Bilberry anthocyanins prevent hyperoxia-induced alterations in human endothelial cells via MAPK phosphorylation

Exposure to elevated concentrations of inspired oxygen (hyperoxia) often is required in the treatment of acute respiratory failure. Increased ROS production, impaired substrate utilization by mitochondria and loss of mitochondrial integrity occur during hyperoxia and can contribute to impaired cell survival. Herein, the effects of polyphenol supplementation on endothelial cells under hyperoxic conditions have been studied. At this scope, a model employing cellular systems and mimicking the complex metabolism occurring in humans and was used (Canali et al., 2007). To this aim, we supplemented 3 healthy subjects (used as bioreactors) with 160 mg of a commercially available anthocyanin-rich berry extract and, after two hours, blood samples were withdrawn and serum (bioreactor-derived serum, BDS) used to enrich the medium of human umbilical vein endothelial cells (HUVECs) cultured in vitro under hyperoxic conditions (32% O2 for 24 h). The transcriptional activity, as shown by change in NRf2 nuclear translocation, appeared decreased in HUVECs under hyperoxic condition, but cell pretreatment with BDS was able to partially prevent these alterations. Interestingly, pretreatment with BDS was able to induce Nrf2 nuclear translocation also under normoxic conditions. These results were also confirmed by evaluating HO-1 gene expression, that is known to be activated by Nrf2. Furthermore we demonstrated that BDS was able to activate ERK1/2 both in normoxia than in hyperoxia. PD98059 (a selective pharmacological inhibitor of mitogen-activated protein kinase kinase, MAPKK) avoided Nrf2 nuclear translocation as well as ARE genes expression in BDS-tretead HUVECs so suggesting an important role for ERK1/2 in BDS protection. The results described herein can contribute, by means of a more physiological approach, to the understanding of molecular mechanisms involved in hyperoxia-induced endothelial alterations and in protective effect of plantderived active