线粒体分裂蛋白Drp1在成骨细胞氧化应激损伤中的调控机制研究

Accumulating evidence demonstrates that oxidative stress is particularly involved in bone pathogenesis including osteoporosis, bone tumor development, diabetes-induced bone complications and joint inflammatory diseases, and manipulation of the redox balance in bone cells, which has been recently highlighted, represents an important new approach in the prevention of bone damage. Drp1, the regulator of mitochondrial fission, plays critical roles in the oxidative stress-induced dysfunctions, but much about the roles of Drp1 in the bone biology remains to be understood. In the previous study, we found Drp1 is greatly upregulated in osteoblasts under oxidative stress, blockade of Drp1 by the pharmacological inhibition could significantly attenuate the ROS formation and accumulation, which suggested that Drp1 might play an important role in regulating the osteoblast dysfunctions induced by oxidative stress, but the underlying mechanisms are not clear. In this project, using the classical oxidative stress model, we will assess the exact oxidative stress-induced dysfunctions in osteoblast. To examine the role of Drp1 in regulating the osteoblast dysfunctions induced by oxidative stress, we will apply the blockade of Drp1 by the pharmacological inhibition with the mitochondrial division inhibitor Mdivi-1, genetic manipulation with a dominant negative Drp1, Drp1 siRNA on osteoblast, as well as the generation of stable cell lines with stable overexpression of wild type and mutant Drp1. Further, we will determine the cellular and molecular mechanisms on how Drp1 involves in the osteoblast dysfunctions induced by oxidative stress. The results of this project may provide new insights in the field of oxidative stress-related bone dysfunctions, and hold promise as a potential novel prevention and therapeutic strategies for oxidative stress-related bone dysfunctions.

氧化应激在骨相关疾病（如骨质疏松、骨肿瘤、糖尿病相关骨并发症、骨关节炎症等）中的作用日益受到重视，已成为研究骨疾病发生发展规律及其防治对策的新热点。线粒体分裂蛋白Drp1在氧化应激损伤中具有关键调控作用，但相关研究尚未涉及骨生物学领域。申请者前期发现氧化应激状态下成骨细胞Drp1表达显著上调，对其药物抑制可明显降低细胞氧应激水平，提示Drp1在成骨细胞氧化应激损伤中有重要调控作用，而其作用机制尚不清楚。基于此，本研究拟构建经典氧化应激模型，明确成骨细胞氧化应激损伤表型，采用Drp1特异性药物抑制剂、Drp1基因沉默及构建Drp1野生型/突变型高表达稳转细胞系等方式正反双重验证，探讨Drp1在成骨细胞氧化应激损伤中的作用，并进一步深入探究其作用途径及分子机制。该项目研究成果有望为骨氧化应激损伤研究开辟新的思路，也可为以Drp1为靶点在调节骨氧化应激损伤及其防治对策方面的应用研究提供理论依据。

氧化应激在骨相关疾病（如骨质疏松、骨肿瘤、糖尿病相关骨并发症、骨关节炎症等）中的作用日益受到重视，已成为研究骨疾病发生发展规律及其防治对策的新热点。线粒体分裂蛋白Drp1在氧化应激损伤中具有关键调控作用，但相关研究尚未涉及骨生物学领域。本项目构建了体外成骨细胞氧化应激损伤相关模型（包括经典氧化应激损伤模型、高糖损伤模型、炎症模型），并采用Drp1特异性药物抑制剂、Drp1基因沉默siRNA/高表达质粒转染等方式，正反双重验证Drp1在成骨细胞应激损伤中的调控作用，证实氧化应激的积累可以促进Drp1的磷酸化及线粒体易位，促进线粒体分裂，进而通过激活MAPK通路致成骨细胞功能损伤；揭示了ROS-Drp1-MAPK(p38,ERK)信号通路，阐明了Drp1在成骨细胞应激损伤中调控作用的分子机制。同时，申请人延伸探索了糖尿病骨组织中线粒体分裂异常情况，并探索了线粒体膜通透性转换孔在内的其他可调控Drp1的潜在分子靶点，为下一步拟申请研究项目提供了研究基础。本项目的完成不仅揭示了Drp1介导的线粒体分裂异常在成骨细胞应激损伤中的调控作用及其分子生物学机制，为骨应激损伤研究开辟新的思路，也可为以Drp1为靶点调节骨应激损伤及其防治对策方面的应用研究提供理论依据。