Parkin 清除功能失调的线粒体可调节 ROS 水平并提高人软骨细胞的存活率。
Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes.
机构信息
Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH 44272, USA.
Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH 44272, USA.
出版信息
Osteoarthritis Cartilage. 2018 Aug;26(8):1087-1097. doi: 10.1016/j.joca.2017.07.020. Epub 2017 Aug 8.
OBJECTIVE
Mitochondrial dysfunction, oxidative stress and chondrocyte death are important contributors to the development and pathogenesis of osteoarthritis (OA). In this study, we determined the expression and role of Parkin in the clearance of damaged/dysfunctional mitochondria, regulation of reactive oxygen species (ROS) levels and chondrocyte survival under pathological conditions.
METHODS
Human chondrocytes were from the unaffected area of knee OA cartilage (n = 12) and were stimulated with IL-1β to mimic pathological conditions. Mitochondrial membrane depolarization and ROS levels were determined using specific dyes and flow cytometry. Autophagy was determined by Western blotting for ATG5, Beclin1, immunofluorescence staining and confocal microscopy. Gene expression was determined by RT-qPCR. siRNA, wild-type and mutant Parkin plasmids were transfected using Amaxa system. Apoptosis was determined by PI staining of chondrocytes and TUNEL assay.
RESULTS
IL-1β-stimulated OA chondrocytes showed high levels of ROS generation, mitochondrial membrane damage, accumulation of damaged mitochondria and higher incidence of apoptosis. IL-1β stimulation of chondrocytes with depleted Parkin expression resulted in sustained high levels of ROS, accumulation of damaged/dysfunctional mitochondria and enhanced apoptosis. Parkin translocation to depolarized/damaged mitochondria and recruitment of p62/SQSTM1 was required for the elimination of damaged/dysfunctional mitochondria in IL-1β-stimulated OA chondrocytes. Importantly we demonstrate that Parkin elimination of depolarized/damaged mitochondria required the Parkin ubiquitin ligase activity and resulted in reduced ROS levels and inhibition of apoptosis in OA chondrocytes under pathological conditions.
CONCLUSIONS
Our data demonstrates that Parkin functions to eliminate depolarized/damaged mitochondria in chondrocytes which is necessary for mitochondrial quality control, regulation of ROS levels and chondrocyte survival under pathological conditions.
目的
线粒体功能障碍、氧化应激和软骨细胞死亡是骨关节炎（OA）发生和发病机制的重要因素。在这项研究中，我们确定了 Parkin 在受损/功能失调的线粒体清除、活性氧（ROS）水平调节和病理条件下软骨细胞存活中的表达和作用。
方法
从膝关节 OA 软骨的未受影响区域（n=12）分离出人软骨细胞，并通过 IL-1β 刺激来模拟病理条件。使用特异性染料和流式细胞术测定线粒体膜去极化和 ROS 水平。通过 Western blot 检测 ATG5、Beclin1、免疫荧光染色和共聚焦显微镜检测自噬。通过 RT-qPCR 测定基因表达。使用 Amaxa 系统转染 siRNA、野生型和突变型 Parkin 质粒。通过 PI 染色和 TUNEL 测定法测定软骨细胞凋亡。
结果
IL-1β 刺激的 OA 软骨细胞表现出高水平的 ROS 生成、线粒体膜损伤、受损线粒体的积累和更高的凋亡发生率。IL-1β 刺激 Parkin 表达耗尽的软骨细胞导致持续高水平的 ROS、受损/功能失调的线粒体积累和增强的凋亡。Parkin 易位到去极化/受损的线粒体以及 p62/SQSTM1 的募集对于 IL-1β 刺激的 OA 软骨细胞中受损/功能失调的线粒体的消除是必需的。重要的是，我们证明 Parkin 消除去极化/受损的线粒体需要 Parkin 泛素连接酶活性，并在病理条件下降低 OA 软骨细胞中的 ROS 水平和抑制凋亡。
结论
我们的数据表明，Parkin 在软骨细胞中起作用以消除去极化/受损的线粒体，这对于线粒体质量控制、ROS 水平调节和病理条件下软骨细胞存活是必需的。
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