RNS-mediated Damage To Endothelial Cell Line And The Protective Mechanisms Of Ginsenoside Rg1 On Beta-amyloid-induced RNS Damages

Chapter1 The mechanisms of ectogenesis RNS donor S-nitrosoglutathione (GSNO)-induced damage in human endothelial cellsThe course of Alzheimer's disease accompanied by a large generation of RNS,but the mechanisms of RNS-induced endothelial cell injury has not been clarified. Furthermore,the pathophysiological relevance of S-nitrosoglutathione(GSNO)-induced endothelial cell injury remains unclear.The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells.Morphological evaluation through DAPI staining and propidium iodide(PI) flow cytometry was used to detect apoptosis.In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade.When intracellular reactive oxygen species(ROS) production was monitored in GSNO-treated cells by diaminofluorescein-FM,we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential,indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway.Importantly,we found that peroxynitrite formation and omi/HtrA2 release from mitochondria were involved in this phenomenon,whereas changes of death-receptor dependent signaling were not detected in the same context.The inhibition of NADPH oxidase activation and omi/HtrA2 by a pharmacological approach provided significant protection against caspase 3 activation as well as GSNO-induced cell death,confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together,ROS overproduction and loss of mitochondrial omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death,and the modulation of these pathways can be of significant therapeutic benefit.Chapter2 The effect of RNS in Aβ-mediated damage to EA.hy926 endothelial cells and the protective mechanisms of ginsenoside Rg1The deposition of beta-amyloid(Aβ) in the brain parenchyma and cerebrovasculature has been characterized in Alzheimer's disease(AD),but the precise mechanisms of Aβ-mediated neuronal and endothelial insult remains unclear.In the present study,we attempted to evaluate the potential mechanisms of Aβ-mediated insult in endothelial cells as well as protective effect of Ginsenocide Rg1.In cultured EA.hy926 endothelial cells,Aβ_25-35(25μM) treatment induced cell toxicity, characterized by decreased cell viability,lactate dehydrogenase(LDH) release,and significant apoptotic characteristics,which is associated with both the overproduction of intracellular ROS and loss of mitochondrial membrane potential.By contrast, cytotoxicity of Aβ_25-35 was significantly reversed by pre-incubation of Rgl in endothelial cells.Furthermore,Rgl prevented Aβ_25-35-induced nitric oxide overproduction and protein tyrosine nitration in same context.Immunoblotting results revealed that upregulation of iNOS and nitrotyrosine,Bcl-2/Bax downregulation, Cytochrome C release and activation of caspase 3 in Aβ_25-35-treated cells were partially blocked by Rg1.Additionally,Aβ25-35 triggered the activation of p38-MAPK and inhibited the phosphorylation of ERK,and these effect were effectively suppressed by Rg1 treatment,whereas glucocorticoid receptor antagonist RU 486 and MEK inhibitor U0126 both weakened the protective effect of Rg1.Taken together,our results suggest that Rgl protects EA.hy926 endothelial cells against Aβ_25-35-induced apoptotic cascade through suppressing peroxynitrite formation and anti-oxidative mechanisms.Summary:1.We demonstrate that excessive ROS generation and peroxynitrite formation resulting from GSNO treatment accounts for initiation of GSNO/NO-induced endothelial injury in vitro.2.Our results indicate that NO-mediated intrinsic pathway signaling constitutes a critical component of apoptotic signaling in Aβ-mediated endothelial cell death.Rg1 is a potential endothelial protective compound against Aβ_25-35 insult which was exerted by suppressing protein tyrosine nitration and anti-oxidative mechanisms.