Elderberry extract inhibits tumour necrosis factor induced monocyte adhesion to endothelial cells via modulation of the NF-κB pathway

Abstract

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): De Montfort PhD funded scholarship scheme Introduction During the early stages of atherosclerosis, monocytes attach to endothelial cells (EC) before differentiating into macrophages causing the accumulation of foam cells and plaque growth. NF-κB and vascular cell adhesion molecule 1 (VCAM-1) are some of the main mediators in EC that are involved in the monocyte adhesion process. Elderberry extract (EE) are rich in anthocyanins a subclass polyphenol with vascular-protective properties. Purpose The main of aim of this study is to examine if EE can prevent TNF-α induced inflammation and monocyte adherence to EC. Methods Primary Human Umbilical Vein Endothelial Cells (HUVEC) were pre-treated for 1-h and stimulated with or without TNF-α 10 ng/ml for western blot. Cell lysates from the treated cells were then subjected to Western blotting and probed for total and phospho-NF-κB. For monocyte adherence cells were pre-treated for 24-h, then stimulated with/or without TNF-α 10 ng/ml for 24-h. Fluorescently labelled THP-1 cells were then added to the HUVECs for an additional 30 minutes, and samples were measured in a fluorescence plate reader. Cells were put through flow cytometry for measuring reactive oxygen species (ROS) using DCFHFDA assay or VCAM-1 levels using Anti-CD106. Results An increase in ROS production and NF-κB phosphorylation was found after stimulation TNF-α 10 ng/ml (p = 0.01). However, cells pre-incubated with EE (50 μg/ml) for 1-hour before TNF-α stimulation caused a reduction in ROS as well as inhibition of NF-κB phosphorylation (p < 0.01). TNF-α 10 ng/ml increased the monocyte adherence to the HUVECs by a 2-fold although, EE prevented TNF-α monocyte adherence (mean value, 589.7 vs 408 p = 0.0033). This was associated with suppressed VCAM-1 expression found in the EE pre-treatment with TNF-α (p = 0.02). Discussion Our Preliminary data demonstrates that EE, can prevent monocytes binding onto EC potentially by inhibiting TNF-α induced NF-κB and VCAM-1 levels. Our findings postulate that NF-κB and VCAM-1 could be the direct link for targeting the prevention of monocyte adherence to EC, although this would need confirmation by blocking key cellular signalling pathways to confirm its role. This preliminary data suggests that EE and potentially other polyphenols could be a useful strategy for targeting the initial stages of atherosclerosis.