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Changes in baseline flow-induced dilation response and interdependence of gene expression caused by the high salt intake in Sprague- Dawley rats

Previous studies have shown that acute salt intake (high salt diet with 4% NaCl), impairs vascular reactivity of blood vessels. High salt intake caused decreased protein expression of antioxidant enzymes, e.g. superoxide dismutase isoforms (McEwen ST. Time-Course and Mechanisms of Restored Vascular Relaxation by Reduced Salt Intake and Angiotensin II Infusion in Rats Fed a High-Salt Diet. Microcirculation 2009 ; 16(3):220- 234.) which also results in increased levels of oxidative stress. Effects of high salt on flow-mediated dilation, as well as the role of particular genesand their interdependencein the development of endothelial dysfunction in middle cerebral arteries (MCA) are not completely understood. The objective of this study is todeterminethe interconnection of genes and whether salt intake changes the interdependence of expression of genes important in the mechanisms of the flow induced dilatation in MCA of rats. 11-weeks old healthy male SD rats were divided in low salt (LS) group (N=16) fed with standard rat chow (0.4% NaCl) and high salt (HS) group fed with high salt food (4% NaCl) for 1 week. After diet protocol and prior decapitation, rats were anesthetized with ketamin-chloride (75 mg/kg) and midazolam (2.5 mg/kg) and than sacrificed. First, MCA were isolated and cannulated for baseline vascular reactivity measurements in response to stepwise increase in pressure (Δ10–Δ100 mmHg). The rest of brain blood vessels was taken for determination of mRNA expression of superoxide dismutase isoforms (Cu/Zn SOD, MnSOD, EC-SOD), cyclooxygenase 1 and 2 (COX1 and COX2), hypoxia inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and glutathione-peroxidase 1 and 4 (GPx1 and GPx4) by Real-time PCR. Differences in gene expression between the groups were determined by t test (SigmaPlot 11.0.) and mutual connection between the investigated genes with Pearson’s correlation coefficient r (MedCalc 10.2.0.0.) Results of functional studies were tested with MANOVA test and analyzed using statistical program IBM SPSS Statistics 20. Results are presented as mean ± SEMand p<0.05 was considered significant. All experimental procedures conformed to the European Guidelines for the Care and Use of Laboratory Animals (directive 86/609) and were approved by the local Ethical Committee. Baseline measurements show significantly decreased flow-induced dilation (FID) of MCA in HS diet group of rats(p = 0.007). 1 week of HS resulted in significantly decreased mRNA level ofCOX1 (p=0.039), COX2 (p=0.011), iNOS (p=0.004) and GPx4 (p=0.041) compared to LS group. Relative expression of Cu/Zn SOD, MnSOD, EC-SOD, GPx1, HIF-1α, VEGF and eNOS genes did not changed significantly between this two groups. In LS group MnSOD positively correlated with GPx1 (r=0.904, p=0.01) and COX1 (r=0.802, p=0.05). VEGF positively correlated with iNOS (r= 0.859, p=0.02) but negatively withHIF-1α (r=-0.903, p= 0.03). Cu/Zn SOD, EC-SOD, eNOS, COX2 and GPx4 are not correlated with any of the tested genes in this group of animals. In HS group, MnSOD positively correlated with COX1 (r= 0.943, p=0.005), GPx1 (r=0.960, p=0.001) and HIF-1α (r=0.935, p=0.002). HIF-1α also positively correlated with GPx1 (r=0.834, p=0.02), GPx1 with COX1 (r=0.818, p=0.04) and GPx4 with VEGF (r=0.960, p=0.01). Cu/Zn SOD, EC-SOD, COX2 and iNOS have not reached statistical significance of correlation with any of the tested genes. High salt intake impairs vasodilation of middle cerebral arteriesand leads to changes in gene expression and their interdependence. Antioxidant enzymes (MnSOD and GPx 1 and 4) are significantly positively correlated with genes essential for maintenance of normal endothelial functions. It is also possible thatMnSOD and glutathione-peroxidases have more important role in changed mechanisms compared to other tested antioxidant enzymes.

flow-induced dilation response ; endothelium ; high salt diet ; Sprague-Dawley rats

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