Role of Neutral Sphingomyelinase-2 in aging

描述(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，至少在一定程度上，这是由于细胞对不同压力诱导剂反应能力的改变。在这项建议中要检验的假设是，衰老诱导的中性鞘细胞鞘-2(NSMase-2)活性上调是肝细胞IL-1β在衰老过程中夸大反应的基础。我们进一步假设，与衰老相关的NSMase-2活性的增加是细胞谷胱甘肽水平下降的结果。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。具体目的如下：1.检测NSMase-2在衰老诱导的IL-1b高反应性中的作用。对这一特定目标的研究将破译NSMase-2在老年动物IL-1b高反应性中的作用。基因沉默方法将在体外、老年大鼠肝细胞中以及在体内老年动物中使用。具体目的2.研究热量限制老年大鼠对IL-1b的高反应性。卡路里限制(CR)被认为可以延长实验动物的寿命，减轻氧化应激，包括防止衰老导致的GSH/GSSH下降。因此，我们假设CR可减弱肝脏对IL-1B的高反应性。这将在体内和体外进行测试。如果NSMase-2通过作用于GSH/GSSH的下游改变来介导IL-1b的高反应性，那么NSMase-2在老年CR中的过度表达应该可以恢复与衰老相关的IL-1b的高反应性。这将通过腺病毒介导的NSMase-2在老年CR大鼠的分离肝细胞和完整肝脏中的表达来验证。具体目的3：研究GSH在衰老相关NSMase-2激活和IL-1b高反应性中的作用。这些研究将询问衰老过程中GSH耗竭和NSMase-2激活是否存在因果联系，并将在老年动物的IL-1b过度反应和氧化应激的开始之间建立联系。这些研究将对衰老发生的细胞机制提供新的理解，并可能在开发新的老年抗炎药物方面具有实际应用价值。

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.