The Permeability Transition in Hepatic Encephalopathy

描述（由申请方提供）：肝性脑病（HE）是重度肝衰竭患者发病和死亡的重要原因。急性肝性脑病合并暴发性肝功能衰竭预后极差，除紧急肝移植外，尚无特异性治疗。虽然其发病机制仍然知之甚少，氨强烈牵连作为一种神经毒素，和星形胶质细胞似乎是氨神经毒性的主要目标。此外，改变的生物能量学和氧化应激被认为在这种疾病中起主要作用。这些事实导致考虑线粒体通透性转换（MPT）的参与作为HE和氨神经毒性的发病机制中的一个因素。MPT是一种Ca 2+依赖性、环孢菌素A（CsA）敏感性过程，由于线粒体内膜中孔的打开导致离子梯度的崩溃并最终导致线粒体功能障碍。我们最近发现，氨诱导的MPT在培养的星形胶质细胞。我们打算使用氨处理的神经细胞培养物和HE/高氨血症（HA）的体内模型来研究MPT在HE和高氨血症中的作用。我们的工作假设是氨诱导星形胶质细胞中的MPT，最终导致线粒体衰竭和星形胶质细胞功能障碍。这一概念的推论是，抑制或干扰星形胶质细胞中MPT的发展可能会改善HE中的CNS功能障碍。本研究的具体目的是：1）确定氨诱导神经细胞MPT的相关因子。我们的重点将是在HE/H的发病机制，也已被证明在其他细胞中诱导MPT牵连的代理。具体而言，我们将研究的作用，钙2+，活性氧，一氧化氮，pH值和谷氨酰胺。我们将确定这些因素是否在氨处理的培养物中升高，以及减少它们的产生或阻止它们的作用是否会减少或消除MPT。此外，我们将研究这些因素之间可能的顺序相互关系。2)为了确定氨诱导的星形胶质细胞异常（形态学改变，神经递质摄取缺陷和细胞肿胀）是否由MPT介导，我们将研究MPT抑制剂（CsA，bongkrekik酸）是否能够减少或阻断氨的有害影响。3)研究线粒体功能障碍作为MPT介导的细胞损伤的潜在因素的参与。我们将确定氨处理后线粒体功能的状态，然后研究改善能量代谢是否会抑制氨诱导的细胞损伤。4)阐明MPT是否发生在HE（硫代乙酰胺治疗）和高氨血症的体内模型中。我们还将确定体外抑制MPT的因子（例如，CsA、三氟拉嗪）也能够在体内这样做。此外，我们将评估MPT阻滞剂改善临床、组织病理学、神经化学异常以及HE/HA中观察到的脑肿胀程度的能力。我们相信，这些研究将产生重要的数据，对HE的发病机制，并可能有助于开发新的治疗策略，治疗这种情况。

DESCRIPTION (provided by applicant): Hepatic encephalopathy (HE) is an important cause of morbidity and mortality in patients with severe liver failure. Acute HE associated with fulminant hepatic failure has an extremely poor prognosis and specific therapy is not available, short of an emergency liver transplantation. Although its pathogenesis remains poorly understood, ammonia is strongly implicated as a neurotoxin, and astrocytes appear to be the primary target of ammonia neurotoxicity. Additionally, altered bioenergetics and oxidative stress are thought to play a major role in this disorder. These facts led to a consideration of the involvement of mitochondrial permeability transition (MPT) as a factor in the pathogenesis of HE and ammonia neurotoxicity. The MPT is a Ca2+-dependent, cyclosporin A (CsA)-sensitive process due to the opening of a pore in the inner mitochondrial membrane leading to a collapse of ionic gradients and ultimately to mitochondrial dysfunction. We have recently shown that ammonia induced the MPT in cultured astrocytes. We intend to examine the role of the MPT in HE and hyperammonemia using ammonia-treated neural cell cultures and in vivo models of HE/hyperammonemia (HA). Our working hypothesis is that ammonia induces the MPT in astrocytes, culminating in mitochondrial failure and astroglial dysfunction. A corollary of this concept is that inhibition or interference in the development of the MPT in astrocytes may ameliorate CNS dysfunction in HE. The Specific Aims of this proposal are: 1) To identify the factors responsible for the ammonia-induced MPT in cultured neural cells. Our focus will be on agents implicated in the pathogenesis of HE/H that have also been shown to induce the MPT in other cells. Specifically, we will examine the role of Ca 2+, reactive oxygen species, nitric oxide, pH and glutamine. We will determine whether these factors are elevated in ammonia-treated cultures, and whether diminishing their production or blocking their actions reduces or abolishes the MPT. Additionally, we will examine possible sequential interrelationships among these factors. 2) To determine whether ammonia-induced abnormalities in astrocytes (morphological alterations, defects in neurotransmitter uptake, and cell swelling) are mediated by the MPT, we will investigate whether inhibitors of the MPT (CsA, bongkrekik acid) are capable of diminishing or blocking the deleterious effects of ammonia. 3) To investigate the involvement of mitochondrial dysfunction as a potential factor in MPT-mediated cell injury. We will determine the state of mitochondrial function after ammonia treatment, and then investigate whether improving energy metabolism will inhibit ammonia-induced cellular injury. 4) To clarify whether the MPT occurs in in vivo models of HE (thioacetarnide treatment) and hyperammonemia. We will also determine whether factors that inhibit the MPT in vitro (e.g., CsA, trifluoperazine) are also capable of doing so in vivo. Additionally, we will assess the ability of MPT blockers to improve the clinical, histopathologic, neurochemical abnormalities, and the extent of brain swelling observed in HE/HA. We believe that these studies will yield critical data bearing on the pathogenesis of HE, and may potentially aid in the development of novel therapeutic strategies for the treatment of this condition.