Alcohol-Induced Bone Resorption: The Role of Oxidative Stress

酒精引起的骨吸收：氧化应激的作用

• 批准号: 7890356
• 负责人: Martin J J Ronis
• 金额: $ 34.56万
• 依托单位: ARKANSAS CHILDREN'S HOSPITAL RES INST
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-10 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7890356
• 关键词: Acetylcysteine; Age; Aging; Alcohol abuse; Alcohol consumption; Alcohols; Antioxidants; Applications Grants; Area; Ascorbic Acid; Bone Growth; Bone Marrow; Bone Marrow Cells; Bone Resorption; Bone remodeling; C57BL/6 Mouse; Cell Culture Techniques; Cell Line; Characteristics; Chronic; Coculture Techniques; Consumption; Coupled; Curcumin; Data; Development; Diet; Disease; Eating; Elderly; Enteral Nutrition; Enzymes; Ethanol; Ethanol toxicity; Family; Family member; Female; Free Radical Scavenging; Free Radicals; Gene Deletion; Generations; Heavy Drinking; Hydrogen Peroxide; Impairment; In Vitro; Injury; Investigation; Knockout Mice; Laboratories; Lead; Ligands; Link; Liquid substance; MAP Kinase Modules; Menopause; Messenger RNA; Metabolism; Modeling; Molecular; Molecular Probes; Morbidity - disease rate; Mus; NADPH Oxidase; NIH Program Announcements; National Institute on Alcohol Abuse and Alcoholism; Nuclear; Osteoblasts; Osteoclasts; Osteopenia; Osteoporosis; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Phosphorylation; Postmenopause; Production; Publishing; Rattus; Reactive Oxygen Species; Research; Risk; Risk Factors; Role; STAT3 gene; Serum Markers; Signal Transduction; Signaling Molecule; Small Interfering RNA; Surface; System; T-Lymphocyte; TNF gene; TNFSF11 gene; Testing; Therapeutic; Tissues; Toxic effect; Transfection; Transgenic Mice; Transgenic Organisms; Wild Type Mouse; Woman; Writing; alcohol effect; animal data; antioxidant therapy; binge drinking; bone; bone cell; bone health; bone loss; bone mass; catalase; cytokine; dietary antioxidant; diphenyleneiodonium; emerging adult; feeding; in vivo; inhibitor/antagonist; insight; loss of function; member; men; mortality; mouse model; novel; osteoclastogenesis; prevent; public health relevance; receptor; reproductive; response

DESCRIPTION (provided by applicant): Although it is well known that women are more susceptible to the toxic effects of ethanol (EtOH) than men, much less is known about the molecular mechanisms underlying alcohol toxicity in women especially as relates to bone. Alcohol abuse during early adulthood results in impaired bone growth and in the U.S.A. approximately 20% of women age 18-30 (4.4 million) binge drink. A resulting reduction in peak bone mass would predispose women to osteoporosis in later life. The molecular mechanisms underlying the toxic effects of EtOH on bone remain poorly understood. EtOH, particularly at higher concentrations characteristic of binge drinking, can increase bone resorption. We have developed a new mouse model to study alcohol-induced bone loss in cycling females. Chronic consumption of EtOH as part of liquid diets using this system produces EtOH-induced bone loss without compromising food intake. Bone loss in female mice was accompanied by increased serum markers of bone resorption and increased osteoclast numbers in ex-vivo bone marrow cultures. These data are consistent with previously published data from our laboratory in cycling female rats fed ethanol via total enteral nutrition. In the rat model, increased bone resorption was accompanied by increased expression of RANKL in bone. RANKL is a member of the TNF family expressed on the surface of osteoblasts which signals through the receptor RANK on the surface of osteoclast precursors to stimulate osteoclastogenesis. EtOH-induction of RANKL occurred in primary osteoblast cultures from rats and mice and in rat and mouse UMR-106 and ST-2 osteoblast-like cell lines. EtOH-induction of RANKL was blocked by the antioxidant N-acetylcysteine (NAC) in vitro. RANKL appears to be regulated via ERK1/2 and STAT3 phosphorylation. In vitro data suggest that the ERK/STAT3/RANKL pathway is stimulated by reactive oxygen species (ROS) produced by an ethanol-induced NADPH oxidase (NOX4). Expression of this enzyme in osteoblasts is increased by EtOH and blocked by NAC. The mouse liquid diet model will be utilized to determine the role of NOX4 and oxidative stress in EtOH-induced bone resorption in vivo using p47phox -/- mice and catalase transgenic mice. In vitro, the mouse ST-2 cell line, differentiated mouse stromal osteoblast cultures and osteoblast/osteoclast precursor co-cultures will be utilized to probe the molecular cascade from EtOH to RANKL and the inhibition of EtOH-induced RANKL by NAC and other antioxidants. NOX gain and loss of function studies will be performed by transient transfection and use of siRNA and the effects of hydrogen peroxide and free radical scavenging on the ERK/STAT3/RANKL cascade will be examined. In addition, in vivo studies will be conducted to examine the ability of antioxidants to block EtOH-induced bone resorption. PUBLIC HEALTH RELEVANCE: Excessive alcohol consumption is an increasing problem in young women. The results from research proposed herein will link the deleterious effects of alcohol consumption on bone in women of reproductive age with osteoporosis, a major cause of morbidity and mortality in postmenopausal women. Alcohol abuse results in osteopenia and increases osteoporosis risk. One potentially important yet under-explored area of bone health is the relationship between bone resorption associated with alcohol abuse in young women and increased risk of osteoporosis which results from impairments in attainment of peak bone mass. In addition, alcohol-induced bone loss shares many features in common with bone loss during menopause and aging. Our animal data suggests that dietary antioxidants may prevent alcohol-induced bone resorption and we will test this in the current application. Molecular studies of alcohol actions on ROS signaling cascades in bone are expected to provide fundamental insights into common pathways underlying bone resorption.

描述（由申请人提供）：虽然众所周知，女性比男性更容易受到乙醇（EtOH）的毒性作用，但对于女性酒精毒性的分子机制（尤其是与骨骼相关的分子机制）知之甚少。成年早期酗酒会导致骨骼生长受损，在美国，大约 20% 的 18-30 岁女性（440 万人）酗酒。由此导致的峰值骨量减少将使女性在以后的生活中容易患骨质疏松症。乙醇对骨骼产生毒性作用的分子机制仍知之甚少。乙醇，特别是在酗酒时的较高浓度下，可以增加骨吸收。我们开发了一种新的小鼠模型来研究酒精引起的女性自行车骨质流失。使用该系统长期食用乙醇作为流质饮食的一部分会导致乙醇引起的骨质流失，而不会影响食物摄入量。雌性小鼠的骨质流失伴随着骨吸收血清标志物的增加以及离体骨髓培养物中破骨细胞数量的增加。这些数据与我们实验室之前发表的通过全肠内营养喂养乙醇的循环雌性大鼠的数据一致。在大鼠模型中，骨吸收的增加伴随着骨中 RANKL 表达的增加。 RANKL 是在成骨细胞表面表达的 TNF 家族成员，通过破骨细胞前体表面的受体 RANK 发出信号以刺激破骨细胞生成。 EtOH 诱导的 RANKL 发生在大鼠和小鼠的原代成骨细胞培养物以及大鼠和小鼠 UMR-106 和 ST-2 成骨细胞样细胞系中。在体外，EtOH 诱导的 RANKL 被抗氧化剂 N-乙酰半胱氨酸 (NAC) 阻断。 RANKL 似乎通过 ERK1/2 和 STAT3 磷酸化进行调节。体外数据表明 ERK/STAT3/RANKL 通路受到乙醇诱导的 NADPH 氧化酶 (NOX4) 产生的活性氧 (ROS) 的刺激。 EtOH 会增加该酶在成骨细胞中的表达，而 NAC 则会阻断该酶的表达。小鼠液体饮食模型将用于使用 p47phox -/- 小鼠和过氧化氢酶转基因小鼠确定 NOX4 和氧化应激在 EtOH 诱导的体内骨吸收中的作用。在体外，小鼠 ST-2 细胞系、分化的小鼠基质成骨细胞培养物和成骨细胞/破骨细胞前体共培养物将用于探测从 EtOH 到 RANKL 的分子级联以及 NAC 和其他抗氧化剂对 EtOH 诱导的 RANKL 的抑制。 NOX 功能获得和丧失的研究将通过瞬时转染和使用 siRNA 进行，并将检查过氧化氢和自由基清除对 ERK/STAT3/RANKL 级联的影响。此外，还将进行体内研究，以检查抗氧化剂阻止乙醇诱导的骨吸收的能力。公共卫生相关性：过量饮酒是年轻女性日益严重的问题。本文提出的研究结果将把饮酒对育龄妇女骨骼的有害影响与骨质疏松症联系起来，骨质疏松症是绝经后妇女发病和死亡的主要原因。酗酒会导致骨质减少并增加骨质疏松的风险。骨骼健康的一个潜在重要但尚未充分探索的领域是年轻女性与酗酒相关的骨吸收与由于达到峰值骨量受损而导致的骨质疏松症风险增加之间的关系。此外，酒精引起的骨质流失与更年期和衰老过程中的骨质流失有许多共同点。我们的动物数据表明，膳食抗氧化剂可以防止酒精引起的骨吸收，我们将在当前的应用中对此进行测试。酒精对骨中 ROS 信号级联反应的分子研究有望为骨吸收的常见途径提供基本见解。