花生四烯酸为靶标的代谢网络介导OPG/RANK/RANKL对绝经后骨重建过程的调控机制研究

The imbalance of bone remodeling plays a pivotal role in postmenopausal osteoporosis (PMO) due to estrogen deficiency. The OPG/RANKL/RANK system as the dominant, final mediator of osteoclastogenesis and PMO represents a major advance in maintaining bone remodeling balance. However, the regulation mechanism of OPG/RANKL/RANK system via estrogen has not been elucidated. Recently, numerous studies have indicated that the abnormal arachidonic acid (AA) metabolic network is closely related to estrogen levels in body and directly involved in the regulation of OPG, RANKL level, which would stimulate osteoblast replication and differentiation, increase bone resorption, and maintain bone remodeling associated with osteoporosis. For this reason, we hypothesized that AA metabolic network disorder led to disturbance of OPG/RANK/RANKL pathway play an important factor in the pathogenesis of PMO.This study was therefore designed as follows based on our previous research: 1) Taking our well developed no-target and target based on AA metabolic network metabolomic technology from the overall perspective to quantitative and qualitative description the metabolic profile in plasma and urine in postmenopausal osteoporosis patients. 2)Subsequently, we further explicated that the systematic alteration of AA metabolic network on regulation of bone remodeling process by OPG/RANKL/RANK system using the combination of molecular biology and metabolic enzyme inhibition technology on animal model, osteoblasts and osteoclasts. The topic will successfully explore pathomechanism of bone loss induced by estrogen deficiency from the perspective of metabolomics based on AA metabolic network, and provide novel targets for the treatment of postmenopausal osteoporosis.

骨重建失衡是雌激素缺乏导致绝经后骨质疏松症(PMO)的重要因素。OPG/RANK/RANKL主导维持骨重建平衡，是诱发PMO的最终通路。但雌激素对该通路的调控机制尚未阐明。最新研究发现花生四烯酸（AA）代谢网络异常与雌激素状态密切相关，又直接参与OPG，RANKL的调控，在维持骨重建内环境，成骨细胞增殖及破骨细胞活化中发挥着重要作用。据此，我们推测：绝经后AA代谢网络紊乱导致OPG/RANK/RANKL通路失衡是PMO的重要原因。本课题采用非靶标和基于AA靶标的代谢组学技术，从整体角度定性定量描述PMO患者AA代谢网络变化特点和规律。并结合分子生物学，代谢酶单一途径抑制等技术在整体和细胞水平上，阐述AA代谢网络系统性变化对OPG/RANK/RANKL维持的骨重建过程的调控作用。本项目提出了整体代谢网路与特定分子通路相统一的研究策略,从新的视觉阐述了PMO的发病机制,为其有效防治提供新靶点。

随着我国人口老龄化，绝经后骨质疏松症（PMO）成为威胁老年妇女生活和健康的主要疾病之一。该病存在着发病隐匿，发病机制不明，诊断指标单一，药物疗效缓慢等诸多难题。代谢组学技术在骨质疏松症生物标记物的发现、早期诊断及发病机制研究等方面发挥的优势作用促使其发展成为应对这些难题的重要方式..（1）基于 GC-MS 技术的骨质疏松患者血清代谢组学研究.收集40~80 岁年龄段绝经后骨质疏松患者，进行代谢组学研究。血清代谢组经 PCA 及 PLS-DA 分析，发现绝经后骨质疏松症体内代谢发生显著改变，异于绝经前正常女性，绝经后骨量正常和骨量减少组的代谢谱。通过代谢通路分析和ROC曲线分析发现：骨质疏松的形成与脂肪酸代谢通路密切相关，ROC曲线分析结果表明AA与骨密度关联性更高。花生四烯酸水平的增加可能是绝经后骨质疏松症潜在的生物标记物。..（2）基于花生四烯酸靶标的代谢组学技术的骨质疏松患者血清研究.通过建立基于AA代谢通路的LC-MS/MS血清样本检测方法，首次用于临床骨质疏松症患者血清样本中花生四烯酸及其代谢产物的检测，从整体角度定性定量描述PMO患者AA 代谢网络变化特点和规律，阐述其可能的发病机理。结果显示：与正常组和绝经后骨量正常组相比，PMO患者体内AA(P=2.6×10−3)和5-HETE (P=9.8×10−4)显著增加。由于绝经后骨质疏松症体内AA水平上升，导致了其下游代谢产物也随之增加，最提示了AA水平的增加可能是导致绝经后骨质疏松症患者体内花生四烯酸代谢紊乱，以及诱发骨代谢过程异常的一个至关重要的因素。因此研究高浓度的AA对骨代谢的调控作用将有助于系统揭示绝经后骨质疏松症的发病机制。..（3）花生四烯酸对骨重建过程的调控作用.采用体外实验阐述AA的增加对骨重建过程的调控作用。结果显示AA（15-60μM）显著地促进成骨细胞增殖，上调碱性磷酸酶和骨钙素的表达，提示增加AA的水平可以促进成骨细胞的增殖与分化。同时AA可以诱导ERK1/2的磷酸化，进而激活RUNX2和 Osterix的基因表达加速骨形成；AA上调RANKL的表达，增加RANKL/OPG的比例，负向调控OPG/RANK/RANKL通路，诱导骨吸收，使骨代谢过程趋向于高转换状态，表现出与临床相似的病理特征。提示：雌激素缺乏后，体内AA水平的增加诱导高转换的骨重建过程是诱发PMO的重要因素。