Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland.
Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland; Baltimore Veterans Affairs Medical Center, Baltimore, Maryland.
Cell Mol Gastroenterol Hepatol. 2020;9(4):611-625. doi: 10.1016/j.jcmgh.2019.12.002. Epub 2019 Dec 18.
BACKGROUND & AIMS: The protective intestinal mucosal barrier consists of multiple elements including mucus and epithelial layers and immune defense; nonetheless, barrier dysfunction is common in various disorders. The imprinted and developmentally regulated long noncoding RNA H19 is involved in many cell processes and diseases. Here, we investigated the role of H19 in regulating Paneth and goblet cells and autophagy, and its impact on intestinal barrier dysfunction induced by septic stress.
Studies were conducted in H19-deficient (H19) mice, mucosal tissues from patients with sepsis, primary enterocytes, and Caco-2 cells. Septic stress was induced by cecal ligation and puncture (CLP), and gut permeability was detected by tracer fluorescein isothiocyanate-dextran assays. The function of Paneth and goblet cells was examined by immunostaining for lysozyme and mucin 2, respectively, and autophagy was examined by microtubule-associated proteins 1A/1B light chain 3 II immunostaining and Western blot analysis. Intestinal organoids were isolated from H19 and control littermate mice and treated with lipopolysaccharide (LPS).
Intestinal mucosal tissues in mice 24 hours after exposure to CLP and in patients with sepsis showed high H19 levels, associated with intestinal barrier dysfunction. Targeted deletion of the H19 gene in mice enhanced the function of Paneth and goblet cells and promoted autophagy in the small intestinal mucosa. Knockout of H19 protected Paneth and goblet cells against septic stress, preserved autophagy activation, and promoted gut barrier function after exposure to CLP. Compared with organoids from control littermate mice, intestinal organoids isolated from H19 mice had increased numbers of lysozyme- and mucin 2-positive cells and showed increased tolerance to LPS. Conversely, ectopic overexpression of H19 in cultured intestinal epithelial cells prevented rapamycin-induced autophagy and abolished the rapamycin-induced protection of the epithelial barrier against LPS.
In investigations of mice, human tissues, primary organoids, and intestinal epithelial cells, we found that increased H19 inhibited the function of Paneth and goblet cells and suppressed autophagy, thus potentially contributing to barrier dysfunction in intestinal pathologies.
保护性肠黏膜屏障由包括黏液和上皮层以及免疫防御在内的多种元素组成；然而，屏障功能障碍在各种疾病中很常见。印迹和发育调控的长链非编码 RNA H19 参与许多细胞过程和疾病。在这里，我们研究了 H19 在调节潘氏细胞和杯状细胞以及自噬中的作用，及其对脓毒症应激诱导的肠屏障功能障碍的影响。
在 H19 缺陷（H19）小鼠、脓毒症患者的黏膜组织、原代肠细胞和 Caco-2 细胞中进行了研究。通过盲肠结扎和穿孔（CLP）诱导脓毒症应激，并通过示踪荧光素异硫氰酸酯-葡聚糖检测法检测肠道通透性。通过免疫染色分别检测溶菌酶和粘蛋白 2 来检测潘氏细胞和杯状细胞的功能，通过微管相关蛋白 1A/1B 轻链 3 II 免疫染色和 Western blot 分析检测自噬。从小鼠中分离出 H19 和对照同窝仔鼠的肠类器官，并进行脂多糖（LPS）处理。
CLP 暴露 24 小时后，小鼠的肠道黏膜组织和脓毒症患者的组织中 H19 水平升高，与肠道屏障功能障碍有关。在小鼠中靶向敲除 H19 基因可增强潘氏细胞和杯状细胞的功能，并促进小肠黏膜中的自噬。H19 敲除可保护潘氏细胞和杯状细胞免受脓毒症应激，维持自噬激活，并促进 CLP 暴露后的肠道屏障功能。与对照同窝仔鼠的类器官相比，从小鼠中分离出的肠类器官具有更多的溶菌酶和粘蛋白 2 阳性细胞，并且对 LPS 的耐受性更高。相反，在培养的肠上皮细胞中异位过表达 H19 可阻止雷帕霉素诱导的自噬，并消除雷帕霉素诱导的上皮屏障对 LPS 的保护作用。
在对小鼠、人组织、原代类器官和肠上皮细胞的研究中，我们发现 H19 的增加抑制了潘氏细胞和杯状细胞的功能，并抑制了自噬，从而可能导致肠道病理中的屏障功能障碍。
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