HERB-INDUCED OXIDANT PRECONDITIONING IN CARDIOMYOCYTES

描述(由申请人提供)：这项提案建立在以下研究的基础上：草药提取物黄芩及其主要成分黄芩素可能模拟心脏预适应(PC)的许多重要组成部分，如触发瞬时线粒体氧化信号，激活存活激酶Akt，抑制再灌注活性氧(ROS)和增加再灌注一氧化氮(NO)的生成。它是我们测试的为数不多的PC化合物之一，在3种致命的心肌缺血/再灌注(I/R)模型中提供显著保护：鸡心肌细胞、小鼠心肌细胞和小鼠心脏骤停。一种已经在流行的替代药物中使用的草药提取物在作为有不良心脏事件风险的患者的药理心脏PC制剂方面具有实际优势。本研究旨在通过确定产生保护性表型的机制，进一步研究SBE和/或黄芩素对小鼠心肌细胞PC的保护作用。我们推测SBE和/或黄芩素可能通过产生一过性ROS，启动PKCE和/或Akt的激活，从而激活eNOS，增加再灌注NO的生成，从而诱导PC对小鼠心肌细胞I/R损伤的保护作用。具体地说，Aim 1将优化触发ROS介导的PC的SBE和/或黄芩素的剂量，并使用各种抗氧化剂和线粒体抑制剂确定这些ROS的来源和重要性。进一步，我们将测试这些瞬时ROS是否诱导再灌流NO的增加和对细胞凋亡的保护。在目标2中，我们将评估最有效的草药诱导PC方案提供保护的途径。我们将通过PKCE易位、Akt磷酸化和Akt活性来评估中药诱导的PC中PKCE和/或Akt的激活。我们还将研究ROS在诱导PKCE和/或Akt激活中的作用，以及这种激酶激活对eNOS磷酸化和NO生成的影响，包括使用激酶抑制剂、siRNA基因沉默和eNOS和Akt-1的敲除策略。这些结果将有助于阐明小鼠心肌细胞中重要的PC通路，重点关注线粒体氧化剂、某些应激蛋白激酶和eNOS如何相互作用来保护I/R损伤。这项工作将被用于计划将草药-PC翻译到我们的心脏骤停小鼠模型中。最后，更好地了解SBE和/或黄芩素PC可能会导致新的氧化剂介导的PC临床治疗。与公共卫生相关：从普通人的角度来看，很少有药物可以预防未来的心脏病发作。此外，许多关于草药化合物作为替代药物的研究都集中在它们的抗氧化作用上。这项提案将研究一种草药混合物，这种混合物实际上会增加心脏中一种类型的氧化应激，这可能有助于心脏的状况，并防止未来的心脏病发作伤害。

DESCRIPTION (provided by applicant): This proposal builds upon work suggesting that the herbal extract Scutellaria baicalensis (SbE) and its major component baicalein may simulate many important components of cardiac preconditioning (PC), i.e. trigger transient mitochondrial oxidant signaling, activate the survival kinase Akt, attenuate reperfusion reactive oxygen species (ROS) and increase reperfusion nitric oxide (NO) generation. It is one of the few PC compounds we have tested that provides significant protection in 3 lethal models of cardiac ischemia/ reperfusion (I/R): chick cardiomyocytes, murine cardiomyocytes and murine cardiac arrest. An herbal extract already used in popular alternative medicine has practical advantages for use as a pharmacologic cardiac PC agent in patients at risk for adverse cardiac events. This proposal seeks to further examine PC protection induced by SbE and/or baicalein in murine cardiomyocytes by determining the mechanisms involved in generating the protective phenotype. We hypothesize that SbE and/or baicalein will induce PC protection against murine cardiomyocyte I/R injury via a transient ROS generation that initiates the activation of PKCe and/or Akt resulting in eNOS activation and increased reperfusion NO generation. Specifically, Aim 1 will optimize the dose of SbE and/or baicalein that trigger ROS-mediated PC, and determine the source and importance of these ROS using various antioxidants and mitochondrial inhibitors. Further, we will test whether these transient ROS induce augmented reperfusion NO and protection against apoptosis. In Aim 2, we will evaluate the pathway by which the most effective herb-induced PC protocol confers protection. We will assess the activation of PKCe and/or Akt in herb-induced PC using PKCe translocation, Akt phosphorylation, and Akt activity. We also will examine the role of ROS in inducing PKCe and/or Akt activation and the effect of this kinase activation on eNOS phosphorylation and NO generation using kinase inhibitors, siRNA gene silencing and knockout strategies for eNOS and Akt-1. These results will help clarify important PC pathways in murine cardiomyocytes, focusing on how mitochondrial oxidants, certain stress kinases, and eNOS may interact to protect against I/R injury. This work will be used to plan translational studies of herb-PC into our mouse model of cardiac arrest. Finally, a better understanding of SbE- and/or baicalein PC could lead to new oxidant-mediated PC clinical therapies. PUBLIC HEALTH RELEVANCE: From a layperson perspective, there are few medicines that protect against future heart attacks. In addition, many studies of herb compounds as alternative medicines have focused on their antioxidant effects. This proposal will study an herb mixture that actually increases a type of oxidant stress in the heart that may help condition it and protect against future heart attack injury.