Role of Neutral Sphingomyelinase-2 in aging

描述（由申请人提供）：衰老的特征是免疫功能和应激反应的改变。越来越清楚的是，这至少部分是由于细胞对不同应激诱导物的反应能力的改变。本提案中待检验的假设是，衰老诱导的中性鞘磷脂酶-2（NSMase-2）活性上调是衰老过程中肝细胞IL-1 β过度反应的基础。我们进一步假设衰老相关的NSMase-2活性增加是细胞谷胱甘肽水平降低的结果。因此，我们研究的长期目标是了解衰老、细胞对炎症反应的改变和氧化应激状态之间的关系。提出了以下具体目标：具体目标1。检测NSMase-2作为衰老诱导的IL-1b高反应性介质的作用。这一特定目标的研究将解释NSMase-2对老年动物IL-1b高反应性的贡献。基因沉默方法将在体外（分离自老年大鼠的肝细胞）和体内（老年动物）中使用。具体目标2。目的：研究热量限制对老年大鼠IL-1b高反应性的影响。热量限制（CR）已知可以延长实验动物的寿命，并减轻氧化应激的发生，包括防止衰老诱导的GSH/GSSH下降。因此，我们假设CR减弱肝脏IL-1B高反应性。这将在体内和体外进行测试。如果NSMase-2通过作用于GSH/GSSH变化的下游来介导IL-1b高反应性，那么NSMase-2在老年CR中的过表达应该恢复与衰老相关的IL-1b高反应性。这将通过在老年CR大鼠的分离肝细胞和完整肝脏中腺病毒介导的NSMase-2表达进行测试。具体目标3：研究GSH在衰老相关的NSMase-2激活和IL-1b高反应性中的作用。这些研究将探讨衰老中GSH耗竭和NSMase-2激活是否存在因果关系，并将建立老年动物氧化应激发作和IL-1b高反应性之间的联系。拟议的研究将提供新的理解的细胞机制参与衰老的发病，并可能在开发新的抗炎药物的老年人的实际应用。

DESCRIPTION (provided by applicant): Aging is characterized by an altered immune function and stress response. It becomes increasingly clear that, at least in part, this is due to alterations in ability of the cells to respond to different stress inducers. The hypothesis to be tested in this proposal is that aging-induced up-regulation of neutral sphingomyelianse-2 (NSMase-2) activity underlies the exaggerated response of hepatocytes IL-1beta during aging. We further hypothesized that aging-associated increases in NSMase-2 activity are consequence of the decreases in the cellular glutathione level. Therefore, the long-term objective of our study is to understand the relation between aging, the altered cellular response to inflammation, and the state of oxidative stress. The following specific aims are proposed: Specific aim 1. To test the role of NSMase-2 as a mediator of aging-induced hyperresponsiveness to IL-1b. Studies in this specific aim will decipher the contribution of NSMase-2 to the IL-1b hyperresponsiveness of aged animals. Gene silencing approach will be used in vitro, in hepatocytes isolated from old rats, and in vivo, in aged animals. Specific aim 2. To study the IL-1b hyperresponsiveness in calorie-restricted aged rats. Calorie-restriction (CR) is known to increase the life span of experimental animals and to attenuate the onset of oxidative stress, including preventing aging-induced GSH/GSSH decline. Thus we hypothesized that CR attenuates IL-1B hyperresponsiveness of liver. This will be tested in vivo and in vitro. If NSMase-2 mediates IL-1b hyperresponsiveness by acting down-stream of GSH/GSSH changes, then NSMase-2 overexpression in aged CR should restore the aging-associated IL-1b hyperresponsiveness. This will be tested by adenoviral-mediated expression of NSMase-2 in isolated hepatocytes and intact liver from aged CR rats. Specific aim 3: To study the role of GSH in aging-associated NSMase-2 activation and IL-1b hyperresponsiveness. These studies will ask whether GSH depletion in aging and NSMase-2 activation are causatively linked and will establish a link between the onset of oxidative stress and the IL-1b hyperesponsivness in aged animals. The proposed studies will provide novel understanding on the cellular mechanisms involved in the onset of aging and may have practical application in the development of new anti-inflammatory drugs for the elderly.