Bulut Kerem, Felderbauer Peter, Deters Susanne, Hoeck Karoline, Schmidt-Choudhury Anjona, Schmidt Wolfgang E, Hoffmann Peter
Department of Internal Medicine I, St. Josef Hospital, Ruhr-University of Bochum, Gudrun Str. 54, 44791, Bochum, Germany.
Int J Colorectal Dis. 2008 May;23(5):535-41. doi: 10.1007/s00384-008-0447-7. Epub 2008 Feb 15.
Calcitonin-gene-related peptide (CGRP) and substance P (SP) are neurotransmitters of extrinsic primary afferent neurons located within the dorsal root ganglia. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on intestinal mucosa was presumed. The mucosal protection partly depends on a CGRP-mediated modulation of mucosal blood flow. Limited data are available regarding CGRP- or SP-mediated effects on epithelial cell restitution. Having shown earlier that SP-stimulated fibroblasts but not CGRP-stimulated fibroblasts induce epithelial cell migration in vitro, the aim of this study was to explore whether mast cells mediate effects of SP and CGRP on epithelial cell restitution in vitro.
Mast cells (C57) were exposed to SP [10(-12)-10(-6 M)] and CGRP [10(-12)-10(-7 M)]. After a 24-h incubation period, the cell supernatants (conditioned media, CDM) were taken from mast cell cultures and directly applied to rat intestinal epithelial cell lines-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h prior to the experiments. Epithelial cell migration was assessed by counting cells across the wound edge and epithelial cell proliferation was measured using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide test.
CGRP significantly induced epithelial cell migration and proliferation via mast cells when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralizing anti-transforming growth factor-beta (TGF-beta) had been added to the cell cultures. SP had no effects on epithelial cells following stimulation of mast cells.
CGRP modulates epithelial cell restitution in vitro mediated by mast cells. The CGRP- and mast-cell-induced epithelial cell migration is TGF-beta dependent. This observation underlines an important role for extrinsic primary afferent neurons in mucosal defence and repair and in keeping the mucosal homeostasis. This knowledge leads to a better understanding of the interaction of the enteric nervous system and wound healing and may, in the future, lead to new therapeutic approaches to inflammatory diseases of the intestine.
降钙素基因相关肽（CGRP）和P物质（SP）是位于背根神经节的外周初级传入神经元的神经递质。在大鼠和兔子的结肠炎实验模型中，推测SP和CGRP对肠黏膜具有保护作用。黏膜保护部分取决于CGRP介导的黏膜血流调节。关于CGRP或SP对上皮细胞修复的影响，现有数据有限。此前研究表明，SP刺激的成纤维细胞而非CGRP刺激的成纤维细胞可在体外诱导上皮细胞迁移，本研究旨在探讨肥大细胞是否介导SP和CGRP对体外上皮细胞修复的影响。
将肥大细胞（C57）暴露于SP[10⁻¹² - 10⁻⁶ M]和CGRP[10⁻¹² - 10⁻⁷ M]。孵育24小时后，从肥大细胞培养物中获取细胞上清液（条件培养基，CDM），并直接应用于大鼠肠上皮细胞系-18或Caco-2单层细胞，这些细胞在实验前24小时已用剃须刀片划伤。通过计数伤口边缘的细胞评估上皮细胞迁移，并使用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基溴化四氮唑试验测量上皮细胞增殖。
当将上清液直接应用于体外上皮细胞时，CGRP通过肥大细胞显著诱导上皮细胞迁移和增殖。在细胞培养物中添加中和性抗转化生长因子-β（TGF-β）后，对上皮细胞迁移的影响消失。刺激肥大细胞后，SP对上皮细胞无影响。
CGRP在体外调节肥大细胞介导的上皮细胞修复。CGRP和肥大细胞诱导的上皮细胞迁移依赖于TGF-β。这一观察结果强调了外周初级传入神经元在黏膜防御、修复以及维持黏膜稳态中的重要作用。这一认识有助于更好地理解肠神经系统与伤口愈合之间的相互作用，并可能在未来导致针对肠道炎症性疾病的新治疗方法。
