Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, 44272, OH, USA.
Osteoarthritis Cartilage. 2022 Feb;30(2):315-328. doi: 10.1016/j.joca.2021.11.003. Epub 2021 Nov 9.
To determine the Dynamin-related protein 1 (DRP1) regulation of mitochondrial fission in chondrocytes under pathological conditions, an area which is underexplored in osteoarthritis pathogenesis.
DRP1 protein expression was determined by immunohistochemistry (IHC) or immunofluorescence (IF) staining of cartilage sections. IL-1β-induced DRP1 mRNA expression in chondrocytes was quantified by qPCR and protein expression by immunoblotting. Mitochondrial fragmentation in chondrocytes was visualized by MitoTracker staining or IF staining of mitochondrial marker proteins or by transient expression of mitoDsRed. Mitochondrial reactive oxygen species (ROS) levels were determined by MitoSOX staining. Apoptosis was determined by lactate dehydrogenase (LDH) release assay, Caspase 3/7 activity assay, propidium iodide (PI), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and IF staining of cleaved caspase 3. Cytochrome c release was determined by confocal microscopy. Surgical destabilization of the medial meniscus (DMM) was used to induce osteoarthritis (OA) in mice.
Expression of DRP1 and mitochondrial damage was high in human OA cartilage and in the joints of mice subjected to DMM surgery which also showed increased chondrocytes apoptosis. IL-1β-induced mitochondrial network fragmentation and chondrocyte apoptosis via modulation of DRP1 expression and activity and induce apoptosis via Bax-mediated release of Cytochrome c. Pharmacological inhibition of DRP1 activity by Mdivi-1 blocked IL-1β-induced mitochondrial damage and apoptosis in chondrocytes. Additionally, IL-1β-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is crucial for DRP1 activation and induction of mitochondrial network fragmentation in chondrocytes as these were blocked by inhibiting ERK1/2 activation.
These findings demonstrate that ERK1/2 is a critical player in DRP1-mediated induction of mitochondrial fission and apoptosis in IL-1β-stimulated chondrocytes.
确定动力相关蛋白 1（DRP1）在病理条件下对软骨细胞中线粒体裂变的调节作用，这是骨关节炎发病机制中研究不足的领域。
通过软骨切片的免疫组化（IHC）或免疫荧光（IF）染色来确定 DRP1 蛋白的表达。通过 qPCR 定量检测 IL-1β诱导的软骨细胞中 DRP1 mRNA 的表达，通过免疫印迹法检测蛋白的表达。通过 MitoTracker 染色或线粒体标记蛋白的 IF 染色或瞬时表达 mitoDsRed 来可视化软骨细胞中的线粒体碎片化。通过 MitoSOX 染色测定线粒体活性氧（ROS）水平。通过乳酸脱氢酶（LDH）释放测定法、半胱天冬酶 3/7 活性测定法、碘化丙啶（PI）和末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）染色以及 cleaved caspase 3 的 IF 染色来测定细胞凋亡。通过共聚焦显微镜测定细胞色素 c 释放。通过内侧半月板（DMM）手术不稳化来诱导小鼠骨关节炎（OA）。
DRP1 和线粒体损伤的表达在人类 OA 软骨中和接受 DMM 手术的小鼠关节中均较高，并且也显示出增加的软骨细胞凋亡。IL-1β 通过调节 DRP1 的表达和活性诱导线粒体网络碎片化和软骨细胞凋亡，并通过 Bax 介导的细胞色素 c 释放诱导凋亡。DRP1 活性的药理学抑制通过 Mdivi-1 阻断了 IL-1β 诱导的软骨细胞中线粒体损伤和凋亡。此外，IL-1β 诱导的细胞外信号调节激酶 1/2（ERK1/2）的激活对于 DRP1 的激活和在软骨细胞中诱导线粒体网络碎片化至关重要，因为通过抑制 ERK1/2 的激活来阻断这些。
这些发现表明 ERK1/2 是 DRP1 介导的 IL-1β 刺激的软骨细胞中线粒体裂变和凋亡诱导中的关键因子。
