Mechanisms of Regulation of Intestinal Cl Absorption in IBD Associated Diarrhea

腹泻是炎症性肠病(IBD)的显著症状。反复发作的腹泻不仅 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 相关腹泻的原因是管腔内氯化钠和水分吸收减少，以及 屏障完整性。人体肠道吸收氯化钠的主要途径是Na/H的协同作用 和Cl-/HCO3-交换剂。SLC26A3或DRA(腺瘤中下调)是参与 肠道氯离子吸收。DRA基因敲除小鼠表现出与人类类似的腹泻表型 CLD(先天性氯化物腹泻)和最近的全基因组关联研究(GWAS)将 DRA表达异常在IBD发病机制中的作用此外，我们最近证明了DRA缺乏 在小鼠体内，提高肠道通透性，损害屏障完整性。因此，DRA已经成为一种 腹泻疾病和IBD干预的重要新靶点。因此，至关重要的是要刻画出 参与DRA调节的分子机制在健康和疾病中的作用以及识别能够激活DRA的因素 DRA和/或抵消其下调监管。在这方面，糖皮质激素(GC)是重要的抗炎药物 这些药物是诱导IBD缓解的一线治疗药物。此外，GC已被证明是稳定的 大鼠上皮屏障功能及通过恢复电解质和水分吸收发挥止泻作用 慢性发炎的上皮细胞。然而，到目前为止，GC对DRA表达和氯离子的直接影响 吸收还没有被研究过。我们目前的初步数据提供了强有力的证据，证明合成GC 如地塞米松(DEX)通过糖皮质激素受体(GR)可通过转录增加DRA的表达 激活。地塞米松还可增加小鼠肠道DRA的表达。因此，我们假设 地塞米松通过上调DRA表达和氯离子吸收发挥止泻作用。此外，在光线下 根据我们的初步数据显示DRA在肠道屏障完整性中的关键作用，我们进一步假设 地塞米松介导的DRA上调不仅能缓解腹泻，还能修复上皮细胞 屏障完整性。因此，目前的应用被设计为研究 DEX对DRA基因的表达、GR受体及其相关共激活因子在正常和 利用体外细胞培养、体外人类根尖外肠样体和体内小鼠的炎症条件 模型包括DRA-KO小鼠和肠道特异性GR缺失小鼠。具体目标是：1. 地塞米松诱导DRA表达和功能的转录调控机制及其作用 对抗肿瘤坏死因子-对DRA基因表达的抑制作用；2.阐明其作用机制 利用野生型和肠道特异性GR基因敲除小鼠体内DEX上调DRA；以及3.检查 地塞米松作为一种新的治疗途径在DRA上调中的作用 实验性结肠炎小鼠的屏障完整性。我们提议的研究将产生对以下方面至关重要的机械信息 更好地理解DEX等GC对DRA功能和表达的调节机制 在发展我们的知识以开发更新和更好的治疗工具方面具有至关重要的作用 改善退伍军人健康状况。

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.