Heat shock proteins and alcohol induced liver injury

描述(由申请人提供)： 活性氧自由基(ROS)诱导的氧化应激在酒精性细胞损伤的发病机制中起重要作用。高度保守的热休克反应导致热休克蛋白的产生，对氧化应激增加和包括TNFa细胞毒性在内的其他类型的细胞损伤具有保护机制。热休克蛋白(HSP)，如HSP-70、27和HSP-90，不仅能抑制细胞凋亡，还能阻断氧化应激引起的炎症反应。因此，热休克蛋白的这些特性使其成为一个极好的操作系统，从而为抗氧化治疗开辟了新的途径。我们推测，急性和慢性酒精暴露于肝巨噬细胞(Kupffer细胞)以及肝细胞(肝细胞)可能激活热休克反应，调节炎症反应和细胞凋亡的增加，从而导致酒精诱导的氧化应激。我们的初步数据显示，慢性酒精会增加单核细胞和巨噬细胞的炎症反应。此外，酒精暴露还会增加NFkB，这是肝细胞中的一种抗凋亡分子。因此，我们认为急性酒精可诱发HSPs，而慢性酒精可抑制HSPs调节炎症反应。我们还提出，在肝细胞中，慢性酒精暴露可能导致热休克蛋白不足以抑制细胞凋亡，从而导致酒精性肝损伤。这些细胞中的NFkB可能被上调，以抵消酒精诱导的变化，并促进存活，尽管效率较低。本建议的具体目的是：1)通过检测急性和慢性酒精暴露对炎症细胞和肝细胞中热休克蛋白HSP-70、HSP-90、HSP-60和小分子HSP-27的表达的影响，探讨急性和慢性酒精暴露对炎症细胞和肝细胞中HSP-70、HSP-90、HSP-60和小分子HSP-27表达的影响。C)热休克转录因子的激活。2)观察急、慢性酒精对巨噬细胞炎症及相关基因表达的影响。 基因芯片技术和DNA片段化、caspase-3、-8激活以及基因芯片技术对HEPA-1-6细胞促/抗凋亡基因的诱导作用及其与热休克蛋白改变的相关性。

DESCRIPTION (provided by applicant): Oxidative stress induced by reactive oxygen species (ROS) plays a causative role in the pathogenesis of alcohol induced cellular injury. The highly conserved heat shock response, resulting in production of heat shock proteins has been implicated in protective mechanisms against increased oxidative stress as well as other types of cellular injuries including TNFa cytotoxicity. Heat shock proteins (hsp) such as the hsp-70, 27 and hsp-90 not only inhibit apoptosis but also block increased inflammatory responses generated by oxidative stress. Therefore, these properties of heat shock proteins make it an excellent system for manipulation resulting in opening of new avenues of anti-oxidant therapies. We hypothesize that acute and chronic alcohol exposure of liver macrophages (Kupffer cells) as well as hepatic cells (hepatocytes) may activate the heat shock response and regulate the increased inflammatory responses and apoptosis resulting in alcohol induced oxidative stress. Our preliminary data shows that chronic alcohol increases inflammatory responses in monocytes and macrophages. Further, alcohol exposure also increases NFkB, an anti-apoptotic molecule in hepatic cells. Therefore, we propose that acute alcohol may induce hsps but chronic alcohol may inhibit the hsps to regulate the inflammatory response. We also propose that in hepatic cells chronic alcohol exposure may induce insufficient hsps to inhibit apoptosis, leading to alcohol-induced liver injury. NFkB in these cells may be up regulated to counteract the alcohol-induced alterations and promote survival though with less efficiency. The specific aims of this proposal are - 1) To investigate the effect of acute and chronic alcohol exposure on expression of heat shock proteins such as hsp-70, hsp-90 hsp-60 and small hsps like hsp-27 in inflammatory cells and hepatocytes by evaluating -a) Protein levels of the hsps in nucleus and cytoplasm of the cells by Western blotting, b) Alterations in gene expression by determining mRNA levels by Northern Blotting. c) Activation of the heat shock transcription factor. 2) To evaluate the effect of acute and chronic alcohol on - Induction of inflammation and related genes in macrophages by the microarray method and induction of apoptosis by DNA fragmentation, caspase-3, -8 activation and pro-/anti-apoptotic genes in HEPA 1-6 cells by the microarray method and correlate changes with hsp alterations.