The Permeability Transition in Hepatic Encephalopathy

描述（由申请人提供）：肝性脑病（HE）是严重肝功能衰竭患者发病和死亡的重要原因。急性HE合并暴发性肝功能衰竭预后极差，缺乏特异性治疗，除非紧急肝移植。尽管氨的发病机制尚不清楚，但氨与神经毒素密切相关，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变（MPT）作为HE和氨神经毒性发病机制的一个因素的参与。MPT是一种Ca2+依赖的环孢素a （CsA）敏感过程，由于线粒体内膜上的孔打开导致离子梯度的崩溃，最终导致线粒体功能障碍。我们最近发现氨可以诱导培养的星形胶质细胞的MPT。我们打算通过氨处理的神经细胞培养和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。这一概念的一个推论是，抑制或干扰星形胶质细胞MPT的发育可能会改善HE患者的中枢神经系统功能障碍。本课题的具体目的是：1)确定氨诱导神经细胞MPT的相关因素。我们的重点将放在与HE/H发病机制有关的药物上，这些药物也被证明可以在其他细胞中诱导MPT。具体来说，我们将研究ca2 +，活性氧，一氧化氮，pH值和谷氨酰胺的作用。我们将确定这些因子在氨处理培养物中是否升高，以及减少它们的产生或阻断它们的作用是否会减少或消除MPT。此外，我们将检查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作用。3)探讨线粒体功能障碍在mpt介导的细胞损伤中的潜在作用。我们将测定氨处理后线粒体功能的状态，进而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的因素（如CsA、三氟拉嗪）在体内是否也能起作用。此外，我们将评估MPT阻滞剂改善HE/HA中观察到的临床、组织病理学、神经化学异常和脑肿胀程度的能力。我们相信，这些研究将提供有关HE发病机制的关键数据，并可能有助于开发治疗这种疾病的新治疗策略。

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.