Age effects on liver inflammation and injury

描述（由申请方提供）：肝缺血/再灌注（I/R）损伤是儿科和成人患者肝功能障碍的主要原因。在之前的资助期间，使用已建立的小鼠肝脏I/R模型，我们发现老年小鼠（12-13月龄）在I/R后的肝损伤明显大于年轻小鼠（4-6周龄）。这些发现与创伤患者的临床研究一致，这些研究表明，儿科患者的多器官功能障碍综合征的发病率远低于成人患者。我们发现，老年小鼠肝细胞损伤的增加与转录因子NF-κ B的活化减少有关。我们以前的工作表明，NF-kB具有不同的功能，这取决于所涉及的细胞类型。例如，枯否细胞中NF-kB的活化似乎驱动炎症反应，而肝细胞中NF-kB的活化似乎在细胞保护机制中起作用。我们最近的研究已经开始探索NF-kB激活在年轻和老年小鼠中不同的机制。我们已经发现了一个主要的缺陷，在处理的细胞质抑制剂NF-κ B，IkBa，在老年小鼠。首先，我们已经表明，在老年小鼠中，26 S蛋白酶体的19 S调节颗粒的特定亚基的基因表达显著降低。这些蛋白质亚基参与将泛素化底物募集到蛋白酶体中进行降解。我们的研究表明，这些亚基的表达减少减少了IkBa的降解，因此减少了老年小鼠肝脏中NF-κ B的活化。其次，我们有初步的数据表明，在老年小鼠中，泛素化IkBa的蛋白酶体的募集模式发生了改变。已知细胞质伴侣蛋白，即含缬氨肽的蛋白（VCP），与泛素化蛋白（包括IkBa）结合，并促进其向蛋白酶体的募集。Akt对VCP的磷酸化调节VCP与泛素化蛋白的结合。我们的初步数据表明，老年小鼠肝脏中Akt激活存在缺陷。因此，该项目的总体假设是，老年小鼠肝细胞中蛋白酶体组分和底物募集机制的年龄依赖性改变导致NF-κ B活化降低，使其比年轻小鼠的肝细胞更容易受到缺血应激的影响，导致氧化性组织损伤和细胞死亡增加。我们将以三个具体目标来检验这一假设。目的1将确定年龄改变VCP分子伴侣功能的机制。目的2将确定19 S调节颗粒的单个亚基对26 S蛋白酶体的底物募集和降解的特定作用。最后，目标3将确定是否在体内蛋白质转染的19 S亚基和/或组成型活性Akt恢复正常的激活NF-κ B和减少肝损伤诱导的I/R在老年小鼠。这些研究可以确定治疗靶点，这些靶点可以帮助导致由许多不同临床事件（包括创伤、手术、移植、血管疾病等）引起的急性肝损伤的新治疗。

DESCRIPTION (provided by applicant): Hepatic ischemia/reperfusion (I/R) injury is a primary cause of liver dysfunction in both pediatric and adult patients. During the previous funding period, using an established model of murine hepatic I/R, we found that old mice (12-13 months of age) have significantly greater liver injury after I/R than do young mice (4-6 weeks of age). These findings are consistent with clinical studies of trauma patients which suggest that pediatric patients have a far lower incidence of multiple organ dysfunction syndrome than do adult patients. We discovered that the increased injury was associated with decreased activation of the transcription factor, NF- kB, in hepatocytes of old mice. Our previous work suggests that NF-kB has divergent functions that are dependent upon the cell type involved. For example, activation of NF-kB in Kupffer cells appears to drive the inflammatory response, while NF-kB activation in hepatocytes appears to function in a cell-protective mechanism. Our more recent studies have begun to explore the mechanism by which NF-kB activation is different in young and old mice. We have found a major defect in the processing of the cytoplasmic inhibitor of NF-kB, IkBa, in old mice. First, we have shown that gene expression of specific subunits of the 19S regulatory particle of the 26S proteasome are significantly reduced in old mice. These protein subunits are involved in recruitment of ubiquitinylated substrates to the proteasome for degradation. Our studies suggest that decreased expression of these subunits reduce the degradation of IkBa and therefore decrease NF-kB activation in the livers of old mice. Secondly, we have preliminary data suggesting that a mode of recruitment of ubiquitinlylated IkBa to the proteasome is altered in old mice. The cytoplasmic chaperone, valosin- containing protein (VCP), is known to bind to ubiquitinylated proteins, including IkBa, and facilitate their recruitment to the proteasome. Phosphorylation of VCP by Akt regulates VCP's association with ubiquitinylated proteins. Our preliminary data demonstrate a defect in Akt activation in the livers of old mice. As such, the global hypothesis of this project is that age-dependent alterations to components of the proteasome and to substrate recruitment mechanisms in hepatocytes of old mice results in decreased activation of NF-kB which renders them more susceptible to ischemic stress than hepatocytes from young mice, resulting in increased oxidative tissue injury and cell death. We will test this hypothesis with three specific aims. Aim 1 will determine the mechanism by which age alters the chaperone function of VCP. Aim 2 will determine the specific roles of individual subunits of the 19S regulatory particle on substrate recruitment and degradation by the 26S proteasome. Finally, Aim 3 will determine whether in vivo protein transfection of 19S subunits and/or constitutively active Akt restores normal activation of NF-kB and reduces liver injury induced by I/R in old mice. These studies may identify therapeutic targets that could help lead to new treatments for acute liver injury resulting from a number of different clinical events including trauma, surgery, transplantation, vascular disease, etc.