COCOA PAD II: Effect of Cocoa Flavanols on the Gut Microbiome and Functional Performance

摘要 下肢外周动脉疾病（PAD）影响全球超过2.3亿人，是一种主要的 残疾的原因。然而，PAD的药物治疗很少。从可可种子中提取的可可黄烷醇 可可，“可可”树，具有治疗特性，可以改善小腿肌肉灌注， 腓肠肌异常导致PAD残疾。我们最近资助的可可PAD II 试验（R01-AG068458）是一项多中心双盲随机试验，将测试可可的影响 黄烷醇与安慰剂对PAD患者6个月随访时6分钟步行距离变化的影响。 次要结局包括腓肠肌灌注、体力活动、肱动脉血流介导的扩张 以及线粒体活性和骨骼肌健康的腓肠肌活检测量。初步 有证据支持我们的假设，即肠道微生物群可能是可可有益效果的关键介质 黄烷醇首先，肠道微生物群代谢可可黄烷醇，包括表儿茶素（主要的可可黄烷醇）， 产生进入循环并到达靶器官的代谢物。代谢物发挥许多有益的 方面的影响.微生物衍生的黄烷醇代谢物增加内皮型一氧化氮合酶的磷酸化， 一氧化氮生成第二，饮食中补充可可黄烷醇可促进肠道微生物类群 与健康有关。因此，我们现在提议对可可PAD II试验进行一项辅助研究， 使我们能够收集和分析基线和6个月随访时收集的粪便和血浆样本， 将入组可可PAD II试验的100例PAD受试者。我们将测试以下具体 目的：首先，在PAD患者中，我们将确定基线肠道微生物多样性和 组合物的改善程度与对可可黄烷醇的反应程度有关， 6个月随访时6分钟步行距离和腓肠肌灌注。第二，我们将确定 与安慰剂相比，可可黄烷醇是否有利地改变了肠道微生物多样性和组成， 月随访。第三，我们将确定可可黄烷醇是否诱导肠道微生物的变化， 与安慰剂相比，基线和6个月随访之间， 6分钟步行距离和腓肠肌灌注改善。最后，我们将进行微生物- 在6个月随访时对血液样本进行相关代谢组学分析，以确定代谢组学特征， 与6个月随访时6分钟步行距离和腓肠肌灌注改善相关， 起来如果我们的假设是正确的，那么这项试验将首次将肠道微生物组确定为一种 改善PAD患者步行能力的关键介质。结果还将描绘一个 改善PAD行走能力的关键生物学途径，从而确定肠道微生物组 PAD未来干预的治疗靶点。

ABSTRACT Lower extremity peripheral artery disease (PAD) affects more than 230 million people worldwide and is a major cause of disability. Yet few medical therapies exist for PAD. Cocoa flavanols, from the seeds of theobroma cacao, the “cocoa” tree, have therapeutic properties that may improve calf muscle perfusion and reverse the gastrocnemius muscle abnormalities that contribute to disability in PAD. Our recently funded COCOA PAD II Trial (R01-AG068458) is a multi-center double-blinded randomized trial that will test the effects of cocoa flavanols vs. placebo on change in six-minute walk distance at six month follow-up in people with PAD. Secondary outcomes include gastrocnemius perfusion, physical activity, brachial artery flow-mediated dilation and gastrocnemius muscle biopsy measures of mitochondrial activity and skeletal muscle health. Preliminary evidence supports our hypothesis that gut microbiota may be key mediators of the beneficial effects of cocoa flavanols. First, gut microbiota metabolize cocoa flavanols including epicatechin (the main cocoa flavanol) to produce metabolites that enter the circulation and reach target organs. The metabolites exert many beneficial effects. Microbe-derived flavanol metabolites increase phosphorylation of endothelial nitric oxide synthase and nitric oxide production. Second, dietary supplementation of cocoa flavanols may promote gut microbial taxa associated with health. Therefore, we now propose an ancillary study to the COCOA PAD II Trial that will enable us to collect and analyze stool and plasma samples collected at baseline and six month follow-up from 100 participants with PAD who will be enrolled in the COCOA PAD II Trial. We will test the following specific aims: First, among people with PAD, we will determine whether baseline gut microbial diversity and composition are associated with the degree of response to cocoa flavanols, measured by greater improvement in six-minute walk distance and gastrocnemius perfusion at 6-month follow-up. Second, we will determine whether cocoa flavanols, compared to placebo, favorably alter gut microbial diversity and composition at six- month follow-up. Third, we will determine whether cocoa flavanol-induced changes in gut microbial composition, compared to placebo, between baseline and 6-month follow-up are associated with greater improvement in six-minute walk distance and gastrocnemius perfusion. Finally, we will perform microbe- associated metabolomic profiling of blood samples at six-month follow-up to identify metabolomic profiles that are associated with improved six-minute walk distance and gastrocnemius muscle perfusion at 6-month follow- up. If our hypotheses are correct, this trial will, for the first time, establish the gut microbiome as a critical mediator of improved walking performance in people with PAD. Results will also delineate a key biologic pathway of improved walking performance in PAD, thereby identifying the gut microbiome a therapeutic target for future interventions in PAD.