不同运动负荷通过TRPV5调控软骨细胞线粒体活性的机制研究

Osteoarthritis (OA) is the most common cause of chronic disability in the elderly. Mitochondrial calcium overload in chondrocytes is the main pathological feature of osteoarthritis, and the balance of calcium in mitochondria is regulated by the mitochondrial unidirectional transporter (MCU) complex. MCU complex includes mitochondrial calcium uniporter (MCU), mitochondrial calcium uptake 1 (MICU1), mitochondrial calcium uptake 2 (MICU2), essential MCU regulator (EMRE) related conditional elements. Our previous studies have found that overexpressed TRPV5 in OA chondrocytes can increase mediation of the extracellular calcium influx, activate type II calcium-binding protein kinase (CAMK II) through cascade phosphorylation. Boinformatics software predicts that CAMK II may bind and activate MCU complex. However, the process and mechanism of exercise affecting osteoarthritis remain unclear. Our previous studies also found that different exercise loads (different intensity and time duration) can change the expression of TRPV5 and the process of OA. It is suggested that the expression of TRPV5 changes under different exercise loads may regulate MCU founction, and then change the amount of calcium ions entering mitochondria, thereby affecting mitochondrial activity. This study intends to elucidate the molecular mechanism of TRPV5 regulating chondrocyte mitochondrial activity and intervening OA with different exercise loads by constructing animal models of OA with different exercise loads, using cell stretching experiment, molecular biology experiment and other experimental techniques. . This study can provide not only a new perspective for etiological therapy of OA, but also provide an important suggestion for exercise rehabilitation of osteoarthritis. At the same time, it can set a scientific basis for the clinical research of exercise therapy of OA, and create enomous future value in reducing the huge economic burden of osteoarthritis.

软骨细胞中线粒体内钙离子超载是骨性关节炎(osteoarthritis，OA)的主要病理特征，而线粒体内钙离子的平衡受线粒体单向转运体（MCU）复合体调控。前期研究发现：OA软骨细胞中高表达的TRPV5可以介导细胞外的钙离子内流增加，级联磷酸化激活Ⅱ型钙离子结合蛋白激酶Ⅱ（CAMKⅡ）；生物信息学软件预测CAMKⅡ可能结合并活化MCU。目前运动影响骨关节炎的进程与机制尚不清楚，前期又发现不同的运动负荷（强度与周期）可以改变OA的进程和TRPV5的表达量。于是提出猜想：TRPV5的表达量在不同的运动负荷作用下发生改变后进一步调控MCU，改变进入线粒体钙离子的量，进而影响线粒体活性。本项目拟通过构建不同运动负荷OA动物模型，采用细胞拉伸、分子生物学等多种实验手段阐明TRPV5调控软骨细胞线粒体活性以及不同运动负荷干预OA的分子机制。旨在揭示OA发病新机制以及探寻指导OA康复的最佳运动负荷。

骨性关节炎(osteoarthritis，OA)是导致中老年人慢性残障的最常见原因，60岁以上人口中其发病率在10%以上，预测到2030年将成为世界上老年致残的第一大疾病，严重降低患者生活质量的同时也消耗了大量的医疗和家庭资源。运动与骨关节炎的关系一直是争论的焦点，设计适合骨关节的患者的最佳运动强度和时间是我国运动医学研究领域中运动科学地指导骨关节炎康复的最大瓶颈。本研究在采用骨关节炎大鼠跑台训练模型和经典的分子生物学的研究方法发现：不同时期骨关节炎的关节软骨组织退变程度呈现明显差别，蛋白定量检测的TRPV5随着骨关节炎的进展表达量呈现明显升高趋势；同时我们检测软骨细胞线粒体MCU及线粒体失功能指标的表达情况，发现骨关节炎时软骨细胞线粒体MCU及线粒体失功能指标的表达量升高，并且升高趋势与骨关节炎的进展呈现正相关。在细胞实验中发现： MIA刺激后模仿骨关节炎软骨细胞的改变，随着MIA刺激浓度的逐渐升高，细胞内钙离子浓度逐渐增加，同时与软骨细胞线粒体失功能的相关通路分子表达水平也表现出响应的增加。在骨关节炎大鼠跑台实验中发现：在低等运动强度（6m/min）、长时间（约6weeks）运动的大鼠骨关节炎病变程度最轻，相比不运动的骨关节炎组，膝关节骨关节炎的改善程度最理想，为大鼠膝关节骨关节炎最佳的运动负荷。同时我们通过细胞拉伸应力实验也得出响应的结果，长周期、微小拉力后，软骨细胞内钙离子最少，产生ROS最少，透射电镜下软骨细胞中线粒体数量和形态改变最佳。此研究不但为OA的对因治疗学补充全新视点，也为运动指导骨关节炎的康复提供重要参考。同时也可以为骨性关节炎运动治疗的临床研究做好科学铺垫，更具有减轻骨性关节炎的巨额治疗负担的巨大未来价值。