Role of Neutral Sphingomyelinase-2 in aging

描述（由申请人提供）：衰老的特点是免疫功能和应激反应的改变。越来越清楚的是，至少部分原因是细胞对不同应激诱导物的反应能力发生了变化。本提案要测试的假设是，衰老引起的中性鞘磷脂酶-2 (NSMase-2) 活性上调是衰老过程中肝细胞 IL-1β 过度反应的基础。我们进一步假设，与衰老相关的 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以及氧化应激状态之间的关系。提出以下具体目标： 具体目标 1. 测试 NSMase-2 作为衰老诱导的 IL-1b 高反应性介质的作用。针对这一特定目标的研究将破译 NSMase-2 对老年动物 IL-1b 高反应性的贡献。基因沉默方法将用于体外（从老年大鼠分离的肝细胞）和体内（老年动物）。具体目的2.研究热量限制老年大鼠的IL-1b高反应性。众所周知，热量限制 (CR) 可以延长实验动物的寿命并减轻氧化应激的发生，包括防止衰老引起的 GSH/GSSH 下降。因此我们假设 CR 减弱肝脏的 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，那么老年 CR 中 NSMase-2 的过度表达应该会恢复与衰老相关的 IL-1b 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进行测试。具体目标 3：研究 GSH 在衰老相关 NSMase-2 激活和 IL-1b 高反应性中的作用。这些研究将探讨衰老过程中 GSH 的消耗和 NSMase-2 的激活是否存在因果关系，并将确定老年动物氧化应激的发生与 IL-1b 高反应性之间的联系。拟议的研究将为衰老发生的细胞机制提供新的理解，并可能在开发针对老年人的新型抗炎药物方面具有实际应用。

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.