Transcriptional Regulaton of Pro-Inflammatory Genes by Gambogic Acid

描述(由申请人提供)：本R21申请是对题为“免疫调节机制”的RFA(#AT-07-005)的回应。我们提出的研究的主要目的是了解一种重要的补充药物--藤黄酸(GA)的分子作用机制，该药物具有悠久的药用历史。GA是从藤黄、藤黄和藤黄的树脂中分离得到的一种聚戊烯化的口山酮，含有？，？-不饱和的羰基。已知许多具有这种活性碳中心的植物具有有趣的药理特性。在这方面，具有抗肿瘤活性的GA被认为具有抗炎活性，但到目前为止还没有得到测试。由于？，？-不饱和羰基的存在，GA是一种有效的Michael受体，可以与蛋白质中的自由硫醇基，包括转录因子，共价相互作用。我们的初步结果表明，GA对细菌脂多糖(LPS)诱导的核因子-B(NF-B)的激活具有抑制作用。核因子？B是一种主要的转录因子，负责炎症驱动的疾病进展。我们将继续这些研究，以阐明GA免疫调节炎症的潜在分子机制，特别是两个核因子-B靶向致炎基因的转录，环氧合酶(COX)-2和肿瘤坏死因子(TNF)-？需要检验的具体假设是GA下调COX-2和肿瘤坏死因子？通过与核因子-？B级联的蛋白质组分形成共价的迈克尔加合物。这一假说将通过脂多糖刺激的小鼠骨髓来源的巨噬细胞进行验证，并在C57/BL6小鼠体内的炎症模型中得到验证，目的有两个：1)检测GA对COX-2和TNF-α转录调控的影响；2)阐明GA抑制NF-B激活和功能的分子机制。研究将集中在GA通过对COX-2和TNF？转录的调节来减轻炎症。并将涉及使用基于荧光素酶的报告和染色质免疫沉淀分析。此外，在质谱学和生化功能研究中，将使用生物素化的GA来评估GA与核因子-B途径的蛋白质组分的相互作用。我们的长期目标是加强对补充和替代医学中使用的植物药物在炎症免疫调节中作用的分子作用机制的了解。 公共卫生：藤黄是藤黄科(Guttiferae)藤黄科(Guttiferae)藤黄(Garcinia Morella)和藤黄(G.hanburyi)的树脂，原产于亚洲、澳大利亚、南部非洲和波利尼西亚，含有丰富的聚戊烯基口山酮--藤黄酸(GA)。藤黄提取物治疗多种疾病的药用历史悠久。有趣的是，GA的抗肿瘤活性已经得到很好的证明，并被认为部分是由于GA的抗炎活性所致。然而，GA的抗炎活性尚未得到测试。我们建议在体外和体内炎症模型中研究GA下调促炎基因NF-B依赖的表达的分子基础。

DESCRIPTION (provided by applicant): This R21 application is submitted in response to an RFA (#AT-07-005) entitled "Mechanisms of Immune Modulation." The primary objective of our proposed studies is to understand the molecular mechanism of action of an important complementary medicine, gambogic acid (GA) that has a long history of medicinal use. GA is a polyprenylated xanthone, with an ?, ?-unsaturated carbonyl group, isolated from gamboges, the resin from Garcinia morella and Garcinia hanburyi. Many botanicals with such a reactive carbon center are known to possess interesting pharmacological properties. In this regard, GA, which possesses anti-tumor activity, is thought to possess anti-inflammatory activities that have so far remained untested. Due to the presence of the ?,?-unsaturated carbonyl group, GA is a potent Michael acceptor that can covalently interact with free thiol groups in proteins, including transcription factors. Our preliminary results demonstrate an inhibitory effect of GA on the bacterial lipopolysaccharide (LPS)-induced activation of nuclear factor-?B (NF-?B). NF-?B is a major transcription factor responsible for inflammation-driven disease progression. We will extend these studies to elucidate the underlying molecular mechanism of immune modulation of inflammation by GA with a specific focus on the transcription of two NF-?B target pro-inflammatory genes, cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)-?. The specific hypothesis to be tested is that GA down-regulates the transcription of COX-2 and TNF? via the formation of covalent Michael adducts with protein components of the NF-?B cascade. The hypothesis will be tested using LPS-stimulated murine bone marrow-derived macrophages and validated in an in-vivo inflammation model, in C57/BL6 mice, in two Specific Aims: 1) To examine the effect of GA on the transcriptional regulation of COX-2 and TNF?, and 2) To elucidate the molecular mechanism of attenuation of NF-?B activation and function by GA. Studies will be focused on attenuation of inflammation by GA via the transcriptional regulation of COX-2 and TNF? and will involve the use of luciferase-based reporter and chromatin immunoprecipitation assays. Furthermore, the interaction of GA with protein components of the NF-?B pathway will be assessed by using biotinylated-GA in mass spectrometric and biochemical functional studies. Our long-term objective is to enhance the understanding of the molecular mechanism of action of botanicals used in complementary and alternative medicine for their role in immune regulation of inflammation. PUBLIC HEALTH REVELANCE: Gamboges, the resin from Garcinia morella and G. hanburyi that belong to the family Clusiaceae (Guttiferae), which are native to Asia, Australia, Southern Africa, and Polynesia, are rich in a polyprenylated xanthone, gambogic acid (GA). There is a long history of medicinal use of Garcinia extracts against many ailments. Interestingly, the anti-tumor activity of GA has been well demonstrated and is thought to arise partly due to the anti-inflammatory activity associated with GA. However, the anti-inflammatory activity of GA has not been tested. We propose to study the molecular basis of the down-regulation of NF-?B-dependent expression of pro-inflammatory genes by GA in in vitro and in vivo models of inflammation.