MECHANISMS OF ACTION IN MENOPAUSE

博尔顿项目的主要目标仍然是提供体外生物测定支持，以发现缓解更年期症状的主要膳食补充剂的活性成分。为了实现这一目标，已经开发了几种基于体外细胞的和动物的测定，以研究这些膳食补充剂的作用机制。 补充剂.具体来说，我们感兴趣的是具有雌激素活性的植物，如啤酒花（啤酒花），葛根（野葛）和kwao keur（野葛）。此外，我们还发现了一种缓解更年期症状的替代性雌激素能机制，这对黑升麻（Cimicifuga racemosa）、当归（Angelica sinensis）和缬草根（Valeriana officinalis）可能很重要。要解决的假设是选择的植物提取物是否含有有效的雌激素和/或雌激素能化合物，用于缓解更年期症状以及雌激素的其他有益特性，如抗氧化/化学预防作用。提出了以下具体目标：1。评价植物提取物和活性化合物的激素活性。我们已经筛选了 许多植物提取物和纯化合物的雌激素效应，并研究其作用机制，使用各种体外试验，包括竞争性ER结合试验，雌激素敏感基因的诱导，和ERE-荧光素酶试验。此外，我们已经使用卵巢切除大鼠模型测试了几种这些提取物的体内激素活性。我们还纳入了两个新的筛选试验，包括雌激素受体辅激活剂测定和测定ERE和AP-1 DNA结合能力的测定。2.评价植物提取物和活性化合物的促肾上腺素活性。我们计划进一步研究黑升麻（目的3），以确定活性化合物，并使用已建立的多巴胺能测定法（与5-羟色胺受体的竞争性结合，环AMP活性的升高和SSRI测定法）测试其他有前途的植物。 这一假说认为5-HT 7受体参与体温调节（即潮热），而5-HT 1A和5-HT 5A受体可能负责抑郁和情绪。将纳入动物模型，包括潮热模型和强迫游泳试验（情绪）。3.评价植物提取物和活性化合物的化学预防/抗氧化特性。在上一个授予期间，我们已经开发了各种抗氧化剂/化学预防试验，包括清除稳定的自由基，抑制DNA损伤的醌甲萘醌诱导的彗星试验，并诱导醌还原酶。为此，将对上述植物膳食补充剂的提取物进行分级，并使用抗氧化剂/化学预防测定法测定活性。这些目标的完成将决定哪些特定的植物具有健康 缓解更年期症状的益处，并确定其作用机制。

The major objective of Project by Bolton continues to be to provide in vitro bioassay support for the discovery of the active components for the major dietary supplements for the relief of menopausal symptoms. To achieve this objective several in vitro cell based and animal assays have been developed in order to investigate the mechanism of action of these dietary supplements. Specifically, we are interested in botanicals with estrogenic activity such as hops (Humulus lupulus), kudzu (Pueraria lobata), and kwao keur (Pueraria mirifica). In addition, we have discovered an alternative serotonergic mechanism for the relief of menopausal symptoms which could be important for black cohosh (Cimicifuga racemosa), dong quai (Angelica sinensis), and valerian (Valeriana officinalis). The hypothesis to be addressed is whether select plant extracts contain potent estrogenic and/or serotonergic compounds useful for the relief of menopausal symptoms as well as other beneficial properties of estrogens such as antioxidant/chemopreventive effects. The following specific aims are proposed: 1. To evaluate the hormonal activity of plant extracts and active compounds. We have already screened a number of plant extracts and pure compounds for estrogenic effects and investigated their mechanism of action using a variety of in vitro assays including competitive ER binding assays, induction of estrogen sensitive genes, and ERE-luciferase assays. In addition, we have tested several of these extracts for hormonal activity in vivo using the ovariectomized rat model. We have also incorporated two new screening assays including the estrogen receptor coactivator assay and an assay to measure ERE and AP-1 DNA binding capacity. 2. To evaluate the serotonergic activity of plant extracts and active compounds. We plan to further fractionate black cohosh (aim 3) in order to determine the active compounds as well as test other promising plants using established serotonergic assays (competitive binding to serotonin receptors, elevation in cyclic AMP activity, and SSRI assays), addition, we plan to further test the hypothesis that the 5-HT7 receptor is involved in thermoregulation (i.e. hot flashes) whereas the 5-HT1A and 5-HT5A receptors are likely responsible for depression and mood. Animal models will be incorporated including the hot flash model and the forced swim test (mood). 3. To evaluate the chemoprevention/antioxidant properties of plant extracts and active compounds. During the previous granting period, we have developed a variety of antioxidant/chemoprevention assays including scavenging of stable free radicals, inhibition of DNA damage induced by the quinone menadione using the comet assay, and induction of quinone reductase. In this aim, extracts of the above mentioned botanical dietary supplements will be fractionated and activity will be determined using the antioxidant/chemoprevention assays. The completion of these aims will determine which specific botanicals have health benefits for relief of menopausal symptoms and determine their mechanism of action.