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

项目总结/摘要 青春期是骨骼特别容易受到损伤的发育阶段， 会产生长期影响。酒精（乙醇）使用在青少年中非常普遍，17人中有1人（约6%） 据估计，12-17岁的男孩和女孩中有20%曾酗酒。乙醇是一种骨毒性物质， 通过增加细胞内活性氧（ROS）产生氧化应激。此外，乙醇 降低胰岛素样生长因子-1（IGF-1）的循环水平。软骨细胞是特化的骨细胞 来源于经历逐步分化过程的间充质谱系，包括 增殖、肥大和凋亡- 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 软骨细胞增殖和肥大，并控制氧化应激，和ROS信号的平衡产生 NADPH氧化酶Nox 2和Nox 4。干扰ROS调节可损害正常生长 板块内稳态和纵向生长。乙醇损害生长板成熟，虽然精确的 在体内尚未阐明这种作用的机制。本申请中提出的研究是 旨在揭示乙醇对青春期小鼠生长板影响的机制方面 模型（C57 B16小鼠，6周龄）和慢性乙醇暴露（Lieber-DeCarli饮食，28%乙醇，10周）。 这些研究的总体假设是，乙醇减少IGF-1信号传导，并诱导多种来源的胰岛素样生长因子受体。 细胞内ROS抑制软骨细胞增殖，破坏生长板形成， 骨骼生长三个具体目标将解决系统和细胞内毒性的互补方面 乙醇对软骨形成的影响具体目标1将使用静脉注射IGF-1补充剂来确定 其中乙醇损害IGF-1信号传导并抑制生长板成熟。具体目标2将使用 线粒体靶向抗氧化剂mitoTEMPO抑制线粒体ROS的产生，并确定其 对乙醇的ROS介导效应的贡献。具体目标3将使用单独的Cre-lox基因型， Nox 2和Nox 4特异性地在软骨细胞中，以确定每种酶在乙醇诱导的ROS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。的答复 这些实验条件将通过基因表达（qPCR，RNAscope杂交）、蛋白质 生物学（蛋白质印迹、免疫组织化学、ELISA）、ROS生成（DMPO自旋捕获）、细胞 形态学（组织学染色）和整体骨结构。这些研究将产生新的见解 激素因子和活性氧在软骨细胞成熟中的作用，并提供了乙醇的综合图片 在发育易感性的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.