Abstract
BackgroundThe blood–brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics. Our group’s previous data has shown that tobacco smoke (TS) and electronic cigarettes (EC) affect the BBB integrity, increase stroke incidence, and are considered a risk factor for multiple CNS disorders. Metformin was also found to abrogate the adverse effects of TS and EC.MethodsWe used sucrose and mannitol as paracellular markers to quantitatively assess TS and EC’s impact on the BBB in-vitro. Specifically, we used a quantitative platform to determine the harmful effects of smoking on the BBB and study the protective effect of metformin. Using a transwell system and iPSCs-derived BMECs, we assessed TS and EC’s effect on sucrose and mannitol permeability with and without metformin pre-treatment at different time points. Concurrently, using immunofluorescence (IF) and Western blot (WB) techniques, we evaluated the expression and distribution of tight junction proteins, including ZO-1, occludin, and claudin-5.ResultsOur data showed that TS and EC negatively affect sucrose and mannitol permeability starting after 6 h and up to 24 h. The loss of barrier integrity was associated with a reduction of TEER values. While the overall expression level of ZO-1 and occludin was not significantly downregulated, the distribution of ZO-1 was altered, and discontinuation patterns were evident through IF imaging. In contrast to occludin, claudin-5 expression was significantly decreased by TS and EC, as demonstrated by WB and IF data.ConclusionIn agreement with previous studies, our data showed the metformin could counteract the negative impact of TS and EC on BBB integrity, thus suggesting the possibility of repurposing this drug to afford cerebrovascular protection.
Highlights
The blood–brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics
Evaluation of cell viability MTT cytotoxicity assay was performed to evaluate the impact of TSe and ECe 24 h exposure and the MF treatments (10 and 20 μM concentration) on our iPSCderived brain microvascular endothelial cells (BMECs) to rule out effect based on cell toxicity and the barrier viability
Vapor extracts, and MF on trans-endothelial electrical resistance (TEER) To assess the effect of tobacco smoke (TS) and electronic cigarettes (EC) extracts on BBB integrity, we adopted a transwell system extensively used as an in-vitro model of BBB for drug development and screening [54]
Summary
The blood–brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics. The BBB plays a vital role in protecting the brain parenchyma from potentially harmful substances, provides a dynamic, effective barrier to the entry of drugs and exogenous compounds into the CNS, and controls the bidirectional transport of biological substances needed to sustain brain metabolism and neuronal function [1]. Multiple studies have shown that tobacco smoke (TS) exposure negatively impacts the cerebrovascular system and BBB integrity [3,4,5,6,7,8], and it is considered a prodromal factor for the onset and progression of many neurological disorders. Chronic subcutaneous infusion of nicotine at a pharmacologically relevant dose produced a significant increase of BBB permeability in-vivo, associated with diminished expression of claudin-3 and altered distribution of ZO-1 mediated by endothelial nicotinic acetylcholine receptors [21]
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