Nighot Prashant K, Moeser Adam J, Ryan Kathleen A, Ghashghaei Troy, Blikslager Anthony T
Department of Clinical Science, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street Raleigh, NC 27606, USA.
Exp Cell Res. 2009 Jan 1;315(1):110-8. doi: 10.1016/j.yexcr.2008.10.001. Epub 2008 Oct 17.
Involvement of the epithelial chloride channel ClC-2 has been implicated in barrier recovery following ischemic injury, possibly via a mechanism involving ClC-2 localization to the tight junction. The present study investigated mechanisms of intestinal barrier repair following ischemic injury in ClC-2(-/-) mice.
Wild type, ClC-2 heterozygous and ClC-2(-/-) murine jejunal mucosa was subjected to complete ischemia, after which recovery of barrier function was monitored by measuring in vivo blood-to-lumen clearance of (3)H-mannitol. Tissues were examined by light and electron microscopy. The role of ClC-2 in re-assembly of the tight junction during barrier recovery was studied by immunoblotting, immunolocalization and immunoprecipitation.
Following ischemic injury, ClC-2(-/-) mice had impaired barrier recovery compared to wild type mice, defined by increases in epithelial paracellular permeability independent of epithelial restitution. The recovering ClC-2(-/-) mucosa also had evidence of ultrastructural paracellular defects. The tight junction proteins occludin and claudin-1 shifted significantly to the detergent soluble membrane fraction during post-ischemic recovery in ClC-2(-/-) mice whereas wild type mice had a greater proportion of junctional proteins in the detergent insoluble fraction. Occludin was co-immunoprecipitated with ClC-2 in uninjured wild type mucosa, and the association between occludin and ClC-2 was re-established during ischemic recovery. Based on immunofluorescence studies, re-localization of occludin from diffuse sub-apical areas to apical tight junctions was impaired in ClC-2(-/-) mice.
These data demonstrate a pivotal role of ClC-2 in recovery of the intestinal epithelium barrier by anchoring assembly of tight junctions following ischemic injury.
上皮氯通道ClC-2参与缺血性损伤后的屏障修复，可能是通过一种涉及ClC-2定位于紧密连接的机制。本研究调查了ClC-2基因敲除（ClC-2(-/-)）小鼠缺血性损伤后肠道屏障修复的机制。
对野生型、ClC-2杂合子和ClC-2(-/-)小鼠的空肠黏膜进行完全缺血处理，之后通过测量体内(3)H-甘露醇的血-腔清除率来监测屏障功能的恢复情况。用光镜和电镜检查组织。通过免疫印迹、免疫定位和免疫沉淀研究ClC-2在屏障恢复过程中紧密连接重新组装中的作用。
缺血性损伤后，与野生型小鼠相比，ClC-2(-/-)小鼠的屏障恢复受损，表现为上皮细胞旁通透性增加，且与上皮修复无关。恢复中的ClC-2(-/-)黏膜也有超微结构细胞旁缺陷的证据。在ClC-2(-/-)小鼠缺血后恢复过程中，紧密连接蛋白闭合蛋白和Claudin-1显著转移至去污剂可溶膜部分，而野生型小鼠的连接蛋白在去污剂不溶部分中的比例更高。在未受损的野生型黏膜中，闭合蛋白与ClC-2共免疫沉淀，且在缺血恢复过程中闭合蛋白与ClC-2之间的关联重新建立。基于免疫荧光研究，在ClC-2(-/-)小鼠中，闭合蛋白从弥漫性的顶下区域重新定位于顶端紧密连接受到损害。
这些数据表明，ClC-2在缺血性损伤后通过锚定紧密连接的组装在肠道上皮屏障恢复中起关键作用。
