Anti-atherogenic mechanism of ethanol extract of Christia vespertilionis (L.f.) Bakh. F. Leaves in vitro

Background: Vascular inflammation is the key event in early atherogenesis. Pro-inflammatory endothelial cells induce monocyte recruitment into the sub-endothelial layer of the artery. This requires endothelial expression of adhesion molecules namely intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), alongside chemokines production. Christia vespertilionis (L.f.) Bakh.f. (CV) possesses antiinflammatory property. However, its potential anti-atherogenic effect in the context of vascular inflammation has yet to be explored. Purpose: To evaluate the anti-atherogenic mechanism of 80% ethanol extract of CV leaves on tumor necrosis factor-alpha (TNF-alpha)-activated human umbilical vein endothelial cells (HUVECs). Methods: Qualitative analysis of the CV extract was carried out by using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The cell viability of HUVECs treated with CV extract was determined by MTT assay. The effect of CV extract on monocyte adhesion was determined by monocyte-endothelial adhesion assay. Protein expressions of ICAM-1, VCAM-1 and nuclear factor-kappa B (NF-kappa B) signaling pathway were determined by western blot while production of monocyte chemoattractant protein-1 (MCP-1) was determined by ELISA. Results: LC-MS/MS analysis showed that CV extract composed of five main compounds, including schaftoside, orientin, isovitexin, 6-caffeoyl-D-glucose, and 3,3 '-di-O-methyl ellagic acid. Treatment of CV extract at a concentration range from 5 to 60 mu g/mL for 24 h maintained HUVECs viability above 90 %, therefore concentrations of 20, 40 and 60 mu g/mL were selected for the subsequent experiments. All concentrations of CV extract showed a significant inhibitory effect on monocyte adhesion to TNF-alpha-activated HUVECs (p < 0.05). In addition, the protein expressions of ICAM-1 and VCAM-1 were significantly attenuated by CV in a concentration dependent manner (p < 0.001). At all tested concentrations, CV extract also exhibited significant inhibition on the production of MCP-1 (p < 0.05). Moreover, CV extract significantly inhibited TNF-alpha-induced phosphorylation of inhibitor of nuclear factor-kappa B kinase alpha/beta (IKK alpha/beta), inhibitor kappa B-alpha (I kappa B alpha), NF-kappa B and nuclear translocation of NF-kappa B (p < 0.05). Conclusion: CV extract inhibited monocyte adhesion to endothelial cells by suppressing protein expressions of cell adhesion molecules and production of chemokines through downregulation of NF-kappa B signaling pathway. Thus, CV has the potential to be developed as an anti-atherogenic agent for early treatment of atherosclerosis.