Efekat grelina na regulaciju antioksidativnih enzima i inducibilne azot-monoksid-sintaze u jetri pacova

Abstract

U poslednjih nekoliko godina, objavljeni naučni podaci sve više ukazuju na antioksidantne i antiinflamatorne efekte grelina. U ovoj doktorskoj disertaciji su izučavani efekti grelina na ekspresiju enzima jetre: superoksid dismutaze (engl. Superoxide Dismutase, SOD), katalaze (engl. Catalase, CAT), glutation peroksidaze (engl. Glutathione peroxidase, GPx) i glutation reduktaze (engl. Glutathione reductase, GR), kao i ekspresiju njihovog uzvodnog regulatora, nuklearnog faktora-kB (engl. Nuclear factor- kappa B, NFκB). Takođe su izučavani efekti grelina na regulaciju enzima inducibilne azot-monoksid-sintaze (engl. Inducible nitric oxide synthase, iNOS; NOS2) u jetri pacova. Jedan od ciljeva istraživanja u okviru ove doktorske disertacije je bio i utvrđivanje uloge kinaze regulisane ekstraćelijskim signalima (engl. Extracellular regulated protein kinase, ERK1/2) i protein kinaze B (engl. Protein kinase B, Akt) u grelinom regulisonoj aktivnosti i ekspresiji antioksidatnivnih enzima i iNOS u jetri pacova. Kontrolni, normalno hranjeni mužjaci pacova, Wistar soja (mase 200-250g) kao i mužjaci pacova koji su bili na dijeti bogatoj mastima (engl. High fat diet, HF), tretirani su grelinom (0,3nmol/5μl) intracerebroventrikularnim (ICV) injeciranjem, u bočne cerebralne komore svaka 24 h, u trajanju od 5 dana. Dva sata nakon poslednjeg tretmana, životinje su žrtvovane i izolovane su jetre. Za utvrđivanje nivoa ekspresije proteina: iNOS, antioksidativnih enzima, fosforilacije Akt na Ser473, ERK 1/2 i NFκB, subjedinica p50 i p65, primenjena je Western blot metoda. Dobijeni rezultati pokazuju da dolazi do povećanja ekspresije proteina za enzime antioksidativne zaštite: Cu/Zn-SOD (p<0,05), Mn-SOD (p<0,05), CAT (p<0,01), GPx, (p<0,001), kao i GR (p<0,01) u grupi normalno hranjenih životinja tretiranih grelinom u poređenju sa kontrolnim životinjama. Grelin značajno smanjuje ekspresiju iNOS (p<0,001) kao i fosforilaciju NFκB subjedinica p65 (p<0,001) i p50 (p<0,05) u grupi normalno hranjenih životinja u poređenju sa kontrolnom grupom pacova. Nivo fosforilacije Akt na Ser473 značajno je veći u grelinom tretiranoj grupi nego kod kontrolnih životinja (p<0,01)...

In recent years, there have been prominent findings suggesting that ghrelin has antioxidant and anti-inflammatory effects. In this study we investigated the effects of ghrelin on protein expression of liver antioxidant enzymes superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) as well as protein expression of their upstream regulator, nuclear factor kappa B (NFκB). We have also studied the effects of ghrelin on liver inducible nitric oxide synthase (iNOS) protein expression. Furthermore, we aimed to investigate whether extracellular regulated protein kinase (ERK 1/2) and protein kinase B (Akt) are involved in ghrelin regulated liver antioxidant enzymes and iNOS protein expression. Male Wistar rats (200-250g) were treated with ghrelin (0,3nmol/5μl) injected into the lateral cerebral ventricle every 24 h for 5 day, 2 h after the last treatment the animals were sacrificed and liver excised. For protein expression of antioxidant enzymes, iNOS, phosphorylation of Akt at Ser473, ERK1/2 and nuclear factor B (NFκB) subunits 50 and 65, Western blot method was used. Results show significantly higher protein expression of Cu/Zn SOD (p<0,05), Mn-SOD (p<0,05), CAT (p<0,01), GPx, (p<0,001), and GR (p<0,01) in the liver isolated from ghrelin treated animals compared with control animals. On the contrary, ghrelin significantly (p<0,001) reduced protein expression of iNOS. In addition, phosphorylation of NFκB subunits p65 and p50 were significantly (p<0,05) decreased by ghrelin when compared with controls. Phosphorylation of ERK1/2 and phosphorylation of Akt at Ser473 were significantly higher in ghrelin treated than in control animals (p<0,05 for ERK1/2; p<0,01 for Akt). In conclusion, results from this study show that activation of Akt and ERK1/2 are involved in ghrelin-mediated regulation of protein expression of antioxidant enzymes and iNOS in the rat liver.