Nur Nadia, Mohd Razali (2022) Protective Effects Of Alternanthera Sessilis Against Selected Pro-Inflammatory Mediators-Induced Oxidative Stress And Endothelial Activation In Human Aortic Endothelial Cells. Master dissertation/thesis, UTAR.
Abstract
Oxidative stress and activation of human aortic endothelial cells (HAEC) have been shown to contribute to the early stage of various pathological conditions such as atherosclerosis and hypertension. In these conditions, excess production of reactive oxygen species (ROS) and increased expression of cell adhesion molecules impair the endothelial barrier function, thereby increasing endothelial permeability. Tumor necrosis factor-α (TNF-α), a key player of atherogenesis, stimulates endothelial activation and oxidative stress in early atherosclerosis. Currently, statins which are widely used in the treatment of atherosclerosis by lowering cholesterol levels can cause adverse side effects and the treatment outcome is often unsatisfactory. Therefore, new therapeutic agents which target endothelial activation needs to be discovered for the prevention of early atherosclerosis. Alternanthera sessilis, popularly known as “sessile joyweed” or “daun keremak”, is a traditional herbal plant previously shown to possess anti-oxidant and anti-inflammatory effects. This study aimed to investigate protective effects of A. sessilis ethanolic extract on HAEC hyperpermeability, vascular cell adhesion-1 (VCAM-1) expression, ROS and hydrogen peroxide (H2O2) productions. Activities of anti-oxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) were also examined. To select a range of doses which were not cytotoxic, effects of 6.25 - 800 µg/ml of A. sessilis ethanolic extract on HAEC viability was first determined using 3-(4,5- iii Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Effects of A. sessilis on endothelial hyperpermeability was then evaluated using fluorescein isothiocyanate (FITC)- dextran permeability assay. Soluble VCAM-1 levels were assessed using enzyme-linked immunosorbent assay (ELISA). ROS and H2O2 productions were studied using 2',7'- dichlorodihydrofluorescein diacetate (H2-DCFDA) and Amplex Red fluorescent dyes, respectively. SOD and CAT activities were evaluated using commercial SOD and CAT assay kits. The MTT result showed that 25-200 µg/ml A. sessilis ethanolic extract did not cause significant HAEC death. A. sessilis ethanolic extract significantly inhibited TNF-α-induced hyperpermeability in HAEC at 200 µg/ml. The ROS assay also showed that 200 µg/ml of A. sessilis ethanolic extract significantly reduced TNF-α-induced increased ROS production in HAEC. However, A. sessilis failed to suppress increased H2O2 production induced by TNF-α. A. sessilis at all concentrations tested did not reduce increased VCAM-1 expression stimulated by TNF-α. Furthermore, A. sessilis ethanolic extract dramatically improved H2O2-induced decreased SOD and CAT activities. In conclusion, A. sessilis protects against selected proinflammatory mediator-induced endothelial activation and this is associated with inhibition of oxidative stress. This study reveals new knowledge on how A. sessilis suppresses endothelial activation and provides understanding on the mechanism by which A. sessilis protects HAEC against detrimental effects caused by TNF- α and H2O2.
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