JanssenDuijghuijsen Lonneke M, Grefte Sander, de Boer Vincent C J, Zeper Lara, van Dartel Dorien A M, van der Stelt Inge, Bekkenkamp-Grovenstein Melissa, van Norren Klaske, Wichers Harry J, Keijer Jaap
Wageningen Food and Biobased Research, Wageningen University and Research, Wageningen, Netherlands.
Human and Animal Physiology, Wageningen University and Research, Wageningen, Netherlands.
Front Physiol. 2017 Oct 11;8:794. doi: 10.3389/fphys.2017.00794. eCollection 2017.
studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an model mimicking the oxidative situation. Human Caco-2 cells were cultured for 10 days in culture flasks or for 14 days on transwell inserts in either glucose-containing or galactose-containing medium. Mitochondria were visualized and cellular respiration and levels of oxidative phosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletion was induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability was assessed using transepithelial electrical resistance (TEER) and fluorescein flux. Gene expression and cellular distribution of tight junction proteins were analyzed. Caco-2 cells cultured in galactose-containing, but not in glucose-containing, medium showed increased mitochondrial connectivity, oxygen consumption rates and levels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP, rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability. In-depth studies with PA showed a six fold decrease in cellular ATP and revealed increased gene expression of tight junction proteins () 1 and 2, occludin, and claudin 1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearly redistributed from the cellular membrane into the cytoplasm, while the others were not (TJP1, occludin) or slightly (claudin 2, actin) affected. studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an model mimicking the oxidative situation. Well-functioning mitochondria are essential for maintaining cellular energy status and monolayer integrity of galactose grown Caco-2 cells. Energy depletion-induced Caco-2 monolayer permeability may be facilitated by changes in the distribution of claudin 7.
研究表明，肠道屏障的完整性依赖于线粒体ATP的产生。在此，我们旨在通过一个模拟氧化应激情况的模型来提供机制支持。将人Caco-2细胞在含葡萄糖或半乳糖的培养基中于培养瓶中培养10天，或在Transwell小室中培养14天。观察线粒体并测定细胞呼吸和氧化磷酸化（OXPHOS）蛋白水平。使用羰基氰化物间氯苯腙（CCCP）、鱼藤酮或杀粉蝶菌素A（PA）诱导线粒体ATP耗竭。使用跨上皮电阻（TEER）和荧光素通量评估单层通透性。分析紧密连接蛋白的基因表达和细胞分布。在含半乳糖而非含葡萄糖的培养基中培养的Caco-2细胞显示出线粒体连接性增加、耗氧率和OXPHOS蛋白水平升高。使用CCCP、鱼藤酮或PA抑制线粒体ATP产生导致Caco-2单层通透性呈剂量依赖性增加。对PA的深入研究表明细胞ATP减少了六倍，并揭示紧密连接蛋白（紧密连接蛋白1和2、闭合蛋白和claudin 1）的基因表达增加，但claudin 2和7的基因表达减少。其中，claudin 7明显从细胞膜重新分布到细胞质中，而其他蛋白（紧密连接蛋白1、闭合蛋白）没有（TJP1、occludin）或仅有轻微（claudin 2、肌动蛋白）影响。研究表明，肠道屏障的完整性依赖于线粒体ATP的产生。在此，我们旨在通过一个模拟氧化应激情况的模型来提供机制支持。功能良好的线粒体对于维持半乳糖培养的Caco-2细胞的细胞能量状态和单层完整性至关重要。能量耗竭诱导的Caco-2单层通透性可能因claudin 7分布的变化而加剧。
