The Synergistic and Neuroprotective Effects of Alcohol–Antioxidant Treatment on Blood–Brain Barrier Endothelial Cells

Abstract

Background: Alcohol (EtOH) is reported to adversely affect one of the most crucial roles of the blood-brain barrier (BBB), the regulation of its permeability, thereby compromising the stability of the homeostatic environment of the brain. The central component of the BBB, endothelial cells (ECs), regulates BBB transcellular transport, while their paracellular pathways are made virtually impermeable by molecular structures called tight junctions (TJs). These TJs are composed of proteins, such as claudin-5, a protein involved in the regulation of paracellular permeability and of key interest in this study. Methods and Results: The working hypothesis of this study postulated that the high levels of antioxidants (AOs) in the fermented Aspalathus linearis (Rooibos; Rf) tincture may protect the ECs of the BBB against oxidative stress induced by EtOH exposure. Cells were exposed for 24 hours to selected concentrations of EtOH (25 and 100 mM), Rf (containing an antioxidant equivalence of 1.9 nM Aspalathin), and cotreatments of EtOH and Rf. Cell viability, live cell number, and toxicity were analyzed using the trypan blue exclusion assay. RT-qPCR was implemented to quantify claudin-5 transcription. In addition, permeability (Transepithelial Electrical Resistance) of bEnd5 monolayers was measured. The experimental timeline for the above-mentioned parameters was 24 and 48 hours. Conclusions: Our study showed that simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability and cell proliferation, but was not able to reverse or reduce the effects of EtOH on claudin-5 transcription and paracellular permeability. Furthermore, a novel finding in this study suggests that very low concentrations of AOs in tinctures such as Rooibos tea could profoundly alter the redox status of brain ECs.

Transcriptomic‐based toxicological investigations of ethanol to human umbilical vein endothelial cells

“…HUVECs were seeded at a density of 4 × 10 4 per well in 24‐well plates and incubated with 5‐, 10‐, 25‐, 50‐, or 100‐mM ethanol (purchased from Sigma‐Aldrich, USA). The highest concentration (100 mM) is comparable with previous in vitro studies (Boncler, Lukasiak, Dastych, Golanski & Watala, 2019; Fisher, Thomas & Abdul‐Rasool, 2020; Mishra, Agas, Schuetz, Kalluru & Haorah, 2020; Xue et al, 2018). Assuming the highest ethanol consumption as 9.8 L per year (World Health Organisation, 2018) and blood volume of adults as 5 L, the ethanol level in blood every day is approximately 92 mM, close to the highest concentration used in this study.…”

“…We used two concentrations. The concentration of 50 mM has been used in previous toxicological studies (Boncler, Lukasiak, Dastych, Golanski & Watala, 2019; Devall et al, 2020; Fisher, Thomas & Abdul‐Rasool, 2020; Mishra, Agas, Schuetz, Kalluru & Haorah, 2020; Xue et al, 2018; Zhu et al, 2017) and could be reached after daily consumption of ethanol according to the calculation described in Section 2. A much lower concentration 5 mM was also used, because researchers suggested that low levels of ethanol could induce hormesis (Calabrese & Baldwin, 2003; Hayes, 2007).…”

“…Interestingly, numerous studies found that environmental toxicants, for example, mercury (Farkhondeh, Afshari, Mehrpour & Samarghandian, 2020), coronavirus (Hayden, 2020), and nanomaterials (Cao et al, 2017), induced toxicological responses to endothelial cells leading to CVD development. Previous studies also revealed that high levels of ethanol induced toxic effects in endothelial cells (Boncler, Lukasiak, Dastych, Golanski & Watala, 2019; Fisher, Thomas & Abdul‐Rasool, 2020; Mishra, Agas, Schuetz, Kalluru & Haorah, 2020; Xue et al, 2018). However, most of the studies were based on traditional toxicological methods, which focused on a specific signaling pathway but may fail to provide the information about the systemic changes in endothelial cells after ethanol exposure.…”

Aspalathus linearis (Rooibos) and Agmatine May Act Synergistically to Beneficially Modulate Intestinal Tight Junction Integrity and Inflammatory Profile

“…Similarly, Fisher et al (2020) reported protective effects of an aspalathin-rich rooibos tincture in an in vitro BBB model. Notably this study utilised bEnd5 cells, which do not express the TJP occludin [62]. In our study, an ethanol extract of green rooibos decreased the total occludin signal significantly under baseline conditions (absence of TA treatment), which, in the context of the report from Fisher et al (2020), suggests more than one potential mechanism of action for rooibos-related beneficial modulation of tightjunction barriers.…”

Translation of preclinical ethnomedicine data in LMICs: the example of rooibos

“…Although rooibos is anecdotally acclaimed for its calming effect few studies have investigated the effect of rooibos on the brain and nervous system and associated behavioural outcomes. Somewhat disappointingly, most in vitro studies in this context–albeit with positive results–seem largely focused on antioxidant effects ( Hong et al, 2014 ; Fisher et al, 2020 ; Ma et al, 2020 ; Lopez et al, 2022 ; Brasil et al, 2023 ). Only two of these expanded their investigation and also reported different modulatory mechanisms in the context of serotonin metabolism ( Hong et al, 2014 ; Lopez et al, 2022 ), which provides some additional mechanistic support for an anxiolytic or calming effect.…”