生理平流剪切应力通过COX-2/MMPs信号通路参与机械损伤诱发骨关节炎发病的作用机制研究

Mechanical injury produces high hydrostatic stress, tensile strain and fluid flow on articular cartilage, which precipitates in destruction of articular cartilage and development of secondary osteoarthritis (OA). As a consequence of the mechanical damage to articular cartilage, expression of matrix degrading enzymes, such as matrix metalloproteinases (MMPs), are highly induced in chondrocytes, which in turn contribute to the pathology of OA. However, the mechanism remains unknown. Using the human T/C-28a2 chondrocytic cell line as a model system, we demonstrate that prolonged application of high fluid shear to human chondrocytes will induce the expression of MMPs relative to static controls, which was reversed by the treatment of COX-2 inhibitor, NS398. In addition, we will elucidate the mechanisms or signaling pathways that COX-2 regulates MMPs expression in response to lamellar fluid shear stress. More importantly, the biological functions of MMPs on the pathogenesis of OA will be further addressed in the OA model of Hartley guinea pigs. Taking the advantage of high fluid shear stress on cartilage in the OA model of Hartley guinea pigs, the activities of signaling molecules and the expression of MMPs will be determined in celecoxib-treated pigs. As compared with non-treatment pigs, the pivotal roles of MMPs in the development and progression of OA will be finally elucidated. In light of establishing the models of OA investigation in vivo and in vitro, we will summarize and reconstruct the signaling network that regulates shear-mediated OA progression, which may refine our understanding of the individual roles of MMPs in OA pathophysiology. This knowledge will be instrumental in dissecting the underlying OA mechanisms as to develop strategies in treating OA disorders.

机械损伤激活基质金属蛋白酶(MMPs)是造成骨关节破坏，进而导致骨关节炎(OA)发生的关键环节。平流剪切应力作为机械损伤的主要致病因素在OA恶化过程中发挥了至关重要的作用，然而其具体机制尚不清楚。鉴于此，本项目拟采用平流剪切应力处理人软骨细胞T/C-28a2，结合信号传导的相关研究手段，研究平流剪切应力激活的COX-2及其代谢产物调控MMPs表达的信号通路，明确COX-2/MMPs信号通路在介导平流剪切应力诱发人软骨细胞炎症反应过程中的作用机制。同时，利用平流剪切应力自发诱导Hartley豚鼠OA发病的特性，分析COX-2抑制剂类药物-塞来昔布对COX-2信号通路中各分子活性及MMPs表达的影响，从而确证MMPs作为OA发病的最终执行者，在OA发生发展过程中的核心作用。通过本项目的实施，建立和完善体内外OA研究的技术体系，揭示OA发生、发展的分子机制，为OA的预防、诊断和治疗提供理论依据。

机械损伤激活基质金属蛋白酶(MMPs)是造成骨关节破坏，进而导致骨关节炎(OA)发生的关键环节。平流剪切应力(FSS)作为机械损伤的主要致病因素在OA恶化过程中发挥了至关重要的作用，然而其具体机制尚不清楚。鉴于此，本项目拟采用平流剪切应力处理人软骨细胞T/C-28a2和COX-2转基因小鼠模型，结合信号传导的相关研究手段，研究平流剪切应力激活的COX-2及其代谢产物调控MMPs表达的信号通路，明确COX-2/MMPs信号通路在介导平流剪切应力诱发人软骨细胞炎症反应过程中的作用机制。通过体内外实验，发现FSS通过激活COX-2的表达，影响MMP-1，-3和-12活性的分子机制。进而在COX-2转基因小鼠中证实COX-2通过PGs，信号通路激酶（包括：PI3-K/AKT、ERK1/2、p38、JNK和PKA）和转录因子（包括：NF-κB和AP-1）调控MMPs产生的分子机制，并最终确定COX-2激活MMPs是诱发OA发病的关键环节。除标书中的内容以外，在该项目实施期间，进一步将研究拓展到一些关键生长因子，包括: 成纤维细胞生长因子（Fibroblast growth factor, FGF）、胰岛素样生长因子（Insulin growth factor, IGF）和血管内皮生长因子（Vascular endothelial growth factor, VEGF）及炎症因子，包括：白介素-1β（Interleukin-1β, IL-1β）通过影响COX-2表达及代谢调控MMP-1, -3和-12的分子机制。通过该项目的实施，系统揭示了生理FSS通过激活COX-2/MMPs信号通路诱发OA发病的分子机制，不仅实现了理论上的创新，还实现了技术上的创新，建立了体内外研究OA发病的实验体系，为后续药物给药系统的开发奠定基础。