Laurate Permeates the Paracellular Pathway for Small Molecules in the Intestinal Epithelial Cell Model HT-29/B6 via Opening the Tight Junctions by Reversible Relocation of Claudin-5
Laurate Permeates the Paracellular Pathway for Small Molecules in the Intestinal Epithelial Cell Model HT-29/B6 via Opening the Tight Junctions by Reversible Relocation of Claudin-5
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DOI：
10.1007/s11095-014-1350-2
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发表时间：
2014-09-01
影响因子：
3.7
通讯作者：
Amasheh, Salah
中科院分区：
文献类型：
--
作者：
Dittmann, Isabel;Amasheh, Maren;Amasheh, Salah
机制分析中链脂肪酸月牙酸酯对肠上皮细胞系HT-29/B6融合单层经上皮通透性的影响，以及作为吸收促进剂改善经上皮药物通透性的应用。用ussing型室测定细胞外通量标记物的经上皮阻力和表观通透性。采用双路阻抗谱法区分细胞间和细胞旁电阻，并进行共聚焦成像和Western blotting。月桂酸盐导致经上皮耐药显著且可逆地减少50%，这归因于细胞旁耐药的减少。同时，荧光素（330 Da）的通透性增加，而4 kDa的fitc -葡聚糖和硫- nhs - ss -生物素（607 Da）的通透性保持不变。共聚焦激光扫描显微镜显示，claudin-5明显减少，而其他紧密连接蛋白，包括阻止大分子细胞旁通道的蛋白tricellulin，没有受到影响。月桂酸盐通过从紧密连接中提取claudin-5而不影响三细胞接触和大分子的细胞旁传代，诱导细胞旁渗透性增加，分子质量达到330 Da。本文首次在分子水平上对月桂酸诱导的通透性增强进行了力学解释。
To mechanistically analyze effects of the medium-chain fatty acid laurate on transepithelial permeability in confluent monolayers of the intestinal epithelial cell line HT-29/B6, in context with an application as an absorption enhancer improving transepithelial drug permeation.Transepithelial resistance and apparent permeability for paracellular flux markers was measured using Ussing-type chambers. Two-path impedance spectroscopy was employed to differentiate between transcellular and paracellular resistance, and confocal imaging and Western blotting was performed.Laurate resulted in a substantial and reversible decrease in transepithelial resistance by 50% which was attributed to a decrease in paracellular resistance. Simultaneously, an increase in permeability for fluorescein (330 Da) was detected, while permeabilities for 4 kDa FITC-dextran and sulpho-NHS-SS-biotin (607 Da) remained unaltered. Confocal laser-scanning microscopy revealed a marked reduction of claudin-5, while other tight junction proteins including tricellulin, a protein preventing the paracellular passage of macromolecules, were not affected.Laurate induces an increase in paracellular permeability for molecules up to a molecular mass of 330 Da by retrieval of claudin-5 from tight junctions without affecting tricellular contacts and the paracellular passage of macromolecules. We hereby provide, for the first time, a mechanistical explanation of laurate-induced permeability enhancement on molecular level.