5ASA REGULATION OF MNSOD IN INTESTINAL CELLS

炎症性肠病(溃疡性结肠炎和克罗恩病)是 一种慢性炎症性肠病，目前尚不清楚 要么导致，要么治愈。据估计，世界上有200万人 美国有IBD。IBD的发病高峰期在15岁至25岁之间 年龄和大多数IBD患者年龄在40岁以下。 美沙拉明(5-ASA)是柳氮磺胺吡啶的有效成分，是 治疗活动性疾病的主要疗法是什么？ 维持减刑。5-ASA的作用机制仍然存在 然而，不清楚的是，我们发现5-ASA诱导了一种细胞保护性 锰超氧化物歧化酶(MnSOD) 口服柳氮磺吡啶的患者在结肠内获得。锰超氧化物歧化酶是 已知的唯一的5-ASA调控基因。5-氨基水杨酸对MnSOD的诱导作用 可能会突显一种新的治疗机制。MnSOD可能会起到保护性作用 在肠道中的作用，预防或减少细胞因子和氧自由基 介入性损害。这在肠道中可能特别重要，因为它 含有低水平的抗氧化剂。 这项研究项目的目标是定义 控制5-ASA对肠道中MnSOD基因表达的调节 上皮细胞。具体的目标旨在准确地回答 关于参与调控的分子机制的几个问题 5-ASA诱导MnSOD基因的表达。在这个提案中，我们使用的是 反义MnSOD证实MnSOD在5-ASA诱导中的作用 细胞培养中的细胞保护。我们将确定5-ASA如何诱导 通过定义参与蛋白质-DNA相互作用的序列来实现MnSOD 这些基因与MnSOD的转录增强有关。在……里面 除了定义启动子中的顺式作用调控元件 区域，我们将识别和定义 锰超氧化物歧化酶的调节。我们的研究将涉及DNA酶I超敏反应 增强子的分析、启动子缺失分析和评价 使用瞬时转染法的元件。虽然MnSOD的诱导 是由连续的核实验确定的转录，我们将 也评估了MnSODmRNA的稳定作用作为一种贡献 MnSOD基因表达水平的诱导。我们的前提是 了解5-ASA诱导MnSOD的机制，我们将 能够识别和克隆相关的转录因子 作为设计其他治疗剂和5-ASA衍生物以发挥更多作用 MnSOD和其他可能尚未确定的5-ASA的有效诱导剂 受调控的基因。通过确定5-ASA等治疗剂如何 对IBD的疾病活动性产生有益的影响，我们可能会获得 进一步认识IBD的发病机制。

Inflammatory bowel disease (ulcerative colitis and Crohn's disease) are chronic inflammatory disease of the gut for which there is no known cause or cure. It has been estimated that 2 million people in the United States have IBD. The peak onset of IBD is between 15 and 25 years of age and the majority of patients with IBD are under age 40. Mesalamine (5-ASA) is the active ingredient in sulfasalazine and is one of the major therapies use to treat active disease and for the maintenance of remission. The mechanism of action of 5-ASA remains unclear, however, we have found that 5-ASA induces a cytoprotective enzyme, manganese superoxide dismutase (MnSOD), at concentrations obtained in the colon of patients taking sulfasalazine orally. MnSOD is the only known 5-ASA regulated gene. The induction of MnSOD by 5-ASA may highlight a new therapeutic mechanism. MnSOD may serve a protective role in the bowel and prevent or reduce cytokine and oxygen radical mediated damage. This may be particularly important in the bowel as it contains low levels of antioxidants. The goal of this research project is to define the mechanisms that control the 5-ASA regulation of MnSOD gene expression in intestinal epithelial cells. The specific aims are designed to answer precise questions on the molecular mechanisms involved in the regulation of MnSOD gene expression by 5-ASA. In this proposal, we are using antisense MnSOD to document the role of MnSOD in 5-ASA induced cytoprotection in cell culture. We will determine how 5-ASA induces MnSOD by defining the sequences involved in the protein-DNA interactions that are responsible for the enhanced transcription of MnSOD. In addition to defining the cis-acting regulatory elements in the promoter region, we will identify and define enhancer elements involved in the regulation of MnSOD. Our studies will involve Dnase I hypersensitivity analysis, promoter deletion analysis, and evaluation of enhancer elements using transient transfection. Although the induction of MnSOD is transcriptional as determined by nuclear run-on experiments, we will also evaluate the role of MnSOD mRNA stabilization as a contributor to the induction of MnSOD mRNA levels. It is our premise that by understanding the mechanisms by which 5-ASA induces MnSOD, we will be able to identify and clone the transcription factors involved as well as design other therapeutic agents and 5-ASA derivatives to act as more potent inducers of MnSOD and possibly other yet to be defined 5-ASA regulated genes. By determining how therapeutic agents such as 5-ASA exert a beneficial influence on the disease activity of IBD, we may gain a further understanding of the pathogenesis of IBD.