Karwad Mustafa A, Couch Daniel G, Theophilidou Elena, Sarmad Sarir, Barrett David A, Larvin Michael, Wright Karen L, Lund Jonathan N, O'Sullivan Saoirse E
School of Medicine, Royal Derby Hospital, University of Nottingham, Nottingham, United Kingdom.
Centre for Analytical Bioscience, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
FASEB J. 2017 Aug;31(8):3267-3277. doi: 10.1096/fj.201601346R. Epub 2017 Apr 12.
The endocannabinoid system has previously been shown to play a role in the permeability and inflammatory response of the human gut. The goal of our study was to determine the effects of endogenous anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) on the permeability and inflammatory response of intestinal epithelium under normal, inflammatory, and hypoxic conditions. Human intestinal mucosa was modeled using Caco-2 cells. Human tissue was collected from planned colorectal resections. Accumulation of AEA and 2-AG was achieved by inhibiting their metabolizing enzymes URB597 (a fatty acid amide hydrolase inhibitor) and JZL184 (a monoacylglycerol lipase inhibitor). Inflammation and ischemia were simulated with TNF-α and IFN-γ and oxygen deprivation. Permeability changes were measured by transepithelial electrical resistance. The role of the CB receptor was explored using CB-knockdown (CBKd) intestinal epithelial cells. Endocannabinoid levels were measured using liquid chromatography-mass spectrometry. Cytokine secretion was measured using multiplex and ELISA. URB597 and JZL184 caused a concentration-dependent increase in permeability CB ( < 0.0001) and decreased cytokine production. Basolateral application of JZL184 decreased permeability CB ( < 0.0001). URB597 and JZL184 increased the enhanced (worsened) permeability caused by inflammation and hypoxia ( < 0.0001 and < 0.05). CBKd cells showed reduced permeability response to inflammation ( < 0.01) but not hypoxia. 2-AG levels were increased in response to inflammation and hypoxia in Caco-2 cells. In human mucosal tissue, inflammation increased the secretion of granulocyte macrophage-colony stimulating factor, IL-12, -13, and -15, which was prevented with treatment with URB597 and JZL184, and was inhibited by a CB antagonist. The results of this study show that endogenous AEA and 2-AG production and CB activation play a key modulatory roles in normal intestinal mucosa permeability and in inflammatory and hypoxic conditions.-Karwad, M. A., Couch, D. G., Theophilidou, E., Sarmad, S., Barrett, D. A., Larvin, M., Wright, K. L., Lund, J. N., O'Sullivan, S. E. The role of CB in intestinal permeability and inflammation.
内源性大麻素系统此前已被证明在人体肠道的通透性和炎症反应中发挥作用。我们研究的目的是确定内源性花生四烯酸乙醇胺（AEA）和2-花生四烯酸甘油酯（2-AG）在正常、炎症和缺氧条件下对肠上皮通透性和炎症反应的影响。使用Caco-2细胞构建人肠黏膜模型。从计划进行的结直肠切除术中收集人体组织。通过抑制其代谢酶URB597（一种脂肪酸酰胺水解酶抑制剂）和JZL184（一种单酰甘油脂肪酶抑制剂）来实现AEA和2-AG的蓄积。用TNF-α和IFN-γ模拟炎症，用缺氧模拟缺血。通过跨上皮电阻测量通透性变化。使用CB基因敲低（CBKd）肠上皮细胞探究CB受体的作用。使用液相色谱-质谱法测量内源性大麻素水平。使用多重检测法和酶联免疫吸附测定法测量细胞因子分泌。URB597和JZL184导致通透性呈浓度依赖性增加（P<0.0001），并减少细胞因子产生。从基底外侧应用JZL184可降低通透性（P<0.0001）。URB597和JZL184增加了由炎症和缺氧引起的增强（恶化）的通透性（P<0.0001和P<0.05）。CBKd细胞对炎症的通透性反应降低（P<0.01），但对缺氧无反应。在Caco-2细胞中，2-AG水平因炎症和缺氧而升高。在人体黏膜组织中，炎症增加了粒细胞巨噬细胞集落刺激因子、IL-12、IL-13和IL-15的分泌，用URB597和JZL184处理可预防这种情况，且CB拮抗剂可抑制这种分泌。本研究结果表明，内源性AEA和2-AG的产生以及CB激活在正常肠黏膜通透性以及炎症和缺氧条件下发挥关键的调节作用。-卡尔瓦德，M.A.，库奇，D.G.，西奥菲利杜，E.，萨马德，S.，巴雷特，D.A.，拉文，M.，赖特，K.L.，伦德，J.N.，奥沙利文，S.E. CB在肠道通透性和炎症中的作用 。
