Moran G W, O'Neill C, McLaughlin J T
Inflammation Sciences Research Group, University of Manchester, Manchester, M13 9PL, UK.
Regul Pept. 2012 Oct 10;178(1-3):95-101. doi: 10.1016/j.regpep.2012.07.002. Epub 2012 Jul 15.
Tight junctions are intercellular permeability seals that regulate paracellular transport across epithelia. Tight junction function, expression and localisation of constituent proteins are significantly altered by cytokines such as TNFα. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic enteroendocrine peptide. It is not known whether GLP-2 regulates the barrier or tight junctions. The aim of this study was to investigate whether GLP-2 has an effect on tight junction function or protein expression, alone or in response to TNFα exposure.
Caco-2 cells were grown to confluence on filters in the presence or absence of GLP-2. The time course of transepithelial electrical resistance developing across the monolayer was measured; tight junction protein expression was quantified by immunoblotting. At day 20, TNFα in the presence or absence of GLP-2 was added. Changes in TEER and tight junction proteins expression were quantified. Both TNFα and GLP-2 were added on the basolateral side.
GLP-2 exposed Caco-2 cell monolayers showed a significant increase in transepithelial electrical resistance compared to that in untreated control cells. At the same time, expression of the tight junction proteins occludin and zona occludens-1 (ZO-1) was increased at day 17 post-seeding (1.6-fold; p=0.037 and 4.7 fold; p=0.039 respectively). Subsequent TNFα exposure induced a significant 9.3-fold (p<0.001) decrease in transepithelial electrical resistance and a corresponding reduction in the expression of ZO-1 (5.3 fold; p<0.01). However, the TNFα-induced reduction in transepithelial electrical resistance in GLP-2-exposed cells was highly attenuated to 1.8-fold (p<0.01). No change in tight junction protein expression was noted in GLP-2 exposed cells after cytokine exposure.
GLP-2 enhances formation of the epithelial barrier and its constituent proteins in Caco-2 cells, and diminishes the effects of TNFα. If these effects are replicated in vivo the GLP-2 receptor may present a therapeutic target in intestinal inflammation.
紧密连接是细胞间的通透性屏障，可调节上皮细胞间的旁细胞转运。诸如肿瘤坏死因子α（TNFα）等细胞因子会显著改变紧密连接的功能、组成蛋白的表达及定位。胰高血糖素样肽-2（GLP-2）是一种肠营养性肠内分泌肽。目前尚不清楚GLP-2是否调节屏障或紧密连接。本研究的目的是调查GLP-2单独或在暴露于TNFα时是否对紧密连接功能或蛋白表达有影响。
将Caco-2细胞在有无GLP-2的情况下培养至在滤膜上汇合。测量单层细胞上跨上皮电阻的时间进程；通过免疫印迹法定量紧密连接蛋白表达。在第20天，加入有无GLP-2存在时的TNFα。对跨上皮电阻（TEER）和紧密连接蛋白表达的变化进行定量。TNFα和GLP-2均添加到基底外侧。
与未处理的对照细胞相比，暴露于GLP-2的Caco-2细胞单层的跨上皮电阻显著增加。同时，在接种后第17天，紧密连接蛋白闭合蛋白和闭合蛋白连接蛋白-1（ZO-1）的表达增加（分别为1.6倍；p = 0.037和4.7倍；p = 0.039）。随后暴露于TNFα导致跨上皮电阻显著降低9.3倍（p < 0.001），ZO-1表达相应降低（5.3倍；p < 0.01）。然而，在暴露于GLP-2的细胞中，TNFα诱导的跨上皮电阻降低高度减弱至1.8倍（p < 0.01）。细胞因子暴露后，暴露于GLP-2的细胞中紧密连接蛋白表达未观察到变化。
GLP-2增强Caco-2细胞中上皮屏障及其组成蛋白的形成，并减弱TNFα的作用。如果这些作用在体内得以重现，GLP-2受体可能成为肠道炎症的治疗靶点。
