5ASA REGULATION OF MNSOD IN INTESTINAL CELLS

炎症性肠病（溃疡性结肠炎和克罗恩病）是 慢性炎症性疾病的肠道，其中没有已知的 原因或治疗。 据估计，200万人在 美国有IBD。 IBD的发病高峰在15 - 25岁之间 大多数IBD患者年龄在40岁以下。 美沙拉嗪（5-阿萨）是柳氮磺胺吡啶中的活性成分， 用于治疗活动性疾病的主要疗法， 维持缓解。 5-阿萨的作用机制仍然是 然而，我们发现5-阿萨诱导了一种细胞保护作用， 酶，锰超氧化物歧化酶（MnSOD），浓度 在口服柳氮磺胺吡啶的患者的结肠中获得。MnSOD是 唯一已知的5-阿萨调控基因。 5-阿萨对MnSOD的诱导作用 可能会突出一个新的治疗机制。 MnSOD可以起到保护作用， 在肠道中的作用，防止或减少细胞因子和氧自由基 介导的损害。 这在肠道中可能特别重要， 含有低水平的抗氧化剂。 本研究项目的目标是确定 控制5-阿萨对小肠MnSOD基因表达的调节 上皮细胞 具体目标旨在回答精确 关于参与调节的分子机制的问题 MnSOD基因表达5-阿萨。 在本提案中，我们使用 反义MnSOD，以证明MnSOD在5-阿萨诱导的 细胞培养中的细胞保护作用。 我们将确定5-阿萨如何诱导 MnSOD通过定义参与蛋白质-DNA相互作用的序列 负责增强MnSOD的转录。 在 除了定义启动子中的顺式作用调节元件之外 区域，我们将确定和定义参与的增强子元件， MnSOD的调节 我们的研究将涉及DNA酶I超敏反应 分析、启动子缺失分析和增强子的评估 使用瞬时转染的元件。 虽然MnSOD的诱导 是转录的核运行实验确定，我们将 还评估了MnSOD mRNA稳定作为一个贡献者的作用， MnSOD mRNA水平的诱导。 我们的前提是， 了解5-阿萨诱导MnSOD的机制，我们将 能够识别和克隆相关的转录因子， 作为设计其他治疗药物和5-阿萨衍生物， MnSOD和可能的其他尚未确定的5-阿萨的有效诱导剂 调控基因 通过确定治疗药物如5-阿萨 对IBD的疾病活动产生有益的影响，我们可能会获得 进一步了解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.