INSERM, UMR913, Nantes, France; Nantes University, Nantes, France; Institut des Maladies de l'Appareil Digestif, IMAD, CHU Nantes, Hopital Hôtel-Dieu, Nantes, France; Centre de Recherche en Nutrition Humaine, Nantes, France.
INSERM UMR1043, Toulouse, France.
Gastroenterology. 2016 Jan;150(1):168-80. doi: 10.1053/j.gastro.2015.09.038. Epub 2015 Nov 11.
BACKGROUND & AIMS: Enteric glial cells (EGCs) produce soluble mediators that regulate homeostasis and permeability of the intestinal epithelial barrier (IEB). We investigated the profile of polyunsaturated fatty acid (PUFA) metabolites produced by EGCs from rats and from patients with Crohn's disease (CD), compared with controls, along with the ability of one of these metabolites, 15-hydroxyeicosatetraenoic acid (15-HETE), to regulate the permeability of the IEB.
We isolated EGCs from male Sprague-Dawley rats, intestinal resections of 6 patients with CD, and uninflamed healthy areas of intestinal tissue from 6 patients who underwent surgery for colorectal cancer (controls). EGC-conditioned media was analyzed by high-sensitivity liquid-chromatography tandem mass spectrometry to determine PUFA signatures. We used immunostaining to identify 15-HETE-producing enzymes in EGCs and tissues. The effects of human EGCs and 15-HETE on permeability and transepithelial electrical resistance of the IEB were measured using Caco-2 cells; effects on signal transduction proteins were measured with immunoblots. Levels of proteins were reduced in Caco-2 cells using short-hairpin RNAs or proteins were inhibited pharmacologically. Rats were given intraperitoneal injections of 15-HETE or an inhibitor of 15-lipoxygenase (the enzyme that produces 15-HETE); colons were collected and permeability was measured.
EGCs expressed 15-lipoxygenase-2 and produced high levels of 15-HETE, which increased IEB resistance and reduced IEB permeability. 15-HETE production was reduced in EGCs from patients with CD compared with controls. EGCs from patients with CD were unable to reduce the permeability of the IEB; the addition of 15-HETE restored permeability to levels of control tissues. Inhibiting 15-HETE production in rats increased the permeability of the IEB in colon tissues. We found that 15-HETE regulates IEB permeability by inhibiting an adenosine monophosphate-activated protein kinase and increasing expression of zonula occludens-1.
Enteric glial cells from patients with CD have reduced production of 15-HETE, which controls IEB permeability by inhibiting adenosine monophosphate-activated protein kinase and increasing expression of zonula occludens-1.
肠胶质细胞（EGCs）产生可溶性介质，调节肠道上皮屏障（IEB）的稳态和通透性。我们研究了大鼠和克罗恩病（CD）患者的 EGC 产生的多不饱和脂肪酸（PUFA）代谢物的特征，并与对照组进行了比较，同时研究了其中一种代谢物 15-羟二十碳四烯酸（15-HETE）调节 IEB 通透性的能力。
我们从雄性 Sprague-Dawley 大鼠、6 例 CD 患者的肠道切除术和 6 例因结直肠癌而行手术的无炎症健康肠组织中分离 EGCs。通过高灵敏度液相色谱串联质谱法分析 EGC 条件培养基以确定 PUFA 特征。我们使用免疫染色鉴定 EGCs 和组织中的 15-HETE 产生酶。使用 Caco-2 细胞测量人 EGCs 和 15-HETE 对 IEB 通透性和跨上皮电阻的影响；使用免疫印迹法测量对信号转导蛋白的影响。使用短发夹 RNA 降低 Caco-2 细胞中的蛋白质水平，或使用药理学抑制剂抑制蛋白质。向大鼠腹腔内注射 15-HETE 或 15-脂氧合酶抑制剂（产生 15-HETE 的酶）；收集结肠并测量通透性。
EGCs 表达 15-脂氧合酶-2 并产生高水平的 15-HETE，增加 IEB 阻力并降低 IEB 通透性。与对照组相比，CD 患者的 EGCs 产生的 15-HETE 减少。CD 患者的 EGCs 无法降低 IEB 的通透性；添加 15-HETE 可将通透性恢复至对照组织水平。在大鼠中抑制 15-HETE 的产生增加了结肠组织中 IEB 的通透性。我们发现，15-HETE 通过抑制腺苷一磷酸激活蛋白激酶和增加闭合蛋白-1 的表达来调节 IEB 通透性。
CD 患者的 EGCs 产生的 15-HETE 减少，通过抑制腺苷一磷酸激活蛋白激酶和增加闭合蛋白-1 的表达来控制 IEB 的通透性。
