Elamin Elhaseen, Masclee Ad, Troost Freddy, Dekker Jan, Jonkers Daisy
Top Institute Food and Nutrition (TIFN) , Wageningen, the Netherlands; Division of Gastroenterology and Hepatology , Department of Internal Medicine, Maastricht University Medical Center, Maastricht, the Netherlands; School for Nutrition, Toxicology and Metabolism , Maastricht University Medical Center, Maastricht, the Netherlands.
Alcohol Clin Exp Res. 2014 Feb;38(2):344-53. doi: 10.1111/acer.12234. Epub 2013 Aug 19.
Acetaldehyde (AcH) is mutagenic and can reach high concentrations in colonic lumen after ethanol consumption and is associated with intestinal barrier dysfunction and an increased risk of progressive cancers, including colorectal carcinoma. Snail, the transcription factor of epithelial-mesenchymal transition, is known to down-regulate expression of tight junction (TJ) and adherens junction (AJ) proteins, resulting in loss of epithelial integrity, cancer progression, and metastases. As AcH is mutagenic, the role of Snail in the AcH-induced disruption of intestinal epithelial TJs deserves further investigation. Our aim was to investigate the role of oxidative stress and Snail activation in AcH-induced barrier disruption in Caco-2 monolayers.
The monolayers were exposed from the apical side to AcH ± L-cysteine. Reactive oxygen species (ROS) generation and Snail activation were assessed by ELISA and immunofluorescence. Paracellular permeability, localization, and expression of ZO-1, occludin, E-cadherin, and β-catenin were examined using transepithelial electrical resistance (TEER), fluorescein isothiocyanate-labeled dextran 4 kDa (FITC-D4), immunofluorescence, and ELISA, respectively. Involvement of Snail was further addressed by inhibiting Snail using small interfering RNA (siRNA).
Exposure to 25 μM AcH increased ROS generation and ROS-dependently induced Snail phosphorylation. In addition, AcH increased paracellular permeability (decrease in TEER and increase in FITC-D4 permeation) in association with redistribution and decrease of TJ and AJ protein levels, which could be attenuated by L-cysteine. Knockdown of Snail by siRNA attenuated the AcH-induced redistribution and decrease in the TJ and AJ proteins, in association with improvement of the barrier function.
Our data demonstrate that oxidative stress-mediated Snail phosphorylation is likely a novel mechanism contributing to the deleterious effects of AcH on the TJ and AJ, and intestinal barrier function.
乙醛（AcH）具有致突变性，在摄入乙醇后可在结肠腔内达到高浓度，且与肠道屏障功能障碍以及包括结直肠癌在内的进展性癌症风险增加有关。Snail是上皮-间质转化的转录因子，已知其可下调紧密连接（TJ）和黏附连接（AJ）蛋白的表达，导致上皮完整性丧失、癌症进展和转移。由于AcH具有致突变性，Snail在AcH诱导的肠道上皮TJ破坏中的作用值得进一步研究。我们的目的是研究氧化应激和Snail激活在AcH诱导的Caco-2单层细胞屏障破坏中的作用。
将单层细胞从顶端暴露于AcH ± L-半胱氨酸。通过酶联免疫吸附测定（ELISA）和免疫荧光评估活性氧（ROS）生成和Snail激活。分别使用跨上皮电阻（TEER）、异硫氰酸荧光素标记的4 kDa葡聚糖（FITC-D4）、免疫荧光和ELISA检测ZO-1、闭合蛋白、E-钙黏蛋白和β-连环蛋白的细胞旁通透性、定位和表达。通过使用小干扰RNA（siRNA）抑制Snail进一步探讨Snail的作用。
暴露于25 μM AcH会增加ROS生成，并以ROS依赖性方式诱导Snail磷酸化。此外，AcH会增加细胞旁通透性（TEER降低和FITC-D4渗透增加），同时伴有TJ和AJ蛋白水平的重新分布和降低，L-半胱氨酸可减弱这种情况。通过siRNA敲低Snail可减弱AcH诱导的TJ和AJ蛋白的重新分布和降低，同时改善屏障功能。
我们的数据表明，氧化应激介导的Snail磷酸化可能是一种新机制，导致AcH对TJ和AJ以及肠道屏障功能产生有害影响。
