Genistein for the Prevention and Treatment of Diabetic Vascular Inflammation

描述(申请人提供)：这项研究的长期目标是通过开发新的生物活性治疗药物来降低与糖尿病血管功能障碍和相关并发症相关的发病率和死亡率。高血糖引起的血管炎症导致单核细胞与血管内皮细胞(ECs)的黏附，是糖尿病患者动脉粥样硬化发病的关键因素。我们的研究发现，植物学金雀异黄素抑制高血糖诱导的人白细胞-内皮细胞相互作用，提示金雀异黄素在糖尿病血管并发症中具有潜在的抗炎作用。白细胞与内皮细胞的黏附是通过内皮细胞上的趋化因子和黏附分子介导的，而这些分子的表达受到核因子kB(NF-kB)的严重上调。进一步分析表明，染料木素抑制高糖诱导的人主动脉内皮细胞(HAECs)产生单核细胞趋化蛋白-1(MCP-1)和白介素8(IL-8)，这两种趋化因子是单核细胞与活化内皮细胞牢固黏附的关键因素。该实验室最近的研究发现，金雀异黄素是内皮细胞中cAMP信号系统的激活剂。有趣的是，我们的研究表明，饮食中摄入染料木素增加了糖尿病小鼠的血浆cAMP，但降低了MCP-1的水平。我们这项建议的具体目的是确定染料木素抑制高血糖诱导的血管炎症的分子机制。我们推测金雀异黄素通过激活cAMP/PKA信号通路，抑制高糖诱导的单核细胞与内皮细胞的黏附，从而抑制核因子-kB的活性，进而抑制趋化因子和黏附分子的表达。目的1利用HAECs研究：1)金雀异黄素是否抑制高血糖诱导的趋化因子和黏附分子的表达；2)金雀异黄素是否抑制高血糖诱导的核因子-kB活性；3)金雀异黄素的抗炎作用是否通过cAMP/PKA途径介导。特殊目的2将使用糖尿病小鼠模型来确定：1)金雀异黄素是否在体内诱导cAMP/PKA信号转导；2)金雀异黄素是否抑制血管产生趋化因子和黏附分子；3)金雀异黄素是否抑制单核细胞向血管内皮细胞募集。金雀异黄素将通过营养补充的方法给化学诱导的和肥胖的糖尿病小鼠。蛋白质和信使核糖核酸的表达将通过蛋白质印迹、酶联免疫吸附试验或实时定量聚合酶链式反应进行评估。用试剂盒测定内皮细胞腺苷环化酶活性、cAMP和PKA活性。用酶联免疫吸附试验和报告基因测定法测定核因子-kB活性。CAMP/PKA级联通路在染料木素抗炎作用中的作用将通过药理学和分子干预方法来确定。从机理上了解染料木素如何预防糖尿病引起的血管炎症，将有助于开发有效的CAM策略，以保护糖尿病患者免受血管并发症的影响。 公共卫生相关性：动脉粥样硬化等心血管疾病是糖尿病患者死亡的主要原因。越来越多的人认识到，糖尿病患者血糖持续升高所介导的血管炎症在动脉粥样硬化的发病机制中起着关键作用。我们的研究表明，植物染料木素是一种很有前途的抗慢性高糖引起的血管炎症的药物。这项研究的结果可能会引导我们开发新的、天然的和具有成本效益的药物来预防和治疗糖尿病引起的动脉粥样硬化，糖尿病是一种影响超过2000万美国人的慢性病。

DESCRIPTION (provided by applicant): The long-term goal of this research is to decrease the morbidity and mortality related to diabetic vascular dysfunction and related complications, by developing novel, bioactive therapeutic agents. Hyperglycemia- induced vascular inflammation, leading to monocyte adhesion to vascular endothelial cells (ECs), is the key factor that initiates the pathogenesis of atherosclerosis in diabetic patients. Our studies found that botanical genistein inhibits hyperglycemia-induced human leukocyte-EC interaction, suggesting a potential anti- inflammatory action of genistein in diabetic vascular complications. Leukocyte adhesion to endothelium is mediated through both chemokines and adhesion molecules on ECs, and the expression of these molecules is critically up-regulated by nuclear factor kB (NF-kB). Further analysis showed that genistein suppressed high glucose-induced production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in human aortic ECs (HAECs), two chemokines that are the key factors in the firm adhesion of monocytes to activated ECs. Recent studies in this laboratory found that genistein is an activator of the cAMP signaling system in ECs. Interestingly, our studies showed that dietary intake of genistein increased plasma cAMP but reduced MCP-1 levels in diabetic mice. Our specific aim of this proposal is to determine the molecular mechanism by which genistein suppresses hyperglycemia-induced vascular inflammation. We hypothesized that genistein, by activation of the cAMP/PKA signaling, inhibits high glucose-induced monocyte adhesion to ECs through suppression of NF-kB activity and subsequent inhibition of chemokine and adhesion molecule expression. Specific aim 1 will use HAECs to investigate: 1) whether genistein inhibits hyperglycemia-induced expression of chemokines and adhesion molecules; 2) whether genistein inhibits hyperglycemia-induced NF-kB activity; and 3) whether the anti-inflammatory properties of genistein are mediated via the cAMP/PKA pathway. Specific aim 2 will use diabetic mouse models to determine: 1) whether genistein induces cAMP/PKA signaling in vivo; 2) whether genistein inhibits chemokine and adhesion molecule production from blood vessels; and 3) whether genistein suppresses monocyte recruitment to vascular ECs. Genistein will be administered through a nutritional supplementation approach to both chemically induced and obese diabetic mice. The protein and mRNA expression will be assessed by Western blot, ELISA or real-time PCR. The adenylate cylase activity, cAMP and PKA activation in ECs will be determined using assay kits. NF-kB activity will be determined with ELISA and reporter gene assay. The role of the cAMP/PKA cascade in the anti-inflammatory action of genistein will be determined by using both pharmacological and molecular intervention approaches. The mechanistic understanding of how genistein protects against diabetes-caused vascular inflammation will facilitate the development of efficacious CAM strategies to protect diabetic patients from vascular complications. PUBLIC HEALTH RELEVANCE: Cardiovascular disease such as atherosclerosis is the main cause of deaths in patients with diabetes mellitus. It is increasingly recognized that vascular inflammation mediated by sustained elevation of blood glucose levels in diabetic patients plays a pivotal role in the pathogenesis of atherosclerosis. Our studies indicate that botanical genistein is a promising agent to protect against chronic high glucose- caused vascular inflammation. The results of this research will potentially lead us to develop novel, natural, and cost-effective agents to prevent and treat atherosclerosis caused by diabetes, a chronic disease that affects over 20 million Americans.