Potential health promoting properties of the triterpenoid ursolic acid as a putative mimetic of caloric reduction - studies in cultured cells and in mice

熊果酸(UA)是一种植物来源的五环三萜类化合物。我们以前的工作表明，UA改善了果蝇的健康和寿命，并改善了与年龄相关的运动活动下降。这一发现指的是斯帕凝胶(Srl)表达的增加。SPARGEL是哺乳动物过氧化物酶体增殖物激活受体γ辅活化物(PGC1)的同源产物。然而，目前还不清楚我们在苍蝇身上的数据是否可以转化为哺乳动物物种。因此，我们将解决的问题是，UA的潜在延寿特性是否也存在于哺乳动物中，以及它们是否通过PGC1活性的变化来调节。为此，我们将遵循三个(I-III)实验方法。由于Pgc1被认为是尿酸介导作用的潜在关键因素，我们将通过siRNA和CRISPR-Cas9沉默Pgc1α的表达来检测Pgc1α在UA孵育后在小鼠肌肉和肝脏细胞中的表达。因此，我们将证明前列腺素C_1α可能参与尿酸介导的体外生物活性(I)。其次，我们将在Pgc1α基因敲除小鼠模型中研究Pgc1α靶基因在特定组织(肌肉、肝脏)以及原代心肌细胞和肝细胞中的表达。尿酸将与γ-环糊精(γCD)形成络合物，以提高其生物利用度。因此，我们将验证前列腺素C_1α在体内可能参与尿酸介导的效应(II)。前列腺素C_1α的表达在缓解期诱导，并随增龄而下降。因此，我们将再次研究UA对Pgc1α表达的影响，以及对老年小鼠健康和寿命的影响，提供自由或有限补充UA-γCd的饮食。我们将反对UA得出的结果与CR相反，并说明任何潜在的协同或相加的生物活性。因此，我们打算澄清UA是否可以被认为是潜在的CR模拟物(III)。广泛地说，在所有三种实验方法中，我们将确定PGC1RNA靶基因、编码线粒体功能和代谢重要蛋白的基因(通过Oroboros呼吸测量法)、关键因子和选定的效应分子以及miRNA的活性。我们将通过芯片分析、电子对接分析和报告基因分析更详细地验证和表征UA介导的结果。除了这些参数，还将对小鼠进行全面的表型鉴定，包括身体成分、间接量热法、口服葡萄糖耐量、肌肉力量和运动活动。

Ursolic acid (UA) is a phytogenic pentacyclic triterpenoid. Our own previous work shows that UA improves health and life span in the fruit fly Drosophila melanogaster and ameliorates the age-related decline in locomotor activity. This finding is referred to an increase in Spargel (srl) expression. Spargel represents the orthologue of mammalian peroxisome proliferator-activated receptor gamma coactivator (PGC1). However, it is unclear whether our data in flies can be translated into mammalian species. Thus, we will address the question whether the potential life-prolonging properties of UA also exists in mammals and whether they are mediated through a change in PGC1 activity. To this end, we will follow three (I-III) experimental approaches. As Pgc1 is considered as the potential key factor for UA-mediated action, we will examine the expression of Pgc1α target genes after UA incubation in murine muscle and liver cells with siRNA and CRISPR-Cas9 silenced expression of Pgc1α. Hence, we will demonstrate a potential involvement of Pgc1α in UA mediated bioactivity in vitro (I). Secondly, we will investigate the expression of Pgc1α target genes in selected tissues (muscle, liver) and primary myocytes and hepatocytes in a Pgc1α-knock-out mouse model in response to dietary UA. UA will be complexed with γ-cyclodextrin (γCD) to increase its bioavailability. Consequently, we will verify a potential involvement of Pgc1α in UA mediated effects in vivo (II). Pgc1α expression is induced under CR and declines with age. Therefore, we will thirdly study the role of UA on Pgc1α expression and on health and life span in old mice providing a UA-γCD-supplemented diet ad libitum or restricted. We will oppose the UA-derived results to CR and illustrate any potential synergistic or additive bioactivity. Thus, we intent to clarify whether UA could be considered as a potential CR mimetic (III). Extensively, in all three experimental approaches, we will determine the activities of Pgc1α target genes, of genes encoding proteins important in mitochondrial function (via Oroboros-respirometry) and metabolism, of key factors and of selected effector molecules as well as miRNA. We will validate and characterize the UA mediated outcome in more detail via ChIP assay, in silico docking analysis and reporter gene assays. These parameters will be complemented by a comprehensive phenotyping of mice including body composition, indirect calorimetry, oral glucose tolerance, muscle strength and locomotor activity.