Metabolic syndrome and diabetes in obese individuals are strong risk factors for development of inflammatory bowel disease (IBD) and colorectal cancer. The pathogenic mechanisms of low-grade metabolic inflammation, including chronic hyperglycemic stress, in disrupting gut homeostasis are poorly understood. In this study, we sought to understand the impact of a hyperglycemic environment on intestinal barrier integrity and the protective effects of small molecular weight (35 kDa) hyaluronan on epithelial barrier function. Intestinal organoids derived from mouse colon were grown in normal glucose media (5 mM) or high glucose media (25 mM) to study the impact of hyperglycemic stress on the intestinal barrier. Additionally, organoids were pretreated with 35 kDa hyaluronan (HA35) to investigate the effect of hyaluronan on epithelial barrier under high glucose stress. Immunoblotting as well as confocal imaging was used to understand changes in barrier proteins, quantitative as well as spatial distribution, respectively. Alterations in barrier function were measured using trans-epithelial electrical resistance and fluorescein isothiocyanate flux assays. Untargeted proteomics analysis was performed to elucidate mechanisms by which HA35 exerts a protective effect on the barrier. Intestinal organoids derived from receptor knockout mice specific to various HA receptors were utilized to understand the role of HA receptors in barrier protection under high glucose conditions. We found that high glucose stress decreased the protein expression as well as spatial distribution of two key barrier proteins, zona occludens-1 (ZO-1) and occludin. HA35 prevented the degradation or loss of ZO-1 and maintained the spatial distribution of both ZO-1 and occludin under hyperglycemic stress. Functionally, we also observed a protective effect of HA35 on the epithelial barrier under high glucose conditions. We found that HA receptor, layilin, was involved in preventing barrier protein loss (ZO-1) as well as maintaining spatial distribution of ZO-1 and occludin. Additionally, proteomics analysis showed that cell death and survival was the primary pathway upregulated in organoids treated with HA35 under high glucose stress. We found that XIAP associated factor 1 (Xaf1) was modulated by HA35 thereby regulating apoptotic cell death in the intestinal organoid system. Finally, we observed that spatial organization of both focal adhesion kinase (FAK) as well as F-actin was mediated by HA35 via layilin. Our results highlight the impact of hyperglycemic stress on the intestinal barrier function. This is of clinical relevance, as impaired barrier function has been observed in individuals with metabolic syndrome. Additionally, we demonstrate barrier protective effects of HA35 through its receptor layilin and modulation of cellular apoptosis under high glucose stress.

中文翻译：

---

高血糖环境直接损害类器官模型中的肠上皮屏障功能，透明质酸 (∼35 kDa) 通过 Layilin 依赖性机制提供保护


肥胖个体的代谢综合征和糖尿病是发生炎症性肠病（IBD）和结直肠癌的强烈危险因素。低度代谢炎症（包括慢性高血糖应激）破坏肠道稳态的致病机制尚不清楚。在这项研究中，我们试图了解高血糖环境对肠道屏障完整性的影响以及小分子量 (35 kDa) 透明质酸对上皮屏障功能的保护作用。来自小鼠结肠的肠类器官在正常葡萄糖培养基 (5 mM) 或高葡萄糖培养基 (25 mM) 中生长，以研究高血糖应激对肠道屏障的影响。此外，用 35 kDa 透明质酸 (HA35) 预处理类器官，以研究高葡萄糖应激下透明质酸对上皮屏障的影响。免疫印迹和共聚焦成像分别用于了解屏障蛋白的变化、定量和空间分布。使用跨上皮电阻和异硫氰酸荧光素通量测定来测量屏障功能的变化。进行非靶向蛋白质组学分析以阐明 HA35 对屏障发挥保护作用的机制。利用来自对各种HA受体具有特异性的受体敲除小鼠的肠类器官来了解HA受体在高葡萄糖条件下的屏障保护中的作用。我们发现高葡萄糖应激降低了两种关键屏障蛋白 zona occlusionns-1 (ZO-1) 和 occludin 的蛋白质表达和空间分布。 HA35 防止 ZO-1 的降解或损失，并在高血糖应激下维持 ZO-1 和 occludin 的空间分布。 在功能上，我们还观察到HA35在高葡萄糖条件下对上皮屏障的保护作用。我们发现HA受体layilin参与防止屏障蛋白丢失(ZO-1)以及维持ZO-1和occludin的空间分布。此外，蛋白质组学分析表明，在高葡萄糖应激下，用 HA35 处理的类器官中细胞死亡和存活是上调的主要途径。我们发现 XIAP 相关因子 1 (Xaf1) 受 HA35 调节，从而调节肠道类器官系统中的凋亡细胞死亡。最后，我们观察到粘着斑激酶 (FAK) 和 F-肌动蛋白的空间组织是由 HA35 通过 Layilin 介导的。我们的结果强调了高血糖应激对肠道屏障功能的影响。这是具有临床意义的，因为在患有代谢综合征的个体中观察到屏障功能受损。此外，我们还证明了 HA35 通过其受体 Layilin 的屏障保护作用以及在高葡萄糖应激下调节细胞凋亡的作用。