ANTIOXIDANT PROPERTIES OF ESTRADIOL AND SAGEBRUSH-DERIVED FLAVONOIDS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 重金属镉是一种广泛存在的环境污染物，积累在体内，对人类健康构成威胁，特别是对包括骨质疏松在内的骨骼疾病的发展产生影响。镉排放到环境中有几个来源；值得注意的是，全球对含有这种有毒金属的电子废物(如手机和电脑)的处置增加。人类通过吸烟、摄入受污染的食物和水而接触到镉。尽管镉作为一种环境毒素被公认为是一种重要的环境毒素，但人们对镉如何直接影响骨细胞，特别是成骨细胞知之甚少。我们曾报道镉通过激活caspase-3诱导人成骨样细胞Saos-2的凋亡。为了阐明镉在骨骼中的影响，本研究利用非肿瘤来源的MC3T3-E1和肿瘤来源的成骨细胞SAOS-2，探索氧化应激在镉诱导的细胞凋亡中的作用，并研究镉对矿化的影响。越来越多的证据表明，氧化应激导致许多与年龄相关的疾病，包括骨质疏松症，抗氧化剂具有减轻氧化应激的破坏性影响的功能。我们的假设是，镉暴露会导致氧化应激，氧化应激导致成骨细胞矿化减少，细胞凋亡增加。我们预测，抗氧化剂的预处理将对镉诱导的骨毒性具有保护作用。具体目的1是确定雌二醇是否具有抗氧化特性，并防止镉诱导的氧化损伤和成骨细胞的凋亡死亡。具体目标2是确定爱达荷州山艾草提取的黄酮类化合物是否具有抗氧化特性，并防止镉诱导的氧化损伤和成骨细胞的凋亡死亡。具体目标3将确定镉暴露是否导致成骨细胞矿化减少，以及雌二醇或爱达荷州山艾草衍生的黄酮类化合物是否对镉诱导的矿化减少具有保护作用。我们将通过实时定量聚合酶链式反应(Real-time PCR)检测氯化镉暴露对成骨细胞关键基因(如RUNX2、骨桥蛋白、骨钙素和碱性磷酸酶)表达的影响，以及包括von Kossa和茜素红染色在内的矿化特异性标记。这项研究将对镉诱导骨毒性的潜在机制，特别是氧化应激的作用提供深入的认识。这些都是“细胞信号”在成骨细胞发育和凋亡中的作用机制的基础研究。了解镉干扰成骨细胞功能的机制，以及雌二醇和山艾黄酮类化合物作为抗氧化剂的能力，将有助于更好地理解环境毒素如何导致骨骼疾病，以及抗氧化剂减少毒素对骨骼健康的负面影响的能力。一个长期的目标是开发一个成年斑马鱼模型，以验证体外研究，并将镉对成骨细胞的影响与净骨丢失联系起来。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The heavy metal cadmium is a widespread environmental contaminant that accumulates in the body and poses a threat to human health, especially its impact on the development of bone diseases, including osteoporosis. There are several sources of cadmium emission into the environment; notable is the global increase in disposal of electronic-waste (e.g., cell phones and computers) containing this toxic metal. Humans are exposed to cadmium through smoking cigarettes and ingesting contaminated food and water. Despite its recognized importance as an environmental toxin, little is known about how cadmium directly impacts bone cells, in particular bone-forming osteoblasts. We previously reported that cadmium induces apoptosis in human osteoblast-like cells, Saos-2, through caspase-3 activation. To clarify the impact of cadmium in bone, the proposed research explores the role of oxidative stress in cadmium-induced apoptosis and investigate cadmium's impact on mineralization using nontumor derived MC3T3-E1 and tumor derived Saos-2 osteoblasts. There is growing evidence that oxidative stress contributes to a number of age-related diseases, including osteoporosis, and that antioxidants function to mitigate the damaging effect of oxidative stress. Our hypothesis is that cadmium exposure results in oxidative stress which leads to reduced osteoblast mineralization and increased apoptosis. We predict that pretreatment with antioxidants will protect against cadmium-induced osteotoxicity. Specific aim 1 is to determine if estradiol has antioxidant properties and protects against cadmium-induced oxidative damage and osteoblast apoptotic death. Specific aim 2 is to determine if Idaho sagebrush-derived flavonoids have antioxidant properties and protect against cadmium-induced oxidative damage and osteoblast apoptotic death. Specific aim 3 will determine whether cadmium exposure leads to reduced osteoblast mineralization and whether estradiol or Idaho sagebrush-derived flavonoids protect against cadmium-induced decrease in mineralization. We will examine the effect of CdCl2 exposure on the expression of key osteoblast genes (e.g., RUNX2, osteopontin, osteocalcin, and alkaline phophastase) by real-time PCR, and specific markers for mineralization including von Kossa and Alizarin red staining. This research will provide insight into mechanisms underlying cadmium-induced osteotoxicity, in particular the role of oxidative stress. These are fundamental studies in the mechanisms of "Cell Signaling" in osteoblast development and apoptosis. Knowing the mechanisms by which cadmium disrupts osteoblast function, and the ability of estradiol and sagebrush-derived flavonoids to act as antioxidants, will provide a better understanding of how environmental toxins contribute to bone disease and the ability of antioxidants to reduce the negative impacts of toxins on bone health. A long term goal is to develop an adult zebrafish model to validate in vitro studies and link cadmium's impact in osteoblasts to net bone loss.