Division of Medical Sciences & Graduate Entry Medicine, School of Medicine, Royal Derby Hospital, University of Nottingham, Nottingham, UK.
Division of Biomedical & Life Sciences, Faculty of Health & Medicine, Lancaster University, Lancaster, UK.
Biochem Pharmacol. 2019 Oct;168:465-472. doi: 10.1016/j.bcp.2019.07.017. Epub 2019 Jul 17.
We have previously reported that endocannabinoids modulate permeability in Caco-2 cells under inflammatory conditions and hypothesised in the present study that endocannabinoids could also modulate permeability in ischemia/reperfusion.
Caco-2 cells were grown on cell culture inserts to confluence. Trans-epithelial electrical resistance (TEER) was used to measure permeability. To generate hypoxia (0% O), a GasPak™ EZ anaerobe pouch system was used. Endocannabinoids were applied to the apical or basolateral membrane in the presence or absence of receptor antagonists.
Complete hypoxia decreased TEER (increased permeability) by ~35% after 4 h (recoverable) and ~50% after 6 h (non-recoverable). When applied either pre- or post-hypoxia, apical application of N-arachidonoyl-dopamine (NADA, via TRPV1), oleamide (OA, via TRPV1) and oleoylethanolamine (OEA, via TRPV1) inhibited the increase in permeability. Apical administration of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) worsened the permeability effect of hypoxia (both via CB). Basolateral application of NADA (via TRPV1), OA (via CB and TRPV1), noladin ether (NE, via PPARα), and palmitoylethanolamine (PEA, via PPARα) restored permeability after 4 h hypoxia, whereas OEA increased permeability (via PPARα). After 6 h hypoxia, where permeability does not recover, only basolateral application PEA sustainably decreased permeability, and NE decreased permeability.
A variety of endocannabinoids and endocannabinoid-like compounds modulate Caco-2 permeability in hypoxia/reoxygenation, which involves multiple targets, depending on whether the compounds are applied to the basolateral or apical membrane. CB antagonism and TRPV1 or PPARα agonism may represent novel therapeutic targets against several intestinal disorders associated with increased permeability.
我们之前报道过内源性大麻素可在炎症条件下调节 Caco-2 细胞的通透性，并假设在内源性大麻素也可调节缺血/再灌注中的通透性。
将 Caco-2 细胞培养在细胞培养插入物上至汇合。跨上皮电阻（TEER）用于测量通透性。为了产生缺氧（0% O），使用了 GasPak™EZ 厌氧菌袋系统。在存在或不存在受体拮抗剂的情况下，将内源性大麻素应用于顶膜或基底外侧膜。
完全缺氧 4 小时后 TEER 降低（通透性增加）约 35%（可恢复），6 小时后降低约 50%（不可恢复）。无论在缺氧前还是缺氧后应用，N-花生四烯酰-多巴胺（NADA，通过 TRPV1）、油酰胺（OA，通过 TRPV1）和油酰乙醇胺（OEA，通过 TRPV1）的顶膜给药均可抑制通透性的增加。给予大麻素（AEA）和 2-花生四烯酰甘油（2-AG）的顶膜给药会加重缺氧的通透性效应（均通过 CB）。NADA（通过 TRPV1）、OA（通过 CB 和 TRPV1）、诺拉汀乙醚（NE，通过 PPARα）和棕榈酰乙醇胺（PEA，通过 PPARα）的基底外侧给药可在 4 小时缺氧后恢复通透性，而 OEA 则增加通透性（通过 PPARα）。在 6 小时缺氧后，通透性无法恢复，只有基底外侧给药 PEA 可持续降低通透性，NE 降低通透性。
各种内源性大麻素和内源性大麻素样化合物在缺氧/再氧合中调节 Caco-2 通透性，这涉及多个靶点，具体取决于化合物是应用于基底外侧还是顶膜。CB 拮抗剂和 TRPV1 或 PPARα 激动剂可能代表针对几种与通透性增加相关的肠道疾病的新型治疗靶点。
