Effect of Flavanol-Rich Cocoa Extract in Acute Neurodegenerative Conditions

描述（由申请人提供）：假设急性和慢性神经退行性疾病中神经元细胞死亡的决定因素是由自由基损伤介导的。表儿茶素和可可（可可可可），已被报道为神经保护和潜在的预防药物，但潜在的细胞机制尚不清楚。我们的初步结果促使我们将注意力集中在富含(-)-表儿茶素和黄烷醇的可可提取物上，并验证血红素加氧酶（HO）活性可能参与可可提取物神经保护功能的假设。HO可以裂解血红素（一种促氧化剂）形成胆绿素/胆红素（抗氧化剂）、一氧化碳（一种血管扩张剂）和铁（通过增加铁蛋白可以起到保护作用），已被证明在氧化应激、缺血、炎症和高血压中发挥保护作用。虽然HO2是组成性表达，但HO1是诱导性的。因此，通过增加HO1水平来增加内源性抗氧化系统的活性，可能是在细胞和器官水平上实现神经保护的一种方法。在我们使用初级神经元培养的初步实验中测试的化合物中，表儿茶素是最有效的HO1诱导剂之一。我们使用脑卒中模型的结果也表明，口服表儿茶素可显著预防脑缺血再灌注损伤。总之，我们的研究结果表明，单化合物表儿茶素（可可提取物中最丰富的黄烷醇）预处理足以提供内源性神经保护，这表明氧化应激期间的共同治疗是不必要的。这些初步结果表明，HO1的特异性诱导可能是可可提取物发挥其神经保护作用的机制，并促使我们提出可可提取物的一些神经保护作用可能是通过刺激内源性抗氧化途径介导的。在目的1中，我们将确定野生型（WT）小鼠在接受表儿茶素和/或富含黄烷醇的可可提取物（急性或慢性）预处理（急性或慢性）后的神经元细胞死亡和行为结果，并测试这些影响是否在敲除小鼠中减弱。在Aim 2中，我们将确定表儿茶素和/或富含黄烷醇的可可提取物诱导的HO1表达变化是否会导致WT和敲除小鼠的神经元培养细胞存活的变化。总之，这些结果将帮助我们确定食用标准化的可可提取物是否有益，以及可可提取物可以通过何种途径为大脑提供抵抗急性衰弱性神经退行性疾病的能力。

DESCRIPTION (provided by applicant): It has been postulated that the determinants of neuronal cell death in acute and chronic neurodegenerative conditions are mediated by free radical damage. Epicatechin and cocoa (Theobroma cacao), has been reported to be neuroprotective and a potential preventive medicine, but the underlying cellular mechanisms are still unclear. Our preliminary results prompted us to focus our attention on (-)-epicatechin and flavanol-rich cocoa extract and test the hypothesis that heme oxygenase (HO) activity could participate in cocoa extract neuroprotective function. HO, which cleaves heme (a prooxidant) to form biliverdin/bilirubin (antioxidants), carbon monoxide (a vasodilator), and iron (which by increasing ferritin would be protective) has been shown to play a protective role in oxidative stress, ischemia, inflammation, and hypertension. Although HO2 is constitutively expressed, HO1 is inducible. Consequently, an increase in the activity of this endogenous antioxidant system via an increase in HO1 levels, could be a way to achieve neuroprotection both at a cellular and organ levels. Of the compounds tested in our preliminary experiments using primary neuronal cultures, epicatechin was one of the most potent HO1 inducers. Our results using stroke models also suggest that epicatechin given orally significantly prevented ischemic-reperfusion brain damage. Together, our results indicate that pretreatment with the single compound epicatechin which is the flavanol most enriched in cocoa extract - and likely to cocoa extract itself - is sufficient to provide endogenous neuroprotection, suggesting that co-treatment during oxidative stress is not necessary. These preliminary results implied that specific induction of HO1 could be a mechanism by which cocoa extract exerts its neuroprotective actions and motivated us to propose that some of the neuroprotective effects attributed to Cocoa extract could be mediated through a pathway leading to stimulation of an endogenous antioxidant pathway. In Aim 1, we will determine neuronal cell death and behavioral outcomes following global ischemia in wildtype (WT) mice pre-treated (acutely or chronically) with epicatechin and/or flavanol-rich cocoa extract and test whether these effects are attenuated in knockout mice. In Aim 2, we will determine whether changes in HO1 expression induced by epicatechin and/or flavanol-rich cocoa extract result in changes in cell survival in neuronal cultures derived from WT and knockout mice. Together, these results will help us determine whether consumption of a standardized cocoa extract could be beneficial and the pathways by which cocoa extract could provide the brain with resistance to acute debilitating neurodegenerative conditions. PUBLIC HEALTH RELEVANCE: For centuries, cocoa (Theobroma cacao) has been reported as preventive medicine to strengthen the nervous system, but the underlying cellular mechanisms are still unclear. Our preliminary results prompted us to focus our attention on epicatechin and cocoa extract and test the hypothesis that the pathway leading to heme oxygenase (HO) enzyme induction could participate in cocoa's neuroprotective function. HO, which cleaves heme (a pro-oxidant) to form biliverdin/bilirubin (anti-oxidants) has been shown to play a protective role in oxidative stress, ischemia, inflammation, and hypertension. Using pre-clinical laboratory mouse models, we will determine whether prophylactic consumption of a standardized flavanol-rich cocoa extract can prevent neurological decline and neuronal cell death following global ischemia; thus providing new pathways by which cocoa could provide brain resistance against acute neurological disorders.