Abstract
Background: Empirical observations have shown that ozonated autohemotherapy markedly improves the symptoms of chronic limb ischemia (muscular pain at rest, intermittent claudication, etc ) in atherosclerotic patients, but mechanisms of action remain unclear.Aims: Human endothelial cells (HUVECs) are known to release nitrogen monoxide (NO) and we investigated the biological effects of human ozonated serum on HUVECs in culture.Methods: We assessed the relevance of peroxidation, the release of NO as nitrite and of three classical cytokines.Results: The treatment of HUVECs with ozonated serum yields a dose dependent increase of thiobarbituric acid reactive substances (TBARS) and of hydrogen peroxide (H2O2) and a decrease of protein thiol groups (PTG). Concomitantly, in comparison to either the control or the oxygenated sample, there is a significant and steady increase of nitric oxide (NO) production; this is markedly enhanced by the addition of L-arginine (20 μM) and inhibited in the presence of the NO inhibitor, L-NAME (20 mM). The main mediator of ozone action is H2O2 as it has been shown either after its direct measurement or by the addition of 20, 40 and 100 μM. Moreover, during 24 hours incubation we have investigated the production of endothelin 1 (ET-1), E-selectin and Interleukin 8 (IL-8) and it appears that ozonation enhances IL-8, inhibits E-selectin and hardly modifies ET-1 production.Conclusions: It appears that reinfusion of ozonated blood, by enhancing release of NO, may induce vasodilation in ischemic areas and reduce hypoxia.