Transcriptional Regulaton of Pro-Inflammatory Genes by Gambogic Acid

描述（由申请人提供）：本R21申请是为了响应题为“免疫调节机制”的RFA （#AT-07-005）而提交的。我们提出的研究的主要目的是了解一种重要的补充药物，具有悠久药用历史的藤黄酸（GA）的分子作用机制。GA是一种聚戊烯基化的山酮，带有？,吗?-不饱和羰基，从藤黄中分离得到，从藤黄和藤黄中提取树脂。许多具有这种活性碳中心的植物药被认为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，迄今为止尚未经过测试。由于？的存在，？-不饱和羰基，GA是一种有效的迈克尔受体，可以与包括转录因子在内的蛋白质中的游离巯基共价相互作用。我们的初步结果表明，GA对细菌脂多糖（LPS）诱导的核因子-?活化有抑制作用。B (NF - ? B)。NF - ?B是炎症驱动疾病进展的主要转录因子。我们将扩展这些研究，以阐明GA免疫调节炎症的潜在分子机制，并特别关注两个NF-？B靶向促炎基因，环氧化酶(COX)-2和肿瘤坏死因子(TNF)-？待验证的具体假设是GA下调COX-2和TNF？通过与NF-?的蛋白质组分形成共价Michael加合物B级联。该假设将在lps刺激的小鼠骨髓源性巨噬细胞中进行验证，并在C57/BL6小鼠体内炎症模型中进行验证，目的有两个：1)研究GA对COX-2和TNF -2转录调节的影响。2)阐明NF-？GA对B的激活和作用。研究将集中于GA通过COX-2和TNF -2的转录调节来减轻炎症。并将涉及使用基于荧光素酶的报告和染色质免疫沉淀测定。此外，GA与NF-?蛋白组分的相互作用B途径将通过在质谱和生化功能研究中使用生物素化ga进行评估。我们的长期目标是加强对补充和替代医学中植物药物作用的分子机制的理解，因为它们在炎症免疫调节中的作用。

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.