EFFECTS OF ETHANOL ON TNF CYTOTOXICITY

乙醇对 TNF 细胞毒性的影响

• 批准号: 6509107
• 负责人: JOHN G PASTORINO
• 金额: $ 11.07万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6509107
• 关键词: biological signal transduction; cell death; cytotoxicity; enzyme activity; ethanol; laboratory rat; liver cells; membrane permeability; mitochondria; mitogen activated protein kinase; pathologic process; phosphatidylinositol 3 kinase; phosphorylation; pore forming protein; tissue /cell culture; tumor necrosis factor alpha

Dr. Pastorino has extensive experience and publications in research on cell injury. The candidates goal is to become an independent investigator in the mechanisms by which ethanol brings about cell injury. Dr. Pastorino is a member of the Alcohol Research Center at Thomas Jefferson University. This environment offers him numerous opportunities to interact with established investigators in alcohol research. The research career development plan involves 6 components 1). Planning of experiments and interpretation of data, 2). Acquisition and training of laboratory personnel, 3). Grant management. 4). Handling of hazardous and radioactive materials, 5). Humane treatment of laboratory animals 6.) preparations of manuscripts and grant applications. Chronic ethanol exposure promotes the development of alcoholic liver disease (ALD) but the mechanisms underlying ethanol- induced hepatotoxicity remain poorly understood. Proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha) have been linked to many of the associated damage and repair processes seen in ALD. TNFalpha prompts the opening of the mitochondrial permeability transition pore (MPT). The MPT is the opening of a large, nonspecific pore across the outer and inner mitochondrial membrane. In preliminary results we demonstrate that a 48h exposure of HepG2 cells to ethanol, enhanced susceptibility to TNFalpha cytotoxicity. A similar enhancement of TNFalpha cytotoxicity is observed in primary cultured hepatocytes isolated from chronically ethanol-fed rats. The data suggest that ethanol enhances TNFalpha cytotoxicity, at least in part, by promoting the MPT. The general goals of this project are to investigate the mechanisms by which ethanol potentiates TNFalpha induced cytotoxicity. Our working hypothesis is that ethanol inhibits signaling pathways that protect against TNFalpha cytotoxicity and activates pathways which cause the mitochondrial permeability transition. This theory is supported by our preliminary observations that ethanol inhibits P13kinase dependent BAD phosphorylation induced by TNFalpha and promotes cell death in a p38 MAPK dependent and caspase 8 independent manner. Our SPECIFIC AIMS are to #1) define the role of ethanol in the alteration of PI3-kinase activity and its influence on TNFalpha cytotoxicity, #2) delineate the role of p38 MAPK activity in ethanol and TNFalpha cytotoxicity; and #3) delineate the mechanism by which TNFalpha plus ethanol activate Group II caspases.

帕斯托里诺博士在细胞损伤研究方面拥有丰富的经验和出版物。候选人的目标是成为乙醇导致细胞损伤机制的独立研究者。帕斯托里诺博士是托马斯·杰斐逊大学酒精研究中心的成员。这种环境为他提供了许多与酒精研究领域的知名研究人员互动的机会。研究生涯发展计划包括6个组成部分1)。2)实验计划和数据解释。3)实验室人员的获取和培训。格兰特管理。4）. 5)危险和放射性物质的处理；实验动物的人道待遇6)准备稿件和拨款申请。慢性乙醇暴露促进酒精性肝病（ALD）的发展，但乙醇诱导的肝毒性机制尚不清楚。促炎细胞因子如肿瘤坏死因子α (TNFalpha)与ALD中许多相关的损伤和修复过程有关。TNFalpha促进线粒体通透性过渡孔（MPT）的打开。MPT是一个大的、非特异性的孔，穿过线粒体内外膜。在初步结果中，我们证明HepG2细胞暴露于乙醇48小时，增强了对TNFalpha细胞毒性的敏感性。从长期乙醇喂养的大鼠分离的原代培养肝细胞中观察到类似的TNFalpha细胞毒性增强。数据表明，乙醇至少在一定程度上通过促进MPT来增强TNFalpha的细胞毒性。该项目的总体目标是研究乙醇增强TNFalpha诱导的细胞毒性的机制。我们的工作假设是，乙醇抑制了防止TNFalpha细胞毒性的信号通路，并激活了导致线粒体通透性转变的途径。我们的初步观察支持了这一理论，即乙醇抑制由TNFalpha诱导的p13激酶依赖的BAD磷酸化，并以p38 MAPK依赖和caspase 8独立的方式促进细胞死亡。我们的具体目标是#1)定义乙醇在pi3激酶活性改变中的作用及其对TNFalpha细胞毒性的影响；#2)描述p38 MAPK活性在乙醇和TNFalpha细胞毒性中的作用；和#3)描述TNFalpha加乙醇激活II组半胱天冬酶的机制。