Molecular Mechanisms of Genistein in the Prevention of Inflammatory Cytokine (TNF

描述（由申请人提供）：本研究的长期目标是鉴定生物活性成分并开发可改善血管功能的新型营养品，从而降低与血管功能障碍和相关并发症相关的发病率和死亡率。血管炎症及其随后的内皮功能障碍在动脉粥样硬化的发生和发展中起着重要作用。据信，肿瘤坏死因子（TNF）-α在动脉粥样硬化的发病机制中起关键作用。研究表明染料木黄酮可能具有血管保护作用。然而，染料木黄酮作用的分子机制还不清楚。本研究的主要目的是评估染料木黄酮在其对（TNF）-α诱导的血管功能障碍的保护作用中的细胞作用机制。我们已经收集了初步数据，这些数据表明染料木黄酮对血管功能障碍具有潜在的抗炎作用：1）在生理浓度下，染料木黄酮显著抑制TNF-α诱导的单核细胞与人脐静脉内皮细胞（HUVEC）的粘附，2）染料木黄酮显著抑制TNF-α诱导的单核细胞趋化蛋白-1（MCP-1）和白细胞介素-8（IL-8）的产生，这两种趋化因子是单核细胞与活化的内皮细胞牢固粘附的关键因子，（3）TNF-α显著增加NF-κ B结合活性，表明转录因子NF-κ B的活化可能是TNF-α诱导的炎症反应的关键。我们最近已经证明，染料木黄酮直接激活cAMP/蛋白激酶A（PKA）信号在内皮细胞，这是不相关的任何已知染料木黄酮行动，如抑制酪氨酸激酶或结合雌激素受体。我们的工作假设是，在生理浓度下，染料木黄酮通过激活cAMP/PKA信号传导（涉及质膜相关G蛋白的调节）抑制TNF-α诱导的单核细胞向内皮细胞的募集，随后抑制NF-κ B活性及其调节的趋化因子和粘附分子表达。因此，本申请的具体目的是：1）确定染料木黄酮是否抑制TNF-α诱导的粘附分子、血管炎症标志物和NF-κ B在HUVEC中的表达，2）确定染料木黄酮对TNF-α诱导的单核细胞与内皮细胞的粘附、趋化因子、粘附分子的表达以及NF-κ B活化的作用是否通过cAMP/PKA途径介导，以及染料木黄酮对cAMP/PKA级联的刺激是否通过G蛋白介导，3）研究膳食摄入染料木黄酮是否保护小鼠免受TNF-α诱导的血管炎症。上述目标的实现将建立染料木黄酮在其对血管功能障碍的保护作用中的细胞和分子作用机制。 公共卫生相关性：动脉粥样硬化性血管疾病是工业化国家发病率和死亡率的主要原因。我们的初步数据表明，染料木黄酮，一种大豆化合物，是一种有前途的药物，以防止血管功能障碍引起的促炎细胞因子肿瘤坏死因子（TNF）-α。染料木黄酮作用的分子机制的阐明将提供临床相关的预防动脉粥样硬化性血管疾病的策略。

DESCRIPTION (provided by applicant): The long-range goal of this research is to identify bioactive components and develop novel nutraceuticals that may improve vascular function and therefore decrease the morbidity and mortality related to vascular dysfunction and related complications. Vascular inflammation and its subsequent endothelial dysfunction play a fundamental role in the initiation and progression of atherosclerosis. It is believed that tumor necrosis factor (TNF)-alpha is critically involved in the pathogenesis of atherosclerosis. Studies show that genistein may have vascular protective effects. However, the molecular mechanism of genistein action is not well understood. The main objective of this study is to evaluate the cellular mechanism of action of genistein in its protective effect on (TNF)-alpha-induced vascular dysfunction. We have collected preliminary data, which suggest a potential anti-inflammatory action of genistein against vascular dysfunction: 1) genistein, at physiological concentrations, significantly inhibited TNF-alpha-induced adhesion of monocytes to human umbilical vein endothelial cells (HUVECs), 2) genistein significantly suppressed TNF-alpha-induced production of monocyte chemoattractant protein -1 (MCP-1) and interleukin-8 (IL-8), two chemokines that are the key factors in the firm adhesion of monocytes to activated endothelial cells, (3) TNF-alpha significantly increased NF-KB binding activity indicating that activation of the transcription factor NF-kB might be critical for the TNF-alpha-induced inflammatory response. We have recently demonstrated that genistein directly activates the cAMP/protein kinase A (PKA) signaling in endothelial cells, which is not related to any known genistein action such as inhibition of tyrosine kinase or binding to estrogen receptors. Our working hypothesis is that genistein, at physiological concentrations, suppresses TNF-alpha-induced recruit of monocytes to endothelial cells via activation of the cAMP/PKA signaling (involving modulation of plasma membrane associated G-proteins) that subsequently inhibits NF-kB activity and its regulated chemokine and adhesion molecular expression. Accordingly, the specific aims of this application are: 1) to determine whether genistein inhibits TNF-alpha-induced expression of adhesion molecules, markers of vascular inflammation and NF-kB in HUVECs, 2) to determine whether the effects of genistein on TNF- alpha-induced monocyte adhesion to endothelial cells, expression of chemokine, adhesion molecules as well as NF-KB activation is mediated through the cAMP/PKA pathway, and whether stimulation of cAMP/PKA cascade by genistein is mediated through G proteins, 3) to investigate whether dietary intake of genistein protects against TNF-alpha-induced vascular inflammation in mice. Fulfillment of the above aims will establish the cellular and molecular mechanisms of action of genistein in its protective effect against vascular dysfunction. PUBLIC HEALTH RELEVANCE: Atherosclerotic vascular disease is a major cause of morbidity and mortality in the industrial world. Our preliminary data indicate genistein, a soybean compound, is a promising agent to protect against vascular dysfunction caused by the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. The elucidation of the molecular mechanism(s) of action of genistein will provide clinically related strategies in protecting against atherosclerotic vascular disease.