Uticaj estradiola na regulaciju enzima antioksidativne zaštite i inducibilne azot-monoksid-sintaze u jetri normalno uhranjenih i gojaznih mužjaka pacova

Abstract

Steroidni hormon 17β-estradiol (E2) ostvaruje zaštitnu ulogu u jetri, održavajući homeostazu masti i glukoze. Njegov nedostatak doprinosi razvoju steatoze jetre i progresiji poremećene funkcije jetre. U stanjima gojzanosti i prisustva rezistencije na insulin (IR), E2 ostvaruje uticaj na regulaciju enzima antioksidativne (AOS) zaštite i inducibilne azot-monoksid-sintaze (iNOS). Kontrolni i gojazni mužjaci Wistar soja pacova su tretirani jednom dozom E2 (40μg/kg) ili 1% etanolom 24 sata pre žrtvovanja. Spektrofotometrijskim metodama su određivane koncentracije malon-dialdehida (MDA), karbonilovanja proteina (PCC) i azot-monoksida (NO), kao i aktivnost enzima CAT u lizatu jetre. Western blot metodom su određivani nivoi proteina SOD1, SOD2, CAT, GPx, iNOS, ERα, ERβ, Src, NFκB-p50, NFκB-p65, ROCK1, ROCK2, RhoA, pERK1/2/ERK1/2, pAkt/Akt, pAMPKα1/AMPKα1 proteina. Metodom koimunoprecipitacije je određivan nivo Src u kompleksu sa ERα, dok je metodom Pull-down eseja određivana aktivnost ukupnih Rho proteina u jetri pacova. Metodom qRT-PCR određivan je nivo iRNK za iNOS i miR-221 u jetri pacova. Rezultati su analizirani korišćenjem Studentovog t-testa. Rezultati prikazani u ovoj disertaciji pokazuju da E2 tretman kod normalno uhranjenih pacova značajno povećava nivoe SOD1, GPx, CAT proteina i nivo asocijacije ERα/Src, a smanjuje nivoe proteina iNOS i ERα, fosforilaciju kinaza ERK1/2 i Akt, i ekspresiju miR-221 u jetri. Kod gojaznih pacova, E2 tretman povećava nivoe SOD1, SOD2, GPx i CAT proteina, aktivnost CAT enzima, nivo NO, fosforilaciju AMPKα1 i ekspresiju miR-221, a smanjuje ekspresiju gena za iNOS i nivo iNOS proteina, nivo p65-NFκB, nivo ERα i nivo asocijacije ERα/Src, fosforilaciju kinaza ERK1/2 i Akt, i aktivnost ukupnih Rho proteina u jetri. Na osnovu dobijenih rezultata se može zaključiti da E2 ima zaštitne efekte u jetri u fiziološkim i patofiziološkim uslovima, koji se ogledaju u povećanju nivoa i aktivnosti enzima AOS zaštite i smanjenju ekspresije iNOS. Osim toga, rezultati istraživanja obuhvaćenih ovom doktorskom disertacijom doprinose sagledavanju molekulskih mehanizama kojima E2 učestvuje u regulaciji ekspresije i aktivnosti enzima AOS zaštite i ekspresije iNOS enzima, u jetri normalno uhranjenih i gojaznih pacova.

The steroid hormone 17β-estradiol (E2) has a protective role in the liver, maintaining fat and glucose homeostasis. Its deficiency contributes to the development of liver steatosis and the progression of chronic liver diseases. In obesity and insulin resistance (IR), E2 exerts effects on the regulation of antioxidant defence system (AOS) enzymes and inducible nitric oxide synthase (iNOS). Control and obese male Wistar rats were treated with a bolus injection of E2 (40μg/kg) or with the same volume of 1% ethanol, 24 hours before euthanasia. The concentrations of malondialdehyde (MDA), protein carbonylation content (PCC) and nitric oxide (NO), as well as CAT enzyme activity in liver lysate were determined by spectrophotometric methods. Protein levels of SOD1, SOD2, CAT, GPx, iNOS, ERα, ERβ, Src, NFκB-p50, NFκB-p65, ROCK1, ROCK2, RhoA, pERK1/2/ERK1/2, pAkt/Akt, pAMPKα1/AMPKα1 were determined by Western blot. The coimmunoprecipitation method was used to determine the ERα/Src association, while the pull-down assay method was used to determine the activity of total Rho proteins in the rat liver. The level of iNOS mRNA and miR-221 level in the rat liver were determined by qRT-PCR. Results were analyzed using Student's t-test. The results presented in this doctoral dissertation show that E2 treatment in normally fed rats significantly increases SOD1, GPx, CAT protein levels and ERα/Src association level, while decreasing iNOS and ERα protein levels, ERK1/2 and Akt kinase phosphorylation, and miR-221 expression in liver. In obese rats, E2 treatment increases SOD1, SOD2, GPx and CAT protein levels, CAT activity, NO level, AMPKα1 phosphorylation and miR-221 expression, and decreases iNOS gene and protein expression, p65-NFκB level, ERα level and ERα/Src association level, phosphorylation of the ERK1/2 and Akt, and the activity of total Rho proteins in liver. Based on the obtained results, it can be concluded that E2 has protective effects in the liver in physiological and pathophysiological conditions, which are mediated by an increase in the level of AOS enzymes and a decrease in iNOS expression. In addition, the results of this doctoral dissertation contribute to the understanding of the molecular mechanisms by which E2 is involved in the regulation of the expression and activity of the AOS enzymes and iNOS expression, in the liver of normally fed and obese rats.