Mechanisms of Neutrophil-induced Liver Toxicity

描述（申请人提供）：缺血再灌注（IRP）损伤是一个重要的临床问题，它决定了肝移植、肝手术和出血性或败血性休克后的发病率和死亡率。IRP损伤在脂肪肝中是一个更大的问题。自我延续的炎症反应是再灌注过程中整体组织损伤的关键组成部分。常驻巨噬细胞（Kupffer细胞）和募集的中性粒细胞是直接导致实质细胞损伤的主要细胞类型。这些炎症细胞产生的活性氧（ROS）扩散到肝细胞中，引发细胞内氧化应激，最终导致坏死细胞死亡。巯基可以提供必要的电子来还原灭活这些活性氧。因此，我们假设增强靶细胞的内源性抗氧化能力可能最有效地预防Kupffer细胞和中性粒细胞对细胞的损伤，从而在不损害这些细胞重要的宿主防御功能的情况下减轻炎症反应。金属硫蛋白是一种富含巯基的多肽，每个分子含有18-23个半胱氨酸残基。MT-1和MT-2是高度诱导的基因，在体外已被证明是有效的活性氧清除剂。然而，对于瘦肝和脂肪肝在肝脏IRP或缺血预处理期间MTs的诱导，几乎一无所知。因此，本研究的总体目标是全面分析金属硫蛋白的化学后基因表达，研究其在肝脏IRP损伤和乙醇诱导的脂肪变性肝中的病理生理作用，以及利用MT基因表达作为治疗靶点的潜力。因此，我们的具体实验目标是：1 /2。在瘦肝和脂肪肝IRP损伤过程中，表征MTs的化学诱导并评估其对Kupffer细胞和中性粒细胞诱导的氧化应激和损伤的潜在保护作用或其对微循环功能障碍的影响。3. 评估缺血预处理过程中MTs的诱导及其对瘦肝和脂肪变性肝脏保护作用的总体影响。4. 研究MT诱导培养小鼠肝细胞的保肝机制。该项目将为开发一种新的治疗策略提供临床前基础，该策略可以选择性地针对再灌注期间过度炎症反应的有害影响，而不会损害中性粒细胞和库普弗细胞的宿主防御功能。公共卫生相关性：库普弗细胞和中性粒细胞激活的炎症反应导致多种肝脏病理，包括肝缺血-再灌注损伤（移植、肿瘤切除）、酒精性肝炎、脂肪性肝炎和阻塞性胆汁淤积。然而，这些白细胞也履行宿主防御入侵微生物的重要功能。因此，确定和测试新的治疗策略至关重要，这些策略可以选择性地针对过度炎症反应的有害影响，而不损害中性粒细胞和库普弗细胞的宿主防御功能。

DESCRIPTION (provided by applicant): Ischemia-reperfusion (IRP) injury is an important clinical problem, which determines morbidity and mortality after liver transplantation, liver surgery and hemorrhagic or septic shock. IRP injury is an even bigger issue in steatotic livers. A self-perpetuating inflammatory response is a key component of the overall tissue injury during reperfusion. Resident macrophages (Kupffer cells) and recruited neutrophils are the main cell types directly responsible for parenchymal cell damage. Reactive oxygen species (ROS) generated by these inflammatory cells diffuse into hepatocytes and trigger an intracellular oxidant stress, which ultimately causes necrotic cell death. Sulfhydryl groups can provide the necessary electrons to reductively inactivate these ROS. Therefore, we hypothesize that strengthening the endogenous antioxidant capacity in target cells may be most effective in preventing cell injury by Kupffer cells and neutrophils and thereby attenuating the inflammatory response without compromising vital host-defense functions of these cells. Metallothioneins (MTs) are sulfhydryl-rich polypeptides, which contain 18-23 cysteine residues per molecule. MT-1 and MT-2 are highly inducible genes and have been shown to be effective scavengers of ROS in vitro. However, virtually nothing is known about the induction of MTs during hepatic IRP or ischemic preconditioning in both lean and steatotic livers. Therefore, the overall aims of this proposal are to provide a comprehensive analysis of the postischemic gene expression of metallothioneins and to study their pathophysiological role during hepatic IRP injury in lean livers and ethanol-induced steatotic livers as well as their potential to use MT gene expression as therapeutic target. Hence our specific experimental aims are: 1./2. Characterize postischemic induction of MTs and evaluate the potential protection against a Kupffer cell and neutrophil-induced oxidant stress and injury or its effects on micro-circulatory dysfunction during IRP injury in lean and steatotic livers. 3. Evaluate the induction of MTs during ischemic preconditioning and its overall impact on the protective effects of this procedure in lean and steatotic livers. 4. Study hepatoprotective mechanisms of MT induction in cultured mouse hepatocytes. The project will provide the preclinical basis for the development of a novel therapeutic strategy, which selectively targets the detrimental effects of an excessive inflammatory response during reperfusion without compromising the host defense function of neutrophils and Kupffer cells. Public Health Relevance: An inflammatory response with activation of Kupffer cells and neutrophils contribute to a wide variety of liver pathologies including hepatic ischemia-reperfusion injury (transplantation, tumor resections), alcoholic hepatitis, steatohepatitis and obstructive cholestasis. However, these leukocytes also fulfill a vital host defense function against invading microorganisms. Therefore, it is critical to identify and test novel therapeutic strategies, which selectively target the detrimental effects of an excessive inflammatory response without compromising the host defense function of neutrophils and Kupffer cells.