Ellagic acid inhibits oxidized LDL-mediated LOX-1 expression, ROS generation, and inflammation in human endothelial cells.
BACKGROUND
LOX-1, a lectin-like receptor on endothelial cells, facilitates the uptake of oxidized low-density lipoprotein (oxLDL). Expression of LOX-1 is involved in the pathobiological effects of oxLDL in endothelial cells, including reactive oxygen species (ROS) generation, suppression of endothelial nitric oxide synthase (eNOS) activity, and leukocytic adhesion. Moderate consumption of phenolic-enriched food may have a protective effect against the development of atherosclerosis via the antioxidant capacity of phenolic compounds at the endothelial level. In this study, we determined whether ellagic acid, a polyphenolic compound widely distributed in fruits and nuts, protects against oxLDL-induced endothelial dysfunction by modulating the LOX-1-mediated signaling pathway.


METHODS
Human umbilical vein endothelial cells (HUVECs) were pretreated with ellagic acid at doses of 5, 10, 15, and 20 μM for 2 hours and then incubated with oxLDL (150 μg/mL) for an additional 24 hours.


RESULTS
LOX-1 protein expression was markedly lower after exposure to oxLDL in HUVECs pretreated with ellagic acid or diphenyleneiodonium, a well-known inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, than in HUVECs exposed to oxLDL alone, suggesting that ellagic acid deactivates NADPH oxidase. We also found that oxLDL activated the membrane assembly of p47phox, Rac1, gp91 and p22phox, and the subsequent induction of ROS generation; however, ROS generation was markedly suppressed in cells pretreated with ellagic acid or anti-LOX-1 monoclonal antibody. In addition, oxLDL down-regulated eNOS and up-regulated inducible NO synthase (iNOS), thereby augmenting the formation of NO and protein nitrosylation. Furthermore, oxLDL induced the phosphorylation of p38 mitogen-activated protein kinase, activated the NF-κB-mediated inflammatory signaling molecules interleukin-(IL) 6 and IL-8 and the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin, and stimulated the adherence of THP-1 (a human acute monocytic leukemia cell line) to HUVECs. Pretreatment with ellagic acid, however, exerted significant cytoprotective effects in all events.


CONCLUSIONS
Findings from this study may provide insight into a possible molecular mechanism by which ellagic acid inhibits LOX-1-induced endothelial dysfunction. Our data indicate that ellagic acid exerts its protective effects by inhibiting NADPH oxidase-induced overproduction of superoxide, suppressing the release of NO by down-regulating iNOS, enhancing cellular antioxidant defenses, and attenuating oxLDL-induced LOX-1 up-regulation and eNOS down-regulation.
Lee
Wen-Jane
WJ
Department of Medical Research, Taichung Veterans General Hospital, Tunghai University, Taichung, Taiwan.
Ou
Hsiu-Chung
HC
Hsu
Wen-Cheng
WC
Chou
Min-Min
MM
Tseng
Jenn-Jhy
JJ
Hsu
Shih-Lan
SL
Tsai
Kun-Ling
KL
Sheu
Wayne Huey-Herng
WH
D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004610:Ellagic Acid / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007249:Inflammation / Q000276:immunology / Q000378:metabolism / Q000517:prevention & control*; D018836:Inflammation Mediators / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism*; D051127:Scavenger Receptors, Class E / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D000975:Antioxidants; C508606:CXCL8 protein, human; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; C508600:IL6 protein, human; D018836:Inflammation Mediators; D015850:Interleukin-6; D016209:Interleukin-8; D008077:Lipoproteins, LDL; D016328:NF-kappa B; C495137:OLR1 protein, human; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D051127:Scavenger Receptors, Class E; C099252:oxidized low density lipoprotein; D013481:Superoxides; D004610:Ellagic Acid; D009569:Nitric Oxide; C007517:diphenyleneiodonium; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019255:NADPH Oxidases; D048051:p38 Mitogen-Activated Protein Kinases