Proceedings of The Physiological Society
Kings College London (2005) J Physiol 565P, PC157
Ability of phenolic compounds to protect the blood-brain barrier against oxidative stress
Patabendige, Adjanie AK; Youdim, Kuresh A; Abbott, N Joan;
1. Centre for Neuroscience Research, King's College London, London, United Kingdom.
The endothelial cells lining cerebral capillaries form the blood-brain barrier (BBB), and help to protect the brain from potentially damaging blood or brain derived substances (Abbott, 2002). Oxidative stress (OS) to endothelium as well as neurons may play a causative or exacerbating role in several neuropathologies including stroke (Gilgun-Sherki et al., 2001). Certain plant-derived phenolic compounds including flavonoids have the potential to protect neurones and endothelial cells from OS (Youdim et al., 2002), but there has been little work on their effects on the BBB. In this study we examined gallic acid lauryl ester (GAL) and two phenolic acids produced by the colonic microflora, 3-hydroxyphenylacetic acid (HPA) and 3-(3-hydroxyphenyl)-propionic acid (HPP) for their ability to protect brain endothelial cells from OS, using an immortalised mouse endothelial cell line bEND5. bEND5 cells (from ECACC) were seeded at 1.0 X 104 cells/200 μl medium per well in 96-well plates and grown to confluence. The cells were incubated with 0 to 1600μM H2O2 for 60 min to generate OS. Cytotoxicity/mitochondrial function was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. H2O2 caused a concentration-dependent induction of cytotoxicity, with over 50% cells viable at 200μM H2O2 (64.51±3.77% (mean ± s.e.m., n = 21). To test protective effects, different concentrations (2μM-200μM) of each phenolic compound were given either alone, with 200μM H2O2 (co-incubation) or before H2O2 (pre-incubation) for 60 min, followed by MTT assay. GAL decreased MTT reduction in a concentration-dependent manner, while HPA significantly increased MTT reduction (P<0.05, Student's t test). HPP had no significant effect. In co-incubation studies, all three compounds increased MTT reduction in a concentration-dependent manner, with HPA showing polyphasic effects (P<0.05). Pre-incubation with GAL resulted in a concentration-dependent decrease in MTT reduction, while HPA and HPP showed polyphasic protective effects (P<0.05). The results show that HPA and HPP significantly protected the cells from H2O2-induced OS, consistent with action by scavenging reactive oxygen species (ROS) and activating protective mechanisms. GAL was protective in co-treatment, but was toxic to cells when applied alone and when given as pre-treatment. These findings suggest that selected phenolic compounds have valuable protective potential against OS at the BBB, while GAL may be more suitable as an anti-tumour agent.
Where applicable, experiments conform with Society ethical requirements