Effects of Ethanol on TNF Cytotoxicity

描述(申请人提供)：接触酒精可促进发育 酒精性肝病(ALD)的发生，但乙醇诱导的机制 肝脏毒性仍然知之甚少。一个重要的蜂窝站点显示 乙醇造成的损伤是线粒体。促炎细胞因子 例如肿瘤坏死因子α(TNFpha或TNF)与许多 在ALD中看到的相关损坏和修复过程。肿瘤坏死因子阿尔法提示 线粒体通透性转换孔(MPT)开放。MPT是 可调节的穿过内外的大的、非特定的孔开口 线粒体膜。MPT的发展牵涉到这两个问题 坏死性和某些形式的凋亡性细胞死亡。抑制MPT可防止 在细胞凋亡过程中出现的许多典型症状。肝脏线粒体 从长期摄入酒精的大鼠分离出来的菌株对MPT毛孔更敏感 与对照组分离的线粒体相比，不同的触发物诱导的开放 动物。在初步结果中，我们展示了对肿瘤坏死因子α的增强 慢性酒精喂养大鼠肝细胞毒性的比较 它们的配对控制系统。同样，将HepG2细胞暴露在乙醇中48小时 增强了对肿瘤坏死因子α细胞毒性的敏感性。数据表明，乙醇 至少部分地通过促进MPT来增强肿瘤坏死因子α的细胞毒性。这个 这个项目的总体目标是研究 乙醇可增强肿瘤坏死因子α诱导的细胞毒作用。我们的工作假设是 乙醇抑制了对抗肿瘤坏死因子α的信号通路 细胞毒性和激活促进线粒体通透性的途径 过渡。这一理论得到了我们初步观察的支持 乙醇抑制肿瘤坏死因子α诱导的P13蛋白依赖的不良磷酸化 并促进依赖p38MAPK和非依赖caspase 8的细胞死亡 举止。此外，酒精暴露会使线粒体对MPT和 导致外周苯二氮卓类受体水平的增加 推测为MPT孔的成分。我们的具体目标是进一步定义 乙醇在P13-激酶活性变化中的作用及其影响 论肿瘤坏死因子α的细胞毒作用及机制(S) 乙醇中p38MAPK的激活加上肿瘤坏死因子α的细胞毒性；#3)定义 肿瘤坏死因子加乙醇诱发MPT的机制(S)。

DESCRIPTION (provided by applicant): Ethanol exposure promotes the development of alcoholic liver disease (ALD) but the mechanisms underlying ethanol-induced hepatotoxicity remain poorly understood. An important cellular site manifesting damage brought on by ethanol is the mitochondria. Proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha or TNF) 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 regulatable opening of a large, nonspecific pore across the outer and inner mitochondrial membrane. Development of the MPT has been implicated in both necrotic and some forms of apoptotic cell death. Inhibition of the MPT prevents many of the typical manifestations seen during apoptosis. Liver mitochondria isolated from chronically ethanol fed rats are more susceptible to MPT pore opening induced by various triggers than mitochondria isolated from control animals. In preliminary results we demonstrate an enhancement of TNFalpha cytotoxicity in hepatocytes isolated from chronically ethanol-fed rats compared to their pair-fed controls. Similarly, a 48h exposure of HepG2 cells to ethanol enhanced susceptibility to TNFalpha cytotoxicity. 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 that promote the mitochondrial permeability transition. This theory is supported by our preliminary observations that ethanol inhibits P13-kinase dependent BAD phosphorylation induced by TNFalpha and promotes cell death in a p38 MAPK dependent and caspase 8 independent manner. In addition, ethanol exposure sensitizes mitochondria to the MPT and causes an increase in the levels of the peripheral benzodiazepine receptor, a putative component of the MPT pore. Our SPECIFIC AIMS are to #1) further define the role of ethanol in the alteration of P13-kinase activity and its influence on TNFalpha cytotoxicity and #2) delineate the role and mechanism(s) of activation of p38 MAPK in ethanol plus TNFalpha cytotoxicity; #3) Define the mechanism(s) by which TNF plus ethanol cause the MPT.