The 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. We 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. Our 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. In 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.

中文翻译：

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暴露于香烟烟雾和电子烟蒸气后体外 BBB 紧密连接完整性的比较评估：使用小分子量细胞旁标记物定量评估二甲双胍的保护作用

血脑屏障 (BBB) 在保护中枢神经系统 (CNS) 免受血源性病原体和可能有害的外源性物质侵害方面发挥着关键作用。我们小组之前的数据表明，烟草烟雾 (TS) 和电子烟 (EC) 会影响 BBB 的完整性，增加中风发生率，并被认为是多种中枢神经系统疾病的危险因素。还发现二甲双胍可以消除 TS 和 EC 的不利影响。我们使用蔗糖和甘露醇作为细胞旁标记来定量评估 TS 和 EC 对体外 BBB 的影响。具体而言，我们使用定量平台来确定吸烟对 BBB 的有害影响，并研究二甲双胍的保护作用。使用 transwell 系统和 iPSC 衍生的 BMEC，我们在不同时间点评估了 TS 和 EC 在使用和不使用二甲双胍预处理的情况下对蔗糖和甘露醇渗透性的影响。同时，我们使用免疫荧光 (IF) 和蛋白质印迹 (WB) 技术评估了紧密连接蛋白的表达和分布，包括 ZO-1、occludin 和 claudin-5。我们的数据显示 TS 和 EC 对蔗糖和甘露醇渗透性产生负面影响，从 6 小时开始一直到 24 小时。屏障完整性的丧失与 TEER 值的降低有关。虽然 ZO-1 和 occludin 的整体表达水平没有显着下调，但 ZO-1 的分布发生了改变，并且通过 IF 成像可以明显看出中断模式。与 occludin 相比，如 WB 和 IF 数据所示，TS 和 EC 显着降低了 claudin-5 的表达。