Gene expression analyses of antioxidative enzymes in rats' tissues and human blood: the influence of external factors and correlation with biochemical markers of oxidative sress

Genska ekspresija je proces koji obuhvata transkripciju gena, obradu transkripta injegovu translaciju u protein. Kvantifikacijom genske ekspresije može se videti da lidolazi do ushodne ili nishodne regulacije specifičnih gena i posledica takve regulacije.Značajnu ulogu u zaštiti od oksidativnog stresa (prekomerne količine stvorenihslobodnih radikala) u intra- i ekstraćelijskom prostoru imaju enzimi antioksidativnezaštite, superoksid-dismutaza (SOD) i paraoksonaza (PON). Interakcije genaantioksidativne zaštite i fizičke aktivnosti, gena i tipa dijete, gena i terapijehipolipemicima mogu da modifikuju ekspresiju tih gena; fizička aktivnost slobodnimradikalima, aterogena dijeta i terapija atorvastatinom svojim komponentama, direktno iliindirektno interagujući sa transkripcionim faktorima.Cilj istraživanja je bio da se ispita uticaj fizičke aktivnosti na gensku ekspresijuizoenzima SOD (Cu/Zn SOD i Mn SOD) u limfocitima sportista i uticaj aterogene dijetei terapije atorvastatinom na gensku ekspresiju izoenzima PON (PON1, PON2, PON3) ujetri, belom masnom tkivu i aorti eksperimentalnih životinja. Pored toga, cilj je bio i dase uporede metode za izolaciju RNK iz krvi i ispita njihov uticaj na vrednosti genskeekspresije izoenzima SOD.Istraživanje je obuhvatilo odrasle osobe (kontrolna grupa), grupu fudbalera pre i nakon12 nedelja intenzivnog treniranja i eksperimentalne životinje koje su podeljene u 3grupe (1. grupa na normalnoj dijeti – ND grupa, 2. grupa na aterogenoj dijeti - AD i 3.grupa na aterogenoj dijeti i terapiji atorvastatinom - ADA grupa). RNK je izolovanaTRIzol™ metodom (TRI) iz mononuklearnih ćelija periferne krvi (TRIMĆPK) i naaparatu ABI Prism™ 6100 Nucleic Acid PrepStation (ABI) iz krvi (ABIKRV) imononuklearnih ćelija periferne krvi (ABIMĆPK). Takođe, RNK je izolovana pomoćuTRIzol™ metode iz ispitivanih organa pacova. Za kvantifikaciju relativne genskeekspresije korišćena je Real time PCR metoda.Fudbaleri su nakon 12 nedelja treniranja bili u oksidativnom stresu. Vrednosti genskeekspresije Cu/Zn SOD se nisu statistički značajno promenile (p=0,106), dok suvrednosti Mn SOD bile značajno veće (p=0,031) nakon treniranja. Vrednosti genske ekspresije Cu/Zn SOD su bile parametar koji nezavisno utiče na ukupnu aktivnost SODu plazmi (p=0,025), a koncentracija tiobarbituratna kiselina-reagujućih supstanci jeimala ulogu prediktivnog faktora (p=0,017) za vrednosti genske ekspresije Mn SOD...Gene expression is a process that includes transcription, transcript processing and itstranslation into protein. Gene expression quantification can be used to see weatherspecific genes are up or down regulated and consequences of such regulation. Enzymesof antioxidative defense system, superoxide dismutase (SOD) and paraoxonase (PON)have important roles in protection against oxidative stress (excessive amount ofgenerated free radicals) in intra- and extracellular space. Interaction of antioxidativedefense genes and physical activity, genes and type of diet, genes and hipolipemic drugscan modify the expression patterns of these genes; physical activity by free radicalsformation, atherogenic diet and atorvastatin therapy with its components, directly orindirectly interacting with transcription factors.The aim of this study was to investigate the influence of physical activity on SODisoenzymes’ (Cu/Zn SOD and Mn SOD) gene expression in sportsmen’ lymphocytesand the influence of atherogenic diet and atorvastatin therapy on paraoxonaseisoenzymes’ (PON1, PON2 and PON3) gene expression in liver, white adipose tissueand aorta of experimental animals. In addition, we compared methods for RNAisolation from blood and examined their influence on SOD isoenzymes’ geneexpression.The study included control group, soccer players before and after 12 weeks of intensivetrainings and rats who were divided into 3 groups (1. group on normal diet - ND group,2. group on atherogenic diet - AD group and 3. group on atherogenic diet withatorvastatin therapy- ADA group). RNA was isolated using Trizol™ method (TRI) fromperipheral blood mononuclear cells (TRIPBMC) and using ABI Prism ™ 6100 NucleicAcid PrepStation (ABI) from blood (TRIBLOOD) and from peripheral blood mononuclearcells (ABIPBMC). RNA was also isolated from rats’ organs using TRI method. Real timePCR was used for relative gene expression quantification.Soccer players were in oxidative stress after 12 weeks of intensive trainings. Cu/ZnSOD gene expression levels did not change significantly after training (p=0.106), whilethose of Mn SOD were significantly higher (p=0.031) after than before training. Cu/Zn SOD expression levels were independent parameter that influenced total SOD activityin plasma (p=0.025). Concentration of thiobarbituric acid reactive substances waspredictive parameter for Mn SOD gene expression values (p=0.017)..