Alcohol-Induced Bone Resorption: The Role of Oxidative Stress

描述（由申请人提供）：尽管众所周知女性比男性更容易受到乙醇（EtOH）毒性作用的影响，但对女性酒精毒性的分子机制知之甚少，尤其是与骨相关的机制。成年早期的酒精滥用会导致骨骼生长受损，在美国，大约20%的18-30岁的女性（440万人）酗酒。由此导致的峰值骨量减少将使妇女在以后的生活中容易患骨质疏松症。EtOH对骨毒性作用的分子机制仍然知之甚少。乙醇，特别是在高浓度下的酗酒特征，可以增加骨吸收。我们开发了一种新的小鼠模型来研究骑自行车的女性中酒精引起的骨丢失。使用该系统长期消耗EtOH作为液体饮食的一部分，产生EtOH诱导的骨丢失，而不影响食物摄入。雌性小鼠的骨丢失伴随着骨吸收的血清标志物增加和体外骨髓培养中破骨细胞数量增加。这些数据与我们实验室先前发表的通过全肠内营养饲喂乙醇的骑自行车雌性大鼠的数据一致。在大鼠模型中，骨吸收增加伴随着骨中RANKL表达增加。RANKL是在成骨细胞表面上表达的TNF家族的成员，其通过破骨细胞前体表面上的受体RANK发出信号以刺激破骨细胞生成。在大鼠和小鼠的原代成骨细胞培养物中以及在大鼠和小鼠UMR-106和ST-2成骨细胞样细胞系中发生RANKL的EtOH诱导。在体外，抗氧化剂N-乙酰半胱氨酸（NAC）可阻断EtOH诱导的RANKL。RANKL似乎通过ERK 1/2和STAT 3磷酸化调节。体外数据表明，ERK/STAT 3/RANKL通路受到乙醇诱导的NADPH氧化酶（NOX 4）产生的活性氧（ROS）的刺激。这种酶在成骨细胞中的表达被EtOH增加并被NAC阻断。使用p47 phox-/-小鼠和过氧化氢酶转基因小鼠，采用小鼠流质饮食模型确定NOX 4和氧化应激在体内EtOH诱导的骨吸收中的作用。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的分子级联反应以及NAC和其他抗氧化剂对EtOH诱导的RANKL的抑制。将通过瞬时转染和使用siRNA进行NOX获得和功能丧失研究，并检查过氧化氢和自由基清除对ERK/STAT 3/RANKL级联的影响。此外，将进行体内研究，以检查抗氧化剂阻断EtOH诱导的骨吸收的能力。

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.