, Mirzoeva OK, Pushkare va MA, Korshunova GA, Surnbatyan NV, Varfolomeev SD. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett. 1993; 329 : Kocatepe Tip Dergisi, Cilt 7 No: 3. Eyliil 2006.
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