Di Maro, Martina (2017) Oleuropein effects on rat and human microcirculation. [Tesi di dottorato]

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Abstract

The aim of the present study was to investigate oleuropein effects on microvascular responses. First, we investigated the in vivo effects of oleuropein on rat pial microcirculation submitted to hypoperfusion-reperfusion injury. Therefore, we studied acute microvascular responses such as arteriolar vasodilation, permeability increase, leukocyte adhesion and capillary perfusion, by fluorescence microscopy. The working hypothesis was that this polyphenol may induce nitric oxide (NO) release from endothelial cells and consequently protect cerebral blood flow distribution and cerebral tissue. Rat cerebral cortical eNOS protein levels were evaluated as well as the impact of oxidative stress induced by hypopefusion and reperfusion on brain tissue, utilizing DCFH-DA. The second part of the study was aimed to evaluate oleuropein effects on skin microvascular blood flow oscillations of hyperlipidemic obese patients, by laser Doppler flowmetry (LDF). Therefore, hyperlipidemic obese females were administered with a hypocaloric and hypolipidic diet plus oleuropein for three months. These data were compared with the response of hyperlipidemic obese patients administered with hypocaloric and hypolipidic diet. Under baseline conditions and at the end of the study, nutritional status and lipid profile were evaluated as well as skin blood flow oscillations and reactive hyperemia by LDF. The results of the experimental study in rats indicate that oleuropein significantly improved in vivo microvascular responses after hypoperfusion-reperfusion injury. In particular, 20 mg/Kg b.w. of oleuropein induced a dilation by 28 ±2% of baseline (p < 0.01 vs. hypoperfused group) in order 3 arterioles and significantly reduced microvascular leakage (NGL: 0.13 ± 0.03; p < 0.01 vs. hypoperfused group) as well as leukocyte adhesion on venular walls (2.0 ± 0.5/100 µm v.l./30 sec; p < 0.01 vs. hypoperfused group), at the end of reperfusion. Moreover, this polyphenol was able to preserve capillary perfusion at the end of reperfusion (-26.0±4.5% of baseline; p<0.01 vs. hypoperfused group). These responses were associated to the increased eNOS expression in cortex and in striatum of treated animals. Oleuropein was also able to reduce neuronal damage and ROS production at the end of reperfusion, compared with hypoperfused animals. On the other hand, the results of the clinical study revealed that three months of hypocaloric and hypolipidic diet associated to oleuropein significantly improved nutritional status and lipid profile of hyperlipidemic obese patients. Total and LDL cholesterol, indeed, decreased by 15.0±1.2 and 16.5±1.3%, respectively, in patients treated with diet (OD group), and by 21.3±1.5 and 21.2±1.4%, respectively, in subjects treated with diet plus oleuropein (OL group). Moreover, laser Doppler measurements showed an increase in skin perfusion, compared to baseline conditions and control group (+25.6±1.4% of baseline), while the spectral analysis of skin blood flow oscillations revealed an increase in the NO-dependent and myogenic-related frequency components. Furthermore, PORH response improved in oleuropein-treated group, compared to controls. In conclusion, oleuropein appeared able to protect rat pial microcirculation from hypoperfusion-reperfusion injury increasing nitric oxide release from endothelial cells, reducing oxidative stress and, consequently, preserving pial blood flow distribution. Interestingly, this polyphenol showed beneficial effects also in humans; three months of hypocaloric and hypolipidic diet plus oleuropein increased smooth muscle cell functions and microvascular responses in hyperlipidemic obese patients, improving tissue perfusion.

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