Učinak glukagona i cikličnog adenozin-monofosfata na akutno oštećenje jetre paracetamolom [Effect of glucagon and cyclic adenosine monophosphate on paracetamol-induced liver injury]

Kelava, Tomislav (2010) Učinak glukagona i cikličnog adenozin-monofosfata na akutno oštećenje jetre paracetamolom [Effect of glucagon and cyclic adenosine monophosphate on paracetamol-induced liver injury]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Recent investigations suggest that glucagon, beside its well known physiological effects, might have a potentially important hepatoprotective activity. Until now, it was shown that glucagon protects against apoptotic and lipopolysaccharide-induced liver injury. Also, it was shown that liver transplants, during the cold preservation, survived longer, when in preservation-solution glucagon was added. Like most of its effects, the hepatoprotective activity of glucagon was shown to be mediated by elevation of cAMP level in liver. In our investigation, we investigated the effect of glucagon in a model of paracetamol-induced liver injury and some of the possible mechanisms of its action. CBA male mice were injected with lethal (300 mg/kg) or sublethal (150 mg/kg) dose of paracetamol, i.p. The liver injury was assessed by observing the survival of mice, by liver histology and by measuring concentration of alanine-aminotransferase (ALT) in plasma. Inducible nitric oxide synthase (iNOS) and NF-κB (p65) protein expressions were determined immunohistochemically. GSH and cAMP levels in liver were also measured. Our results show that glucagon has a protective effect in this model of liver injury and that the effect depends on a dose (the strongest protection was achieved with a dose of 0.5 mg/kg) and on a time of application - glucagon alleviated liver injury when given 15 min before or 1 h after paracetamol, whereas in earlier or later treatment it had no effect. Rolipram, an inhibitor of phosphodiesterase and db-cAMP, stabile analog of cAMP, had protective effects similar to that of glucagon. In comparison with normal, untreated animals, animals treated with saline and paracetamol (control group) had elevated nitrite plus nitrate concentration in plasma, enhanced iNOS and NF-κB expression in liver and reduced GSH and cAMP concentration in liver. Animals treated with glucagon and paracetamol had higher hepatic cAMP level, lower nitrite plus nitrate concentration in plasma and lower expression of iNOS in liver cells than animals in control group, whereas there was no difference in NF-κB expression. Glucagon did not prevent a loss of GSH content caused by paracetamol, but it did accelerate its recovery toward the normal levels. To conclude, our investigation indicates that glucagon and its secondary messenger cAMP have a moderately strong protective effect against paracetamol-induced liver injury. The protection is partially mediated through the downregulation of iNOS, but other mechanisms of protection are also probably present. The protective effect of glucagon in this model appears not to be mediated by a modulation of NF-κB activity.

Abstract in Croatian

U posljednjih desetak godina su objavljena istraživanja koja pokazuju da bi glukagon, osim dobro poznatih fizioloških učinaka, mogao imati i, potencijalno važnu, hepatoprotektivnu ulogu. Dosad je pokazano da glukagon štiti jetru od apoptotičkog oštećenja i od oštećenja lipopolisaharidom. Također, pokazano je da presadci jetre, za vrijeme hladne prezervacije organa, dulje preživljavaju ako se čuvaju u otopini kojoj je dodan glukagon. Pokazano je i da je protektivan učinak glukagona, poput većine njegovih drugih učinaka, posredovan povećanjem razine cikličnog adenozin-monofosfata (cAMP-a). U našem istraživanju smo ispitali ima li glukagon učinka u modelu oštećenja jetre paracetamolom i neke od mogućih mehanizama kojima te učinke ostvaruje. Pokusi su obavljeni na visokosrodnim mišjim sojevima CBA (mužjaci) kojima je uštrcana letalna (300 mg/kg) ili visoka subletalna (150 mg/kg) doza paracetamola, i.p. Stupanj oštećenja jetre je procijenjen praćenjem preživljenja životinja, mjerenjem koncentracije alanin aminotransferaze (ALT) u plazmi i određivanjem histološkog stupnja oštećenja jetre. Imunohistokemijskim metodama je određena aktivnost čimbenika NF-κB i izraženost inducibilne NO-sintaze (iNOS), a mjerena je i razina reduciranog glutationa (GSH) i cAMP-a u jetri. Naši rezultati pokazuju da glukagon ima protektivan učinak u ovom modelu i da je učinak ovisan o dozi (najjači učinak smo dobili s dozom 0.5 mg/kg, i.p.) i vremenu primjene - glukagon je smanjio oštećenje jetre kada je uštrcan 15 min prije ili 1 h nakon paracetamola, dok u ranijem ili kasnijem razdoblju nije imao učinka. Rolipram, inhibitor fosfodiesteraze i db-cAMP, stabilni analog cAMP-a su imali učinak sličan glukagonovom. Nakon otrovanja paracetamolom došlo je do povećanja razine nitrita i nitrata u plazmi, povećane izraženosti iNOS-a i NF-κB, i pada razine GSH i cAMP-a u jetri kontrolnih životinja (koje su uz paracetamol primili fiziološku otopinu). Životinje koje su uz paracetamol primile glukagon imale su u usporedbi s kontrolnom skupinom veću razinu cAMP-a u jetri, manju razinu nitrita i nitrata u plazmi i manju izraženost iNOS-a u jetri. Glukagon nije spriječio pad razine GSH u jetri, ali je oporavak prema normalnim vrijednostima bio brži, dok u aktivnosti NF-κB nije bilo razlike. Zaključno, naša su istraživanja pokazala da glukagon i njegov sekundarni glasnik cAMP imaju, umjereno jak i vremenski ograničen, protektivan učinak u modelu oštećenja jetre paracetamolom. Moguće je da je jedan dio protektivnog učinka posredovan smanjenjem izraženosti i aktivnosti iNOS-a u jetri, ali u obzir dolaze i drugi mehanizmi. Čini se da glukagon svoje učinke u ovom modelu ne ostvaruje modulacijom aktivnosti NF-κB.

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