Signalni putevi estradiola uključeni u regulaciju ekspresije i aktivnosti inducibilne azot-monoksid-sintaze i natrijum-kalijum adenozintrifosfataze u srcu gojaznih ženki pacova

Abstract

Gojaznost je oboljenje povezano sa nizom patoloških stanja kao što su: rezistencija na insulin (IR), kardiovaskularne bolesti (KVB) i Diabetes Mellitus tipa 2 (DMT2). Povećana ekspresija i aktivnost inducibilne azot-monoksid-sintaze (iNOS; engl. Inducible Nitric Oxide Synthase) u srcu u stanju gojaznosti, moţe dovesti do apoptoze kardiomiocita i hipertrofije srca, dok sa druge strane gojaznost zdruţena sa IR doprinosi smanjenoj aktivnosti Na+/K+-ATPaze, što dovodi do smanjenja kontraktilnosti vaskulature i razvoja sistemske hipertenzije. Endogeni estradiol svojim delovanjem sprečava nastanak IR i hiperglikemije i ostvaruje pozitivne efekte na kardiovaskularni sistem (KVS), ali sinteza i kardioprotektivni uticaj estradiola mogu biti smanjeni usled razvoja gojaznosti. Estradiol ostvaruje kardioprotektivne tako što utiče na smanjenje ekspresije i aktivnosti iNOS, kao i povećanje ekspresije i aktivnosti Na+/K+-ATPaze, posredstvom različitih signalnih molekula i kinaza, kao što su: supstrat receptora za insulin 1 (IRS-1; engl. Insulin Receptor Substrate)/ fosfatidil-inozitol-3-kinaza (PI3K; engl. Phosphatidylinositide 3-Kinase)/ protein kinaza B (Akt; engl. Protein Kinase B), ekstracelularnim signalima regulisane kinaze 1 i 2 (ERK1/2; engl. Extracellular Signal-Regulated Protein Kinases 1 i 2) kao i RhoA (engl. Ras homolog gene family, member A)/ROCK (engl. Rho-associated protein kinase). Pored direktnog efekta na srce, estradiol posredno reguliše i njegovu funkciju tako sto utiče na metabolizam i transport glukoze i SMK, preko transportera glukoze (GLUT; engl. Glucose Transporters) i translokaze masnih kiselina (FAT; CD36 ; engl. Fatty Acid Translocase). Za izradu ove doktorske disertacije je korišćeno 16 adultnih ţenki pacova soja Wistar, podeljenih u dve eksperimentalne grupe. Prva grupa pacova je tokom 10 nedelja hranjena standardnom laboratorijskom hranom za pacove, dok je druga grupa pacova tokom 10 nedelja hranjena standardnom laboratorijskom hranom obogaćenom sa 42% masti (HF reţim ishrane). Nakon 10 nedelja pacovi su ţrtvovani, sakupljena je krv i izolovan je serum, a srca su ekstrahovana i delovi tkiva su korišćeni za izolovanje proteina i RNK. U serumu pacova je odreĎivana koncentracija estradiola, dok su u lizatu srca pacova odreĎivane koncentracije L-Arginina (L-Arg), NO i slobodnih masnih kiselina (SMK). Metodom qRT-PCR odreĎivan je nivo iRNK iNOS u srcu pacova...

Obesity is associated with several pathological conditions such as: insulin resistance (IR), cardiovascular disease (CVD) and Diabetes Mellitus type 2 (DMT2). Obesity related inducible nitric oxide synthase (iNOS) overexpression could lead to cardiac hypertrophy, while on the other hand obesity induced IR contributes to the reduced Na+/K+-ATPase activity, leading to vascular contractility impairment and development of systemic hypertension. Endogenous estradiol prevents IR and hyperglycaemia and has protective effects on the cardiovascular system (CVS), but the synthesis and cardioprotective effect of estradiol can be reduced due to the development of obesity. Estradiol exerts cardioprotective effects by reducing the expression and activity of iNOS, as well as by increasing the expression of Na+/K+-ATPase activity by regulating the activity of signaling molecules and kinases, such as: insulin receptor substrate 1 (IRS-1)/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt, PKB), extracellular signal regulated kinase 1 and 2 (ERK1/2) as well as RhoA (Ras homolog gene family, member A)/ROCK (Rho-associated protein kinase). Additionally, estradiol indirectly regulates CVS function by influencing the metabolism and transport of glucose and free fatty acids (FFA), through glucose transporter (GLUT) and FAT; CD36 (Fatty Acid Translocase). In this doctoral dissertation 16 adult female Wistar rats were used and divided into two experimental groups. The first group of rats was fed a standard chow for laboratory rats for 10 weeks, while the second group of rats was fed a standard chow for laboratory rats enriched with 42% fat (HF diet) for 10 weeks. After 10 weeks, the rats were sacrificed, blood was collected and the serum isolated, the hearts were collected, and its tissue fragments were used to isolate proteins and RNA. The concentration of estradiol was measured in serum, while concentrations of L-Arginine (L-Arg), NO and free fatty acids (FFA) were measured in the heart lysates. qRT-PCR method was used to measure the level of iNOS mRNA in rats’ hearts. Na+/K+-ATPase was measured in plasma membranes of rats’ hearts. Western blot was used to measure the level of the following proteins: iNOS, NFkB-p50, ERα, p85 and p110 subunits of PI3K, NDRG2, RhoA, ROCK1 and ROCK2 in heart lysates. The level of phosphorylation and expression of Akt and ERK1/2 kinases, as well as the IRS-1/PI3K associations, were determined in heart lysates...