Abstract
BackgroundDysfunction of vascular endothelium is implicated in many pathological situations. Cytoskeleton plays an importance role in vascular endothelial permeability barrier and inflammatory response. Many Chinese herbs have the endothelial protective effect, of which, “Astragalus membranaceus” is a highly valued herb for treatment of cardiovascular and renal diseases in traditional Chinese medicine, In this study, we tested whether calycosin-7-O-β-D-glucoside (Calycosin), a main effective monomer component of “Astragalus membranaceus”, could protect endothelial cells from bacterial endotoxin (LPS)-induced cell injury.MethodsEndothelial cell injury was induced by exposing human umbilical vein endothelial cells (HUVECs) to LPS. The effects of calycosin on LPS-induced changes in cell viability, apoptosis rate, cell migration, nitric oxide synthase (NOS), generationof intracellular reactive oxygen species (ROS) and cytoskeleton organization were determined. Microarray assay was employed to screen the possible gene expression change. Based on the results of microarray assay, the expression profile of genes involved in Rho/ROCK pathway and AKT pathway were further evaluated with quantitative real-time RT-PCR or western blot methods.ResultsCalycosin improved cell viability, suppressed apoptosis and protected the cells from LPS-induced reduction in cell migration and generation of ROS, protein level of NOS at a comparable magnitude to that of Y27632 and valsartan. Similar to Y27632 and valsartan, Calycosin, also neutralized LPS-induced actomyosin contraction and vinculin protein aggregation. Microarray assay, real-time PCR and western blot results revealed that LPS induced expression of FN, ITG A5, RhoA, PI3K (or PIP2 in western blotting), FAK, VEGF and VEGF R2, and inhibited expression of MLCP. We believed multiple pathways involved in the regulation of calycosin on HUVECs. Calycosin are considered to be able to activate MLCP through promoting the generation of NO, decreasing PMLC, suppressing the cytoskeleton remodeling caused by activation of Rho/ROCK pathway and inhibiting AKT pathway by decreasing VEGF, VEGF R2 and PI3K level.ConclusionCalycosin protected HUVEC from LPS-induced endothelial injury, possibly through suppression of Rho/ROCK pathway and regulation of AKT pathway.
Highlights
Dysfunction of vascular endothelium is implicated in many pathological situations
Endothelial cells of different sources are heterogeneous, human umbilical vein endothelial cells (HUVECs) are shown to respond to LPS and are extensively used for exploration of the mechanism implicated in endothelial cell injury [4]
HUVECs were preincubated for 30 min with the 10 μg/ml calycosin-7-O-β-D-glucoside
Summary
Dysfunction of vascular endothelium is implicated in many pathological situations. Cytoskeleton plays an importance role in vascular endothelial permeability barrier and inflammatory response. The disruption of semipermeable barrier of endothelial cell (EC) during inflammatory states causes extravasation of endovascular liquid, tissue edema, thrombosis and aggravated atherosclerosis [2]. Cytoskeleton plays a central role in maintaining the integrity of endothelial structure, vascular permeability barrier and cell signaling transduction. The endothelial cytoskeleton damage has been reported in various pathologic situations, including inflammation, oxidative stress and abnormal hemodynamics. Bacterial endotoxin (LPS), one of the major proinflammatory constituents of the cell walls of gram-negative bacteria, induces inflammatory processes, oxidative stress and production of cytokines. LPS activates vascular endothelial cells and induces leukocyte infiltration within the vascular wall and promote vascular permeability [3]. Endothelial cells of different sources are heterogeneous, human umbilical vein endothelial cells (HUVECs) are shown to respond to LPS and are extensively used for exploration of the mechanism implicated in endothelial cell injury [4]
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