Age effects on liver inflammation and injury

描述（由申请人提供）：肝缺血/再灌注（I/R）损伤是儿童和成人患者肝功能障碍的主要原因。在之前的资助期内，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，表明儿科患者的多器官功能障碍综合征发生率远低于成人患者。我们发现，损伤的增加与老年小鼠肝细胞中转录因子NF- kB活性的降低有关。我们之前的工作表明，NF-kB具有不同的功能，这取决于所涉及的细胞类型。例如，Kupffer细胞中NF-kB的激活似乎驱动炎症反应，而肝细胞中NF-kB的激活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激活在年轻和年老小鼠中不同的机制。我们在老年小鼠中发现了NF-kB细胞质抑制剂IkBa加工的主要缺陷。首先，我们已经证明，在老年小鼠中，26S蛋白酶体的19S调控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招募泛素化底物到蛋白酶体进行降解。我们的研究表明，这些亚基的表达减少减少了IkBa的降解，从而降低了老年小鼠肝脏中NF-kB的激活。其次，我们有初步数据表明，在老年小鼠中，泛素化IkBa向蛋白酶体的募集模式发生了改变。胞质伴侣蛋白，含缬素蛋白（VCP），已知与泛素化蛋白结合，包括IkBa，并促进它们募集到蛋白酶体。Akt磷酸化VCP调控VCP与泛素化蛋白的关联。我们的初步数据表明，老年小鼠肝脏中Akt的激活存在缺陷。因此，该项目的总体假设是，老年小鼠肝细胞中蛋白酶体成分和底物募集机制的年龄依赖性改变导致NF-kB激活降低，使其比年轻小鼠肝细胞更容易受到缺血应激，导致氧化组织损伤和细胞死亡增加。我们将用三个具体目标来检验这一假设。目的1将确定年龄改变VCP伴侣功能的机制。目的2将确定19S调节颗粒的各个亚基在26S蛋白酶体的底物募集和降解中的具体作用。最后，Aim 3将确定在体内转染19S亚基和/或组成型活性Akt是否能恢复NF-kB的正常激活，并减轻老龄小鼠I/R诱导的肝损伤。这些研究可能会确定治疗靶点，有助于为创伤、手术、移植、血管疾病等不同临床事件引起的急性肝损伤找到新的治疗方法。

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. PUBLIC HEALTH RELEVANCE: This proposal will identify biochemical changes in liver cells that occur with age. A better understanding of these changes may lead to the development of therapeutic treatments that could be applied to patients undergoing liver resection and transplantation that would enhance organ and graft function and reduce morbidity and mortality.