Mechanisms of Nitrite Mediated Hepatic Preconditioning: Role of the Mitochondria

描述(由申请人提供)：肝脏缺血/再灌注(I/R)导致的肝功能障碍或衰竭是肝移植手术患者发病率和死亡率的主要原因。目前，尚无治疗策略，供需之间日益扩大的供需缺口迫使人们考虑身体或脂肪变性图表，这些图表非常容易受到I/R的影响。由于可能需要多个层面的干预才能恢复细胞和器官衰竭，最近几年探索的针对I/R损伤的最有前途的保护策略是预适应。因此，模拟这些效应的缺血预适应或药物干预可能最有可能改善肝移植和肝外科的临床结果。最近研究表明，亚硝酸盐通过产生一氧化氮(NO)在肝脏I/R过程中具有细胞保护作用，提示亚硝酸盐可能作为NO的生物储存库，在组织缺血损伤保护中发挥重要作用。此外，我们实验室的初步数据表明，在I/R前24小时给予亚硝酸盐可以保护肝脏免受损伤。此外，亚硝酸盐预适应可减轻L/R对线粒体呼吸的抑制作用。因此，本研究的目的是探讨线粒体在亚硝酸盐介导的肝脏预适应中的作用，其核心假设是亚硝酸盐预适应保留了肝I/R后线粒体的结构和功能，从而减轻了肝细胞的损伤。这项研究的理论基础是，确定亚硝酸盐预适应的机制可能为将其临床应用扩展到面临肝移植或肝脏手术的患者提供基础。因此，我们计划检验我们的中心假设，并通过追求两个具体目标来实现拟议研究的目标。具体目标1将进一步扩展我们的初步数据，通过评估线粒体基质体积、线粒体膜电位、线粒体对钙的摄取、不同复合体的线粒体呼吸、ATP产生、氧化还原电位、ROS产生，以及最终细胞色素C从线粒体释放到胞浆中来探索线粒体的保护作用。《特殊目的2》将退后一步，通过研究线粒体K+通道的作用来探讨亚硝酸盐预适应的信号机制。我们认为，亚硝酸盐通过转化为NO，分别通过PKC和PKA的分子信号转导线粒体K+通道mKATP和mKCa的开放，从而保护线粒体免受随后的I/R损伤。

DESCRIPTION (provided by applicant): Liver dysfunction or failure, as a result of hepatic ischemia/reperfusion (I/R), is a leading cause of morbidity and mortality for patients who have undergone liver transplantation surgery. Currently, no therapeutic strategy exists and the ever growing gap between supply and demand for donors has forced the consideration of cadaveric or steatotic graphs, which are very susceptible to I/R. Because intervention on more than one level is likely needed to allow for the recovery of cellular and organ failure, the most promising protective strategy against I/R injury explored in the last few years is preconditioning. Therefore, ischemic preconditioning or pharmacological interventions that mimic these effects may have the greatest potential to improve clinical outcome in liver transplantation and liver surgery. Recently, nitrite through the generation of nitric oxide (NO) has been shown to have cytoprotective effects in the setting of hepatic I/R, suggesting that nitrite may serve as a biological storage reserve of NO subsurving a critical function in tissue protection from ischemic injury. Furthermore, preliminary data from our lab indicates that the administration of nitrite 24 hours prior to I/R protects the liver against injury. Moreover, nitrite preconditioning was found to attenuate the l/R-induced suppression of mitochondrial respiration. Therefore, the objective of this proposal is to delve into the role of the mitochondria in nitrite mediated hepatic preconditioning with the central hypothesis being that nitrite preconditioning preserves the structure and function of the mitochondria following hepatic I/R, thereby attenuating hepatocellular injury. The rationale for the proposed research is that identifying the mechanisms of nitrite preconditioning may provide a basis for extending the clinical application to patients facing liver transplantation or liver surgery. So, we plan to test our central hypothesis and accomplish the objective of the proposed study by pursuing two specific aims. Specific aim 1 will further expand on our preliminary data by exploring the-protection of the mitochondria through the evaluation of mitochondrial matrix volume, mitochondrial membrane potential, mitochondiral uptake of calcium, mitochondrial respiration of the different complexes, ATP production, redox potential, ROS production, and finally the release of cytochrome C from the mitochondria into the cytosol. Specific aim 2 will take a step back and explore the signaling mechanisms of nitrite preconditioning by examining the role of mitochondrial K+ channels. We believe that nitrite, through its conversion to NO, protects the mitochondria from a subsequent I/R injury by mediating the opening of the mitochondrial K+ channels, mKATP and mKCa, via the molecular signaling of PKC and PKA, respectively.