Efekat estradiola na regulaciju endotelne i inducibilne azot-monoksid-sintaze u srcu gojaznih pacova

Abstract

Estradiol ostvaruje pozitivan efekat na kardiovaskularni sistem (KVS), sprečavanjem nastanka ateroskleroze i endotelne i/ili vaskularne disfunkcije, a deluje i direktno na srce smanjujući njegovu hipertrofiju. Estradiol ostvaruje pozitivne efekte u KVS regulacijom azot-monoksid (NO)-sintaza (NOS), endotelne (eNOS) i inducibilne (iNOS) forme, koje su odgovorne za sintezu NO, aktivacijom sloţene mreţe unutarćelijskih signalnih puteva, u koje su uključeni signalni molekuli supstrat receptora za insulin 1 (IRS-1), fosfatidil-inozitol-3-kinaza (PI3K) i protein kinaza B (Akt). Estradiol utiče na signalnu transdukciju angiotenzina II (Ang II), delujući na ekspresiju receptora za Ang II tipa 1 (AT1R) i tipa 2 (AT2R), kao i na nivo RhoA proteina. Estradiol utiče i na transport energetskih supstrata regulišući translokazu masnih kiselina (CD36) i transportere za glukozu tipa 1 (GLUT1) i tipa 4 (GLUT4). Kontrolni i gojazni muţjaci pacova soja Wistar tretirani su estradiolom (40μg/kg) ili 1% etanolom 24 sata pre ţrtvovanja. Spektrofotometrijskim metodama određivane su koncentracije NO u plazmi i koncentracije L-Arginina (L-Arg), NO i slobodnih masnih kiselina (SMK) u lizatu srca. Western blot metodom određivani su nivoi proteina eNOS, iNOS, NF-κB-p65, pIRS-1/IRS-1, p85 i p110 subjedinice PI3K, pAkt/Akt, RhoA, AT1R, AT2R, GLUT1, GLUT4 i CD36 proteina, kao i asocijacija IRS-1 sa p85-PI3K proteinom u srcu pacova, dok je imunohistohemijskom metodom određivana ekspresija i lokalizacija eNOS i iNOS. Metodom qRT-PCR određivan je nivo iRNK za eNOS i iNOS u srcu pacova. Rezultati su analizirani korišćenjem Studentovog t-testa. Rezultati prikazani u ovoj doktorskoj disertaciji pokazuju da tretman estradiolom kod kontrolnih pacova povećava nivo p85 subjedinice PI3K i fosforilaciju Akt na Thr308, dok smanjuje ekspresiju gena za iNOS...

Estradiol has a positive effect on the cardiovascular system (CVS) by preventing the development of atherosclerosis and endothelial and/or vascular dysfunction. Estradiol exerts many of its effects on the CVS by regulation of nitric oxide synthase (NOS), endothelial (eNOS) and inducible (iNOS) forms that are responsible for the synthesis of nitric oxide (NO), through activation of the complex network of intercellular signal pathways that include signaling molecules: insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt.) Estradiol also affects the signal transduction of angiotensin II (Ang II) by affecting the expression of Ang II receptors: type 1 (AT1R) and type 2 (AT2R), as well as the level of RhoA protein. Finally, estradiol indirectly regulates the transport of energy substrates in heart, by regulating transporters of free fatty acids (FFA) and glucose, CD36 and glucose transporters type 1 (GLUT1) and type 4 (GLUT4). Control and obese male Wistar rats were treated with one dose of estradiol (40 μg/kg) or with the same amount of 1% ethanol in saline 24 hours before sacrificed. The concentration of NO was measured in the plasma and in lysates, while the concentrations of L-Arginine and FFA were determined in the lysates of the rats‘ hearts. The Western blot method was used to determine the level of eNOS, iNOS, NF-kB p65, IRS-1, p85 and p110 subunits of PI3K, Akt, AT1R, AT2R, RhoA, CD36, GLUT1 and GLUT4 in the rats‘ heart, as well as association of IRS-1 with p85 subunit of PI3K. Immunohistochemical analysis was used for localization and expression of eNOS and iNOS proteins in heart. qRT-PCR was used to determen the level of iRNA of eNOS and iNOS in rats‘ heart. Results were analyzed using Student‘s t test...