Josip Juraj Strossmayer University of Osijek. Faculty of Medicine.
Abstract
Cilj istraživanja: Istražiti modulira li hiperbarična oksigenacija (HBO) vaskularnu reaktivnost na angiotenzin II (ANG II) i angiotenzin-(1-7) (ANG-(1-7)) u zdravih i dijabetičnih štakora te ispitati ulogu epoksieikozatrienoičnih kiselina (EETs) i CYP enzima u modulaciji vaskularne reaktivnosti putem HBO. Također, cilj je bio provjeriti imaju li eventualne promjene arterijskog tlaka ili oksidativnog stresa ulogu u modulaciji vaskularnih odgovora. Metode: Istraživanje je provođeno ispitivanjem vaskularnih odgovora izoliranih torakalnih aortalnih prstenova (funkcionalne studije) na ANG II i ANG-(1-7) u zdravih ili dijabetičnih štakora (tretiranih protokolom s intermitentnom HBO u odnosu na netretirane) te uporabom visoko selektivnog inhibitora sinteze EETs (MS-PPOH), radi funkcionalnog ispitivanja uloge EETs. Nadalje, provedene su in vivo studije utjecaja HBO na arterijski tlak te molekularne studije utjecaja HBO na pokazatelje oksidativnog stresa, serumske koncentracije ANG-(1-7) (ELISA), mRNA ekspresije CYP enzima (kvantitativna PCR) i proteinske ekspresije CYP enzima (Western blot) u zdravih i dijabetičnih štakora. Pomoću streptozocina izazvan je animalni model dijabetesa tipa 1. u Sprague-Dawley štakora. Rezultati: HBO nije značajno mijenjala aortalnu reaktivnost na sam ANG II u zdravih i dijabetičnih štakora, ali je vaskularni odgovor na kombinaciju ANG II+ANG-(1-7) bio značajno niži u odnosu n ANG II kontrakciju u zdravih HBO štakora, dok takve razlike nije bilo u netretiranih štakora. HBO je značajno pojačavao aortalni odgovor na sam ANG-(1-7) (nakon prekontrakcije noradrenalinom) u zdravih i dijabetičnih životinja. MS-PPOH poništio je sve učinke HBO na facilitaciju vaskularnih odgovora na ANG-(1-7). Nije bilo značajnih promjena arterijskog tlaka, oksidativnog stresa ili serumske razine ANG-(1-7). HBO je imala tendenciju povećavati ekspresiju CYP enzima, a značajno povećanje mRNA ekspresije izmjereno je za CYP4A1 u zdravih i CYP2J3 u dijabetičnih. Proteinska ekspresija bila značajno povećana za CYP2C11 u dijabetičnih štakora. Zaključak: HBO značajno pojačava vaskularni odgovor na ANG-(1-7), što može objasniti njezine pozitivne učinke u patološkim stanjima. EETs imaju važnu ulogu u posredovanju ove modulacije. HBO povećava ekspresiju pojedinih CYP izoformi, a moguće je da povećava i osjetljivost žile na EETs. Promjene vaskularne reaktivnosti nisu posljedica promjena arterijskog tlaka, oksidativnog stresa ili razine ANG-(1-7). HBO ne mijenja značajno reaktivnost na ANG II u zdravih niti u dijabetičnih štakora.Objectives: To investigate whether hyperbaric oxygenation (HBO) modulates vascular reactivity to angiotensin II (ANG II) and angiotensin-(1-7) (ANG-(1-7)) in healthy and diabetic rats and to assess the role of epoxy eicosatrienoic acids (EETs) and CYP enzymes in the modulation of vascular reactivity by HBO. Additionally, the goal was to examine if possible changes in arterial blood pressure and oxidative stress play a role in such modulation of vascular responses. Methods: The study was conducted using isolated thoracic aortic ring preparations to test vascular reactivity (functional studies) to ANG II and ANG-(1-7) in healthy and diabetic rats (treated with an intermittent HBO protocol compared to nontreated), and using a highly selective inhibitor of EETs synthesis (MS-PPOH) to assess the functionl role of EETs. Furthermore, in vivo studies of HBO effects on arterial blood pressure were conducted, as well as molecular studies of the effects of HBO on oxidative stress indicators, serum concentration of ANG-(1-7), mRNA expression of CYP enzymes (quantitative PCR) and protein expression of CYP enzymes (Western blot) in healthy and diabetic rats. With the use of streptozocin, an animal model of type 1 diabetes was caused in Sprague-Dawley rats. Results: HBO did not significantly change aortic reactivity to ANG II alone in healthy and diabetic rats, but vascular responses to the combination of ANG II + ANG-(1-7) was significantly lower compared to ANG II contractions in healthy HBO rats, whereas such a difference was not present in untreated rats. HBO significantly increased aortic responses to ANG-(1-7) alone (after precontraction with noradrenaline) in healthy and diabetic animals. MS-PPOH reversed all effects of HBO on facilitation of vascular responses to ANG-(1-7). There were no significant changes in arterial pressure, oxidative stress and serum ANG-(1-7) levels. HBO had a tendency to increase CYP enzyme expression, and a statistically significant increase of mRNA expression was measured for CY4A1 in healthy and CYP2J3 in diabetic rats, while protein expression was significantly increased by HBO for CYP2C11 in diabetic rats. Conclusion: HBO significantly increases vascular responses to ANG-(1-7) , what could explain some of the observed positive effects of HBO in pathologic conditions. EETs seem to play an important role in the mechanism of this modulation. HBO increases the expression of specific CYP isoforms, but it is also possible that it increaees the vascular sensitivity to EETs. The changes in vascular reactivity were not a consequence of a possible change in arterial pressure, oxidative stress or ANG-(1-7) blood levels. HBO does not significantly alter reactivity to ANG II in either healthy or diabetic rats
