Identifying the role of reactive oxygen species in alcohol-induced murine growth plate pathology

项目摘要/摘要 青春期代表着骨骼特别容易受到损害的发育阶段 可能会产生长期影响。酒精(酒精)的使用在青少年中非常普遍，每17人中就有1人(~6%) 据估计，12-17岁的男孩和女孩都有酗酒行为。乙醇是一种已知的骨骼毒物 通过增加细胞内的活性氧(ROS)来产生氧化应激。此外，乙醇 降低循环中胰岛素样生长因子-1(IGF-1)的水平。软骨细胞是特化的骨细胞 来源于经历了逐步分化过程的间充质谱系--包括 增殖、肥大和凋亡--在哺乳动物长骨中维持生长板 软骨形成的过程。这一过程在很大程度上受到IGF-1的影响，它支持 软骨细胞的增殖和肥大和控制氧化应激，以及产生的ROS信号的平衡 NADPH氧化酶NOX2和NOX4。干扰ROS调节会损害正常生长 板块动态平衡和纵向生长。乙醇损害生长板成熟，尽管精确的 这种效应背后的机制还没有在体内阐明。本申请中建议的研究包括 旨在用一只青春期小鼠揭示酒精对生长板影响的机械方面 模型(C57BL6小鼠，6周龄)和慢性酒精暴露(Lieber-DeCarli饮食，28%乙醇，10周)。 这些研究的总体假设是乙醇减少了IGF-1信号转导，并诱导了多种来源的 细胞内ROS抑制软骨细胞增殖，扰乱生长板的形成，并损害纵向 骨骼生长。三个具体目标将解决全身毒性和细胞内毒性的互补方面 乙醇对软骨形成的影响。特定目标1将使用静脉注射IGF-1来确定程度 乙醇会损害IGF-1信号转导，抑制生长板成熟。《特定目标2》将使用 线粒体靶向抗氧化剂MitoTEMPO抑制线粒体ROS的产生及其检测 对乙醇的ROS介导效应的贡献。特定目标3将使用单独的Cre-lox基因型，删除 NOX2和NOX4在软骨细胞中的特异性表达，以确定各酶在乙醇诱导的ROS、AS中的作用 以及每种酶在软骨细胞分化的不同阶段中各自的作用。对以下问题的回应 这些实验条件将由基因表达(qPCR、RNAScope杂交)、蛋白质 生物学(Western blotting、免疫组织化学、ELISA)、ROS生成(DMPO自旋捕获)、细胞 形态(组织学染色)和整体骨骼结构。这些研究将产生对 激素因素和ROS在软骨细胞成熟中的作用并提供了乙醇的综合图景 发育易感性重要窗口期对生长板发育的作用机制。

PROJECT SUMMARY/ABSTRACT Adolescence represents a developmental phase where the skeleton is particularly susceptible to damage that can have long-term effects. Alcohol (ethanol) use is highly prevalent among adolescents, where 1 in 17 (~6%) of boys and girls aged 12-17 are estimated to have engaged in binge drinking. Ethanol is a bone toxicant known to generate oxidative stress by increasing intracellular reactive oxygen species (ROS). Additionally, ethanol decreases circulating levels of insulin-like growth factor-1 (IGF-1). Chondrocytes are specialized bone cells derived from the mesenchymal lineage that undergo a stepwise differentiation process – consisting of proliferation, hypertrophy, and apoptosis – to maintain the growth plate in mammalian long bones through the process of chondrogenesis. This process is influenced to a significant extent by IGF-1, which supports chondrocyte proliferation and hypertrophy and controls oxidative stress, and a balance of ROS signals generated by the NADPH oxidase enzymes Nox2 and Nox4. Interference with ROS regulation can impair normal growth plate homeostasis and longitudinal growth. Ethanol impairs growth plate maturation, although the precise mechanisms underlying this effect have not been elucidated in vivo. The studies proposed in this application are designed to uncover the mechanistic aspects of ethanol’s effects on the growth plate using an adolescent mouse model (C57Bl6 mice, aged 6 weeks) and chronic ethanol exposure (Lieber-DeCarli diet, 28% ethanol, 10 weeks). The overall hypothesis of these studies is that ethanol reduces IGF-1 signaling and induces multiple sources of intracellular ROS to suppress chondrocyte proliferation, disrupt growth plate formation, and impair longitudinal bone growth. Three specific aims will address complementary aspects of the systemic and intracellular toxicity of ethanol on chondrogenesis. Specific Aim 1 will use intravenous IGF-1 supplementation to determine the extent to which ethanol impairs IGF-1 signaling and suppresses growth plate maturation. Specific Aim 2 will use the mitochondria-targeted antioxidant mitoTEMPO to suppress mitochondrial ROS generation and determine their contribution to ethanol’s ROS-mediated effects. Specific Aim 3 will use separate Cre-lox genotypes that delete Nox2 and Nox4 specifically in chondrocytes to determine the role of each enzyme in ethanol-induced ROS, as well as each enzyme’s respective role in the distinct stages of chondrocyte differentiation. The responses to these experimental conditions will be determined by gene expression (qPCR, RNAscope hybridization), protein biology (western blotting, immunohistochemistry, ELISA), ROS generation (DMPO spin trapping), cellular morphology (histological staining), and overall bone structure. These studies will generate new insights into the role of hormonal factors and ROS in chondrocyte maturation and provide an integrated picture of ethanol’s mechanism of action on growth plate development during an important window of developmental susceptibility.