Oral deoxynivalenol exposure of pigs modulates the pathophysiological impact of a subsequent LPS-stimulus

在完成的项目期间，我们能够证明脱氧雪腐镰刀菌烯醇(DON)和脂多糖(LPS)对体内蛋白质合成以及体内和体外肠道屏障完整性的不利影响。DON和内毒素的相互作用及其对急性时相反应(APR)的影响、相关细胞因子的表达以及随后诱导的病理生理效应，特别是对肠、肝组织和免疫细胞的影响，主要取决于它们的使用途径。在内毒素攻击前5周喂饲DON的猪，其肝脏出血、淋巴细胞浸润等病理组织学损伤明显减轻。然而，作为免疫活性器官的肝脏(清除内毒素、调节APR)对DON的代谢和消除(结合、胆汁排泄)的贡献无法深入研究，因为在以前的实验中只能获得外周静脉血。为了估计DON和内毒素从胃肠道(GIT)进入门静脉的量，门静脉内DON和内毒素的时间依赖性浓度是必不可少的。此外，测定门静脉血中口服的跨细胞和旁细胞标记物(例如，聚乙二醇乙二醇酯)将能够评估体内的肠上皮通透性，从而评估肠屏障的完整性。我们的研究(体外和体内)表明，与顶端相比，肠壁更容易受到基侧(即全身性)毒素的影响，因此了解DON和内毒素在体内的实际动脉流入GIT是至关重要的。在体外实验中，我们可以显示，当猪肠上皮细胞暴露于基侧DON时，与能量代谢、转录、翻译以及细胞通讯相关的基因(微阵列)都有显著的调控，而相同浓度的顶端暴露不会产生任何变化。有必要研究这些在mRNA水平上发现的变化是否反映在它们相应的体外结构和功能中，以及这是否也适用于活体肠道上皮细胞。我们在本项目提案中的目标是在一个先进的动物模型中帮助阐明随后的内毒素刺激与DON相关的调节机制。我们将特别关注肠道屏障和肝脏在毒素吸收和代谢中的综合作用，以及反映在局部和外周免疫系统的免疫细胞群中的全身炎症反应。

In the completed project period we were able to demonstrate the detrimental effect of deoxynivalenol (DON) and lipopolysaccharide (LPS) on the in vivo protein synthesis as well as the intestinal barrier integrity in vivo and in vitro. The interaction of DON and LPS and its impact on the acute phase reaction (APR), their related cytokine expression and the subsequently induced pathophysiological effects, in particular on intestinal and hepatic tissue and immune cells, depended primarily on their route of application. LPS-induced pathohistological lesions such as haemorrhage and lymphocyte infiltration in the liver seemed to be ameliorated in pigs received a diet containing DON 5 weeks prior to LPS challenge. However, the contribution of the liver, an immunologically active organ (LPS-clearance, mediation of APR), to metabolisation and elimination of DON (conjugation, biliary excretion) could not be investigated in depth due to the fact that only peripheral venous blood was obtainable in the previous experimental setup. In order to estimate the portal influx of DON and LPS from the gastrointestinal tract (GIT) their time-dependent concentrations in the portal vein are essential. Moreover, determining orally administered trans- and paracellular markers (e.g. PEG) in the portal blood would enable an estimation of the intestinal epithelial permeability and thus of intestinal barrier integrity in vivo. Our investigations (in vitro and in vivo) demonstrated that the intestinal lining is more vulnerable to basolateral (i.e. systemic) exposure to toxins as compared to the apical side, thus knowledge of the actual arterial influx of DON and LPS to the GIT in vivo is crucial. In vitro trials we could show a significant regulation of genes (microarray) related to energy metabolism, transcription, translation as well as cell communication when porcine intestinal epithelial cells were exposed to basolateral DON whereas apical exposure of the same concentration yielded no changes. It is essential to investigate whether these changes found on a mRNA-level are reflected in their corresponding structure and function in vitro and whether this holds also true for the intestinal epithelium in vivo.Our objectives in the present project proposal are to contribute to the elucidation of the mechanisms underlying DON-associated modulation of a subsequent LPS stimulus in an advanced animal model. Particularly we will focus on the integrated action of intestinal barrier and liver in toxin absorption and metabolisation as well as on the systemic inflammatory response, reflected in the immune cell population in local and peripheral immune system.