荞麦 Potato I 型抑制剂延缓秀丽隐杆线虫衰老的机制研究

In recent years, human species is faced with a sever challenge of aging. How to delay the process of aging safely and effectively has become a topic receiving extensive attention. The physiological functions and medicinal properties of buckwheat have been widely studied. As one medicinal and edible crop, buckwheat may have beneficial effects on preventing from high cholesterol, hyperglycemia and cardiovascular disease. All these properties are bound up with aging. Potato I type protease inhibitors play an important role in plant resistance to various external stimuli including wounding, insect feeding and microbial infections. Buckwheat trypsin inhibitor (BTI) is a bioactive peptide and belongs to Potato I type protease inhibitors. Our present experiment data indicates that BTI could significantly extend lifespan and increase oxidative stress tolerance in Caenorhabditis elegans (C. elegans). However, the potential molecular mechanisms of how BTI delay aging and extend healthspan remain unclear in C. elegans. In addition, our previous results showed BTI-mediated longevity was dependent on daf-2 and daf-16, and interactions existed between BTI and DAF-2. Based on these findings, we speculate that BTI may delay the process of aging in C. elegans by targeting DAF-2 and regulating Insulin/IGF-1 signaling pathway ( IIS pathway ). In this project, we are going to comprehensively evaluate the anti-aging effect of BTI in C. elegans with a series of health indicators besides extending the lifespan. Then, the regulation effect on IIS pathway mediated by BTI will be explored by qRT-PCR and western blot. The change of reactive oxygen species (ROS) induced by BTI will be also investigated. Further, the interaction between BTI and DAF-2 will be explored by site-directed mutagenesis and Co-IP. It is hoping to lay the theoretical and experimental foundation for the application of BTI and the research of insulin receptor antagonists, and provide new ideas and methods for the development and modification of new anti-aging substances.

近年来，人口老龄化问题日益突出，如何安全有效地延缓衰老引起人们广泛关注。荞麦药食同源，是“三高”患者的理想食物。Potato I 型蛋白酶抑制剂在植物抵御各种不利条件时都发挥出重要作用。课题组近期研究发现，荞麦中的一种 Potato I 型蛋白酶抑制剂 （BTI） 可以显著延长秀丽隐杆线虫（C. elegans）的寿命，但具体机制还不清楚。本项目拟采用 qRT-PCR、Western Blot、免疫共沉淀、定点突变等方法，明确 BTI 对胰岛素样信号通路的调控作用，揭示 BTI 延长 C. elegans 寿命的效应机制，进一步研究 BTI 与类胰岛素受体 DAF-2 的相互作用，寻找其抗衰老功能的关键氨基酸位点或特征序列，初步阐明 BTI 的构效关系，为 BTI 的应用和抗衰老药物的开发提供科学的理论依据。

荞麦中的一种Potato I型蛋白酶抑制剂（BTI）不仅具有调节蛋白酶的活性，在生物体内对信号转导及新陈代谢调节等也发挥着重要作用。本项目研究了BTI对模式生物秀丽隐杆线虫寿命的影响及其作用机制，主要取得了以下结果。（1）明确了BTI可以延长秀丽隐杆线虫的健康寿命。在2.5-10 μM浓度范围内，BTI能明显延长秀丽隐杆线虫寿命，且具有浓度依赖性，10 μM BTI处理组线虫平均寿命比对照组延长了21.18%。BTI可以明显降低线虫体内衰老标志物-半乳糖苷酶活性及褐脂质的积累。BTI对年轻线虫的运动能力和产卵能力没有显著影响，但可以改善老年虫体的运动能力。（2）明确了BTI对秀丽隐杆线虫的延寿作用依赖于IIS通路。BTI对daf-2和daf-16突变体线虫寿命没有延长作用，降低了IIS通路主要成员daf-2、age-1、akt-1的转录水平，提高了daf-16的转录水平，促进了DAF16的核转位，增强DAF-16靶基因sod-3, gsh-px的表达，提高了线虫抗氧化应激能力。（3）明确了BTI 诱导 DAF-16 产生的效应机制。BTI下调了糖酵解关键酶的转录，降低了果糖磷酸激酶和己糖磷酸激酶活性；BTI下调了脂肪酸及甘油三酯合成相关基因的转录，降低了乙酰辅酶A羧化酶及脂肪酸合成酶的活性，提高了脂肪酶活性，明显降低了线虫中脂肪积累，表明BTI作用后线虫产能方式从糖代谢到脂代谢转变。qRT-PCR及转基因线虫荧光检测显示BTI可以提高线虫自噬水平。BTI诱导产生一个瞬时ROS信号激活虫体抗氧化防御，BTI介导的ROS信号依赖IIS通路。（4）BTI的延寿作用与其适当的胰蛋白酶活性有关。构建了不同胰蛋白酶抑制活性的BTI突变体（rBTI-R45A, rBTI-R45F, rBTI-P44T, rBTI-W53R），突变体对线虫的延寿作用均较BTI明显降低，不能改善老年线虫行为能力，对脂肪积累也没有显著影响。突变体不能促进转录因子DAF-16的核转位，DAF-16靶基因Sod-3和Gsh-p.