Molecular Mechanism Of Aspirin Eugenol Ester Against Vascular Endothelial Cell Oxidative Injury

On the basis of the oxidative stress of HUVECs induced by H₂O₂,the mechanism of AEE protecting vascular endothelial from oxidative injury was clarified by cell metabolomics techniques and molecular biology methods including western blotting,immunofluorescence,flow cytometry and gene silencing or overexpression.Moreover,in vivo,with the rat atherosclerosis model of rat established by feeding HFD,the protective effect of AEE on vascular endothelial injury was confirmed and its regulation mechanism was probed by biochemistry,immunohistochemistry or fluorescence techniques.1.Treating HUVECs with 300μM H₂O₂ for 6 h,there were 70%early apoptosis of HUVECs.Pre-treating HUVECs with different concentrations of AEE（0.5,1.0,2.0μM）,the H₂O₂-induced apoptosis of HUVECs significantly decreased.Feeding rats with HFD for 12 weeks,the levels of low density lipoprotein（LDL）,total cholesterol（TCH）,and advanced glycation endproducts（AGEs）in serum were significantly increased,while the level of high density lipoprotein（HDL）was significantly decreased.After treatment with AEE,the pathological changes of biochemical indicators were significantly ameliorated.The results of in vivo and in vitro pharmacodynamics suggested that AEE can protect HUVECs from H₂O₂-induced oxidative injury and prevent the development of HFD-induced atherosclerosis in rats.2.AEE can prevent effectively the H₂O₂-induced dysfunction of mitochondria and lysosomes by reducing ROS generation,increasing the membrane potential of mitochondria,enhancing the stability of lysosomal membranes,decreasing the activities of cathepsins,caspases3 and caspases9,and preventing the occurrence of apoptosis.The results of overexpression/silencing of Bcl2 suggested that AEE might regulate H₂O₂-induced mitochondrial and lysosomal dysregulation by regulating Bcl2 expression.3.AEE can reduce the accumulation of ROS and NO in the oxidative injury HUVECs.AEE prevents the overproduction of NO and the imbalance of Ca homeostasis and energy by reducing the expression of iNOS and the decoupling of eNOS in oxidative injury HUVECs.AEE can also increase the expression of Nrf2 in oxidative injury HUVECs,enhance the antioxidant capacity of HUVECs,and prevent effectively the occurrence of oxidative injury.Further,the results of overexpression/silencing of Nrf2 gene suggested that Nrf2 played a key role on AEE preventing NO imbalance of oxidative injury vascular endothelium.In vivo experiments also showed that AEE can reduce the expression of iNOS in the aorta induced by HFD.4.ROS and reactive nitrogen（RNS）act as inducers of cell adhesion factors,and AEE can prevent effectively the accumulation of ROS and RNS in injury cells.In order to further investigate whether AEE can affect the expression of adhesion factors in oxidative injury vascular endothelium,the changes of adhesion factors in the vascular endothelium were detected among different treatment groups in vivo and in vitro,respectively.The results showed that AEE could significantly reduce the expression of E-selectin induced by H₂O₂ and decrease adhesion of HUVECs to human acute leukemia cell line（THP-1）.Besides,AEE could ameliorate effectively the accumulation of soluble vascular cell adhesion molecule 1（sVCAM-1）,soluble E-selectin（sE-selectin）in serum and overexpression of VCAM-1 and E-selectin in the aorta induced by HFD.5.The results of cells and its culture supernatant metabolomics suggested that the metabolic profiles of AEE group,normal group and H₂O₂ group were distinct.AEE might perform the function of antioxidant injury by interfering with amino acid metabolism,energy metabolism,glutathione metabolism,cofactor and vitamin metabolism.In a conclusion,AEE might be against vascular endothelial dysfunction by enhancing Nrf2 and Bcl2 expression in vascular endothelial cells,improving endothelial antioxidant capacity,reducing ROS and RNS accumulation,and decreasing expression of vascular endothelial cell adhesion molecules.