Cancer Research Program, Julius L Chambers-Biomedical Biotechnology Research Institute, North Carolina Central University, , Durham, North Carolina, USA.
Gut. 2014 May;63(5):711-9. doi: 10.1136/gutjnl-2012-303731. Epub 2013 May 15.
As a major cellular defence mechanism, the Nrf2/Keap1 pathway regulates expression of genes involved in detoxification and stress response. Here we hypothesise that Nrf2 is involved in oesophageal barrier function and plays a protective role against gastro-oesophageal reflux disease (GERD).
Human oesophageal biopsy samples, mouse surgical models and Nrf2(-/-) mice were used to assess the role of the Nrf2/Keap1 pathway in oesophageal barrier function. Trans-epithelial electrical resistance (TEER) was measured with mini-Ussing chambers. HE staining and transmission electron microscopy were used to examine tissue morphology, while gene microarray, immunohistochemistry, western blotting and chromatin immunoprecipitation (ChIP) analysis were used to assess gene expression.
Nrf2 was expressed in normal oesophageal epithelium and activated in GERD of both humans and mice. Nrf2 deficiency and gastro-oesophageal reflux in mice, alone or in combination, reduced TEER and increased intercellular space in oesophageal epithelium. Nrf2 target genes and gene sets associated with oxidoreductase activity, mitochondrial biogenesis and energy production were downregulated in the oesophageal epithelium of Nrf2(-/-) mice. Consistent with the antioxidative function of Nrf2, a DNA oxidative damage marker (8OHdG) dramatically increased in oesophageal epithelial cells of Nrf2(-/-) mice compared with those of wild-type mice. Interestingly, ATP biogenesis, Cox IV (a mitochondrial protein) and Claudin 4 (Cldn4) expression were downregulated in the oesophageal epithelium of Nrf2(-/-) mice, suggesting that energy-dependent tight junction integrity was subject to Nrf2 regulation. ChIP analysis confirmed the binding of Nrf2 to Cldn4 promoter.
Nrf2 deficiency impairs oesophageal barrier function through disrupting energy-dependent tight junction.
作为一种主要的细胞防御机制，Nrf2/Keap1 通路调节参与解毒和应激反应的基因表达。在这里，我们假设 Nrf2 参与食管屏障功能，并在胃食管反流病 (GERD) 中发挥保护作用。
使用人食管活检样本、小鼠手术模型和 Nrf2(-/-) 小鼠来评估 Nrf2/Keap1 通路在食管屏障功能中的作用。使用迷你 Ussing 室测量跨上皮电阻 (TEER)。使用 HE 染色和透射电子显微镜检查组织形态，同时使用基因微阵列、免疫组织化学、western blot 和染色质免疫沉淀 (ChIP) 分析评估基因表达。
Nrf2 在正常食管上皮中表达，并在人类和小鼠的 GERD 中被激活。Nrf2 缺失和小鼠的胃食管反流，单独或联合，降低了 TEER 并增加了食管上皮的细胞间隙。Nrf2 靶基因和与氧化还原酶活性、线粒体生物发生和能量产生相关的基因集在 Nrf2(-/-) 小鼠的食管上皮中下调。与 Nrf2 的抗氧化功能一致，Nrf2(-/-) 小鼠食管上皮细胞中的 DNA 氧化损伤标志物 (8OHdG) 明显增加，与野生型小鼠相比。有趣的是，ATP 生物发生、Cox IV（一种线粒体蛋白）和 Claudin 4（Cldn4）表达在 Nrf2(-/-) 小鼠的食管上皮中下调，表明能量依赖的紧密连接完整性受 Nrf2 调节。ChIP 分析证实了 Nrf2 与 Cldn4 启动子的结合。
Nrf2 缺失通过破坏能量依赖的紧密连接来损害食管屏障功能。
