P-1：Protective effects of eugenol against oxidized LDL-induced cytotoxicity and adhesion molecule expression in endothelial cells

Hsiu-Chung Ou, Fen-Pi Chou, Ching-Hwa Yang, Wayne H-H Sheu

Divison of Endocrinology and Metabolism, Department of Medical Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan

Background: Interaction between oxidized LDL (oxLDL) and endothelium plays a critical role in the initiation and progression of atherosclerosis.  Eugenol, a natural constituent of a number of aromatic plants and their essential oil fractions, has several biological effects.  However, its protective effects against endothelial injury remain unclarified.  In this study, we examined whether eugenol prevents oxLDL-induced vascular endothelial dysfunction.

Methods and Results:  Incubation of LDL with eugenol (100 M-12.5 M) inhibited copper-induced oxidative modification by using diene formation, thiobarbituric acid reactive substances (TABRS) assay and electrophoretic mobility assay.  Eugenol (100 M-12.5 M) reduced reactive oxygen species (ROS) generation in a dose-dependent manner (63%, 51%, 46% and 31% inhibitions, respectively; all P<0.01).  Additionally, 100 M eugenol inhibited oxLDL-induced surface expression of adhesion molecules, ICAM and VCAM, as well as E-selectin (179±14% to 133%±19%; 257±28% to 161%±31% and 268±33% to 160%±40%, respectively; all P<0.01), and adherence to human THP-1 monocytic cells (276±34% vs. 117±38%, 143±33%, 154±15%, respectively; P<0.01).  We also found that eugenol ameliorated the oxLDL-impaired endothelial NO synthase (eNOS) significantly at a lower concentration of 12.5 M by Western blotting.    Additionally, eugenol prevented mitochondrial dysfunction, membrane disturbance and apoptosis when HUVECs were exposed to cytotoxic concentration of oxLDL (200 g/ml), as manifested byMTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (21.9±1.5% vs. 99.7±2.1%, 103.7±4%, 95±2.2%, respectively; P<0.01), LDH release (354±10% vs. 102±11%, 106±3.5%, 120±10.6%, respectively; P<0.01) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain.

Conclusions:  We demonstrated that eugenol prevents the copper-induced oxidative modification of LDL and the oxLDL-induced ROS generation in HVUECs.  Eugenol also reduced the oxLDL-induced expression of adhesion molecules and adherence of THP-1 cells to HUVECs.  In addition, eugenol ameliorated the oxLDL-impaired eNOS protein expression and prevented apoptosis when HUVECs were exposed to cytotoxic concentration of oxLDL.  Our results support that eugenol may have clinical implications against atherosclerotic vascular disease.

Key words: endothelium, oxLDL, eugenol, adhesion molecule, apoptosis