Objective

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.

Design

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.

Results

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.

Conclusions

These findings demonstrate that ERK1/2 is a critical player in DRP1-mediated induction of mitochondrial fission and apoptosis in IL-1β-stimulated chondrocytes.

中文翻译：

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ERK1/2 介导的 DRP1 激活调节软骨细胞的线粒体动力学和凋亡

客观的

确定病理条件下软骨细胞线粒体裂变的动力学相关蛋白 1 (DRP1) 调节，这是骨关节炎发病机制中未充分探索的领域。

设计

通过软骨切片的免疫组织化学 (IHC) 或免疫荧光 (IF) 染色确定 DRP1 蛋白表达。软骨细胞中 IL-1β 诱导的 DRP1 mRNA 表达通过 qPCR 和免疫印迹法定量蛋白质表达。通过线粒体标记蛋白的 MitoTracker 染色或 IF 染色或通过 mitoDsRed 的瞬时表达来观察软骨细胞中的线粒体碎片。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、Caspase 3/7 活性测定、碘化丙啶 (PI) 和末端脱氧核苷酸转移酶 dUTP 缺口末端标记 (TUNEL) 染色和裂解的半胱天冬酶 3 的 IF 染色测定细胞凋亡。测定细胞色素 c 释放通过共聚焦显微镜。

结果

DRP1 的表达和线粒体损伤在人 OA 软骨和接受 DMM 手术的小鼠关节中高，这也显示出软骨细胞凋亡增加。IL-1β 通过调节 DRP1 表达和活性诱导线粒体网络断裂和软骨细胞凋亡，并通过 Bax 介导的细胞色素 c 释放诱导细胞凋亡。Mdivi-1 对 DRP1 活性的药理学抑制阻断了 IL-1β 诱导的软骨细胞线粒体损伤和凋亡。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 激活和软骨细胞线粒体网络断裂的诱导至关重要，因为这些通过抑制 ERK1/2 激活而被阻断。

结论

这些发现表明，ERK1/2 是 DRP1 介导的 IL-1β 刺激的软骨细胞线粒体分裂和凋亡诱导的关键参与者。