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）的毒性作用的影响，但对女性酒精毒性的分子机制知之甚少，尤其是与骨骼有关的机制。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）酗酒。由此导致的骨量峰值降低会使女性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尚不清楚。EtOH，特别是酗酒时高浓度的EtOH，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骑车女性骨质流失。长期消耗EtOH作为液体饮食的一部分，使用该系统产生EtOH诱导的骨质流失，而不影响食物摄入。在离体骨髓培养中，雌性小鼠的骨质流失伴随着骨吸收血清标志物的增加和破骨细胞数量的增加。这些数据与我们实验室先前发表的通过全肠内营养循环喂食乙醇的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的表达增加。RANKL是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，刺激破骨细胞发生。etoh诱导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成骨样细胞系中。体外抗氧化剂n -乙酰半胱氨酸（NAC）可阻断乙酸乙酯对RANKL的诱导作用。RANKL似乎是通过ERK1/2和STAT3磷酸化调控的。体外实验数据表明，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。该酶在成骨细胞中的表达可通过EtOH增加，并被NAC阻断。采用小鼠液体饮食模型，以p47phox -/-小鼠和过氧化氢酶转基因小鼠为实验对象，研究NOX4和氧化应激在etoh诱导的骨吸收中的作用。在体外，我们将利用小鼠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来探索EtOH到RANKL的分子级联以及NAC和其他抗氧化剂对EtOH诱导的RANKL的抑制作用。将通过瞬时转染和使用siRNA进行NOX增益和功能损失的研究，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，以检验抗氧化剂阻止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.