טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShai Yehosua Schubert
SubjectPlant Derived Antioxidants - Isolation, Characterization
and their Effect on NFkB Activation in Vascular
Endothelial Cells
DepartmentDepartment of Biotechnology
Supervisors Ms. Resnick Nitzan
Mr. Neeman Ishak (Deceased)


Abstract

Background: Many vascular diseases result from or accompanied by inflammatory processes, where the activation of the transcription factor Nuclear Factor k B (NFkB) plays a pivotal role. This activation results in the induction of inflammatory genes, is accompanied by elevation in reactive oxygen species and can be inhibited by preconditioning with antioxidants. Methods and results: In the present study the antioxidants pomegranate wine, red wine and N-Acetyl Cysteine (NAC) were tested for their ability to inhibit NFkB activation in vascular endothelial cells, and a novel mechanism of this inhibition is reported. Minute concentrations of pomegranate or red wine significantly reduced the oxidation of 2’,7’-dichloroflurescin diacetate (H2DCFDA) in TNFa treated endothelial cells. The reduced oxidative activity was accompanied by inhibition of TNFa or shear stress mediated p65 nuclear translocation, as well as inhibition of p65 DNA binding. Surprisingly, neither serine phosphorylation on residue 32 nor degradation of the inhibitor of k Ba (IkBa) was inhibited by pomegranate wine or NAC, in TNFa treated endothelial cells. Treatment of the cells with these antioxidants alone lead to enhanced IkBa serine phosphorylation (on residues other then 32 and 36), which was not accompanied by IkBa degradation, or NFkB nuclear translocation. Furthermore, NAC but not PW inhibited the phsophorylation of TNFa -mediated p65 serine 536 phosphorylation. Conclusion: The three antioxidants tested - pomegranate wine, red wine and NAC are potent inhibitors of TNFa or shear stress mediated NFkB activation in vascular endothelial cells. While both NAC and PW had no effect on IkBa phosphorylation or degradation, NAC alone inhibited TNFa-mediated p65 phosphorylation. Thus, these antioxidants act in the cells through unique but divergent pathways.