Abstract
Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis.
Highlights
Pathogenesis of atherosclerosis and neo-intimal thickening post angioplasty involves excessive migration and proliferation of smooth muscle cells (SMCs) from media into the lumen of blood vessels
We investigated the role of G lutea root extract on platelet-derived growth factor (PDGF)-BB induced proliferation of primary cultures of rat aortic smooth muscle cells (RASMCs)
We first determined the concentration of the extract at which it will inhibit proliferation by 50% (IC50) for primary cultures RASMCs, rat and human specific aortic smooth muscle cell lines A7r5 and ATCC CRL-1999 respectively
Summary
Pathogenesis of atherosclerosis and neo-intimal thickening post angioplasty involves excessive migration and proliferation of smooth muscle cells (SMCs) from media into the lumen of blood vessels. Increased expression of several growth factors such as basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF)-BB contribute to atheroma formation [1] These agonists by activating the PI-3kinase and/or the mitogen activated protein kinase (MAPK) pathways promote migration and proliferation of vascular smooth muscle cells leading to their subsequent accumulation in the plaque [2,3]. Work done with purified isogentisin, has demonstrated that it protects endothelial cells from cigarette smoke induced cell death [12] while gentiopicriocide exhibits smooth muscle relaxing effects [13] These studies suggest that Gentiana species may have beneficial cardio-vascular effects the molecular mechanisms employed by these compounds are currently ill-understood. We examined the effects of G lutea extract on PDGF-BB induced cell cycle progression and signal transduction involving ERK1/2 activation and iNOS expression
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