In vitro modulatory effects of fermented rooibos extract (Aspalathus linearis) against ethanol-induced effects on the mouse blood-brain barrier
Alcohol abuse is a growing crisis within South Africa, with severe health and socio-economic implications. Alcohol compromises the function of the blood-brain barrier (BBB), and thus its ability to regulate the homeostatic environment of the CNS is interrupted. In this study, an in vitro model of the BBB was utilized to study the effects of selected concentrations of alcohol (25mM-200mM) and the ameliorating effects of fermented rooibos (Aspalathus linearis) (0.003125%-1%), in an attempt to reverse the harmful oxidative effects of alcohol. The literature clearly states that alcohol (ethanol) compromises the BBB by reactive oxygen species (ROS) production and, therefore, rooibos, a shrub high in antioxidants and widely utilized nationally, was added to alcohol-exposed mouse brain endothelial (bEnd5) cells with the view to reverse the alcohol-induced effects on the BBB model. Alcohol-treated (25mM-400mM) bEnd5 monolayers expressed no toxicity, however, cell numbers were significantly suppressed (P<0.0274). To validate this finding, the activity of the mitochondria was investigated in order to understand if the cell’s metabolism was related to the decrease in cell division. Results showed that for both acute and chronic exposure there was a decrease in mitochondrial activity (MA) for a period of 24-48 hours, thereafter, the MA of the bEnd5 cells returned to normality. However, in experiments which chronically (600mM and 800mM) exposed cells to alcohol over a period of 96 hours, MA was suppressed and did not return to normal. Fermented rooibos caused a biphasic response to cellular proliferation at 24-72 hours, where the lower concentrations (0.0625-0.125 %) caused an increase in cellular proliferation and the higher concentrations (0.5-1%) resulted in a relative decrease in cellular proliferation. The long-term effect, after acute exposure, however, resulted in cell suppression at 96 hours (P<0.0073). With respect to the MA, bEnd5 cells exposed to fermented rooibos showed that lower concentrations (0.003125-0.0125%) were suppressed at 24 hours and was elevated at 48 hours and96 hours for all concentrations. The exception being the highest concentration (0.1%), which showed a depression in MA (P<0.05). Treating cells with both alcohol and rooibos, resulted in exacerbated suppressing of the MA. The physiological function of the BBB model was investigated by monitoring the permeability using transendothelial electrical resistance (TEER) studies and the in vitro model used in this study was endorsed for the first time using high resolution scanning electron microscopy. TEER indicated incidental changes in the permeability, only at 24 hours, for both acute and chronic exposure to alcohol and rooibos. A novel finding, within this study, was the increase in electrical resistance across the formation of the cell monolayer, after treatment with alcohol. The data lead to the hypothesis for the effect of ROS on resistivity and provides a rationale to explain the effects of combinatory treatments that were expected to ameliorate the negative effect of alcohol, however, this study showed synergistically negative effects on the bEnd5 cells. In summary the main findings in this study were: (a) alcohol was not toxic on bEnd5cells, (b) alcohol increased the permeability across monolayers of bEnd5 cells and(c) rooibos did not significantly reverse the ROS-induced effects of alcohol, but exacerbated the effects. Rooibos treatment caused the following: (i) biphasic effect on cellular proliferation, (ii) an increase in MA, and (iii) a cyclic effect in TEER studies.