Uloga prostanoida u mehanizmima akutnog toksičnog oštećenja jetre [The role of prostanoids in acute acetaminophen hepatotoxicity]

Ćavar, Ivan (2010) Uloga prostanoida u mehanizmima akutnog toksičnog oštećenja jetre [The role of prostanoids in acute acetaminophen hepatotoxicity]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Prostanoids are lipid compounds that mediate variety of physiological and pathological functions in almost all body tissues and organs. Prostaglandin E2 (PGE2), which is synthesized by many cell types, and prostaglandin I2 (PGI2), which is synthesized by the vascular endothelium, has a strong cytoprotective and vasodilatory effects in the gastrointestinal tract and in several other tissues and cells. Thromboxane A2 (TxA2) is a major product of arachidonate metabolism in platelets. It is a powerful inducer of platelet aggregation and vasoconstriction and it shows ulcerogenic activity in gastrointestinal tract. These observations prompted us to investigate whether PGE2, PGI2 or TxA2 play a role in host defence to toxic effect of acetaminophen (paracetamol, APAP), which overdose or chronic use of a high dose is a major cause of acute liver failure in the western world. CBAT6T6, C57Bl/6 and (CBAT6T6xC57Bl/6)F1 satybrid mice of both sexes were intoxicated with a single lethal (250-300 mg/kg) or high sublethal (150-200 mg/kg) dose of APAP, which was administered to animals by oral gavage. Stabile analogues of PGE2 (dm-PGE2, 0,2 mg/kg) and PGI2 (iloprost, 0,2 or 0,5 mg/kg), PGI2 (10 μg/kg), inhibitors of PGE2 production through the selective modulation of mPGES-1 expression (CAY10526, 2 mg/kg), PGI receptor (CAY10441, 2 mg/kg) and TxA receptor (daltroban, 5 mg/kg), TxB2 (2 mg/kg) and inhibitor of TxA2-synthase – benzyl imidazole (BZI, 50 mg/kg) were given to mice intraperitoneally (i.p.) 30 min before or 2 h after APAP administration. Anti-TxB2 antibodies (anti-TxB2, 40 μg/kg) were given i.p. 3 h before, while stabile analogue of TxA2 (U-46619, 0,2 and 0,8 mg/kg) was injected into mice intravenously (i.v.) 30 min before APAP. The toxicity of APAP was determined by observing the survival of mice during 48 h, by measuring concentration of alanine-aminotransferase (ALT) in plasma 20-22 h after APAP administration and by liver histology. The results have shown that PGE2 and PGI2 exhibit a strong hepatoprotective effect when they are given to mice either before or after APAP, while CAY10526 and CAY10441 demonstrated mainly the opposite effect. Iloprost had no significant influence on APAP toxicity. Anti-TxB2 and BZI were significantly hepatoprotective and daltroban showed mainly the same (but non significant) action on APAP-induced hepatotoxicity. TxB2 and U-46619 were ineffective in this model of acute liver injury. Immunohistochemistry and immunofluorescence to NF-κB revealed that administration of dm-PGE2 down regulated, in a significant manner, the activation of NF-κB and its translocation from the cytoplasm to the nucleus of hepatocytes. The present results also showed that PGE2 significantly diminished expression of iNOS in hepatocytes and reduced NO production, which was reflected by lower (albeit statistically non significant) nitrite/nitrate concentration in plasma. Our findings support the hypothesis that TxA2 has a pathogenic role in liver toxicity induced with APAP, while PGE2 and PGI2 have a cytoprotective effect and are involved in the defence of the organism to noxious effects of xenobiotics on liver. According to our results, this protection is mediated, at least partially, through down regulation of NF-κB activation and iNOS expression. Therefore, further work is needed to elucidate precise mechanisms underlying cytoprotective effects of PGs in acute liver injury.

Abstract in Croatian

Prostanoidi su spojevi koji nastaju razgradnjom lipida stanične membrane te posreduju u mnogim fiziološkim i patofiziološkim zbivanjima u gotovo svim organima i tkivima u organizmu. Prostaglandin E2 (PGE2), kojeg sintetizira mnogo vrsta stanica, i prostaglandin I2 (PGI2), kojeg stvara endotel krvnih žila, imaju jak citoprotektivni i vazodilatacijski učinak u probavnom sustavu te u nekoliko drugih vrsta tkiva i stanica. Tromboksan A2 (TxA2), kojeg uglavnom stvaraju trombociti, je snažan induktor agregacije trombocita, ima jak vazokonstrikcijski učinak i posreduje u nastanku ulkusa u probavnom sustavu. Cilj ovog istraživanja bio je ispitati ulogu PGE2, PGI2 i TxA2 u obrani organizma od toksičnog učinka paracetamola (acetaminofen, APAP) čiji su predoziranje ili kronična uporaba visokih doza najčešći uzroci akutnog zatajenja jetre u zapadnom svijetu. Pokusi su bili obavljeni na visokosrodnim mišjim sojevima CBAT6/T6, C57Bl/6 i (CBAT6T6xC57Bl/6)F1 u kojih je gastričnom sondom primijenjena letalna (250-300 mg/kg) ili visoka subletalna (150-200 mg/kg) doza APAP-a. Stabilni analozi PGE2 (dm-PGE2, 0,2 mg/kg) i PGI2 (iloprost, 0,2 i 0,5 mg/kg), PGI2 (10 μg/kg), kemijski antagonisti mikrosomalne PGE-sintaze 1 (CAY10526, 2 mg/kg), IP-receptora (CAY10441, 2 mg/kg) i TP-receptora (daltroban, 5 mg/kg), TxB2 (2 mg/kg) te inhibitor TxA2-sintaze – benzilimidazol (BZI, 50 mg/kg) primijenjeni su intraperitonealno (i.p.) 30 min prije ili 2 sata nakon APAP-a. Anti-TxB2 protutijela (anti-TxB2, 40 μg/kg) su primijenjena i.p. 3 sata prije, dok je stabilni agonist TxA2 (U-46619, 0,2 i 0,8 mg/kg) primijenjen intravenski (i.v.) 30 min prije APAP-a. Toksičnost APAP-a se određivala na temelju 48-satnog praćenja preživljenja životinja, mjerenja koncentracije alanin aminotransferaze (ALT) u plazmi i određivanja histološkog stupnja oštećenja jetre 20-22 sata nakon primjene APAP-a. Dm-PGE2 i PGI2 su pokazali snažan hepatoprotektivni učinak kad su bili primijenjeni 30 min prije i 2 sata nakon APAP-a. Iloprost nije pokazao značajan učinak na toksičnost APAP-a, dok su CAY10526 i CAY10441 povećali hepatotoksični učinak kad su bili primijenjeni 2 sata nakon APAP-a. Anti-TxB2 i BZI su također imali snažan hepatoprotektivni učinak, a daltroban je djelimično smanjio toksičnost APAP-a. TxB2 i U-46619 nisu imali učinka u ovom modelu akutnog hepatitisa potaknutog APAP-om. Imunohistokemijskom i imunofluorescencijskom analizom jetrenog tkiva je pokazano da PGE2 smanjuje aktivaciju transkripcijskog čimbenika NF-κB i citoplazmatski izražaj inducibilne NO-sintaze (iNOS). Ovo istraživanje potkrjepljuje hipotezu da TxA2 ima patogenetski učinak, dok su PGE2 i PGI2 endogeni posrednici u obrani organizma od štetnog djelovanja hepatotoksičnih agenasa. Protektivno djelovanje PGE2 je, barem dijelom, posredovano smanjenom aktivacijom NF-κB i izražajem iNOS-a. Potrebna su daljnja istraživanja da bi se ustanovio precizan mehanizam na kojem se temelji citoprotektivni učinak prostaglandina u akutnom toksičnom oštećenju jetre.

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