Mechanisms of Regulation of Intestinal Cl Absorption in IBD Associated Diarrhea

腹泻是炎症性肠病（IBD）的标志性症状。反复发作的痔疮不仅 显著影响治疗选择，也影响美国退伍军人IBD患者的生活质量。IBD 相关的腹泻是由于腔内NaCl和水吸收的减少以及 屏障完整性人体肠道吸收NaCl的主要途径是Na+/H+协同作用 和Cl-/HCO 3-交换器。SLC 26 A3或SLC 26 A3（在腺瘤中下调）是参与细胞凋亡的关键转运蛋白。 肠氯吸收。已经显示出E10基因敲除小鼠表现出类似于人类的E10表型。 CLD（先天性氯化物腹泻）和最近的全基因组关联研究（GWAS）将 IBD发病机制中表达失调的EST 1。此外，我们最近证明， 增强了肠道通透性并损害了屏障完整性。因此， 重要新靶点，用于干预结肠炎性疾病和IBD。因此，至关重要的是， 参与健康和疾病中的细胞因子调节的分子机制，并鉴定可以激活 抑制和/或抵消其下调。在这方面，糖皮质激素（GC）是重要的抗炎 是诱导IBD缓解的一线治疗药物。此外，GC已被证明可以稳定 上皮屏障功能，并通过恢复电解质和水的吸收发挥抗炎症作用， 慢性发炎的上皮然而，到目前为止，GC对表达和氯化物的直接影响还没有得到证实。 吸收尚未研究。我们目前的初步数据提供了强有力的证据， 例如地塞米松（DEX）通过糖皮质激素受体（GR）可以通过转录途径增加CRP 1表达， activation. DEX处理还增加了小鼠肠中的p53表达。因此，我们假设 DEX通过上调β-淀粉样蛋白表达和氯离子吸收发挥抗肿瘤作用。还可以根据 我们的初步数据表明，在肠道屏障完整性的关键作用，我们进一步假设， DEX介导的E2表达上调不仅可以改善腹泻， 屏障完整性因此，本申请被设计为研究在细胞内表达的蛋白质的调节机制。 DEX诱导的GR基因表达，GR受体和相关共激活因子在正常和 利用体外细胞培养物、离体人顶出肠样组织和体内小鼠肠样组织两者的炎性病症 包括DRA-KO小鼠和肠道特异性GR缺失小鼠。 阐明地塞米松诱导的转录调控ESTs表达和功能的机制及其作用 对抗TNF-α对TNF-α基因表达的抑制作用; 2.阐明潜在的机制 利用野生型和肠特异性GR敲除小鼠体内DEX的上调;和3.审查 DEX在逆转腹泻和恢复腹泻中作为一种新治疗方法的上调中的作用 实验性结肠炎小鼠的屏障完整性。我们提出的研究将产生重要的机械信息， 更好地理解GCs（如DEX）调节EMP 3功能和表达的机制 并且在推进我们的知识以开发更新和更好的治疗工具方面至关重要， 改善退伍军人的健康。

Diarrhea is the hallmark symptom of inflammatory bowel diseases (IBD). Recurring diarrheal episodes not only significantly affect treatment options, but also the quality of life of the US veteran patients with IBD. IBD associated diarrhea results from a decrease in luminal NaCl and water absorption as well as derangements in barrier integrity. Major route of NaCl absorption in the human intestine involves concerted operation of Na+/H+ and Cl-/HCO3- exchangers. SLC26A3 or DRA (Down-Regulated in Adenoma) is the key transporter involved in intestinal Cl- absorption. DRA knockout mice have been shown to exhibit diarrheal phenotype resembling human CLD (congenital chloride diarrhea) and recent genome-wide association studies (GWAS) have linked the dysregulated DRA expression to IBD pathogenesis. Further, we have recently demonstrated that DRA deficiency in mice enhances intestinal permeability and compromises barrier integrity. Thus, DRA has emerged as an important novel target for intervention in diarrheal disorders and IBD. Therefore, it is vital to characterize molecular mechanisms involved in DRA regulation in health and disease and to identify agents that can activate DRA and/or counteract its downregulation. In this regard, glucocorticoids (GCs) are important anti-inflammatory agents and are first line therapeutics for the induction of remission in IBD. Also, GCs have been shown to stabilize epithelial barrier function and exert antidiarrheal effects by restoring electrolyte and water absorption in chronically inflamed epithelium. However, to date, the direct effects of GCs on DRA expression and chloride absorption have not been investigated. Our current preliminary data provide strong evidence that synthetic GC such as Dexamethasone (DEX) via glucocorticoid receptor (GR) can increase DRA expression via transcriptional activation. DEX treatment also increased DRA expression in mouse intestine. Therefore, we hypothesized that DEX exerts antidiarrheal effects by upregulating DRA expression and chloride absorption. Also, in light of our preliminary data demonstrating a key role of DRA in intestinal barrier integrity, we further hypothesized that DEX mediated upregulation of DRA will not only ameliorate diarrhea but will also restore epithelial barrier integrity. The current application is, therefore, designed to investigate the mechanisms of regulation of DRA gene expression by DEX, role of GR receptor and the associated co-activators under normal and inflammatory conditions utilizing both in-vitro cell culture, ex-vivo human apical-out enteroids, and in-vivo mouse models including DRA-KO mice and mice with intestine specific deletion of GR. The Specific Aims are: 1. Elucidate the mechanisms of DEX-induced transcriptional regulation of DRA expression and function and its role in counteracting inhibitory effects of TNF- on DRA gene expression; 2. Elucidate the mechanisms underlying DRA up-regulation by DEX in-vivo utilizing wild type and intestine specific GR knockout mice; and 3. Examine the role of DEX in DRA upregulation as a novel therapeutic approach in reversal of diarrhea and restoration of barrier integrity in mice with experimental colitis. Our proposed studies will yield mechanistic information vital for a better understanding of the mechanisms of regulation of DRA function and expression by GCs such as DEX and are of critical importance in advancing our knowledge to develop newer and better therapeutic tools to improve the health of veterans.