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.
Osteoarthritis Cartilage. 2021 Jan;29(1):100-112. doi: 10.1016/j.joca.2020.08.014. Epub 2020 Nov 6.
Lysosomes are the major catabolic organelle of the cell and regulate the macromolecular and organelle turnover and programmed cell death. Here, we investigated the lysosome dysfunction in cartilage and its role in chondrocytes apoptosis and the associated mechanism.
Lysosomal acidification in Osteoarthritis (OA) and aged cartilage was determined by LysoSensor staining. Lysosomal function in chondrocytes was blocked by siRNA mediated depletion of Lysosomal Associated Membrane Protein 2 (LAMP2) or with lysosome inhibitors. Chondrocyte apoptosis was determined by LDH release, Caspase-3/7 activation, TUNEL and PI uptake assays. Loss of mitochondrial membrane potential (MMP/ΔΨM) and mitochondrial superoxide level was determined by JC-1 and MitoSOX staining, respectively. Colocalization of mitochondria with BCL2 associated X (BAX) and Cytochrome c was determined by immunostaining. Destabilization of medial meniscus (DMM) was performed to induce OA in mice.
Lysosomal acidification was found to be significantly decreased in aged mouse and human and mouse OA cartilage which also showed increased chondrocyte apoptosis. Inhibition of lysosomal function resulted in increased oxidative stress, accumulation of dysfunctional mitochondria and apoptosis in chondrocytes in monolayer and in cartilage explant cultures. Depletion of LAMP2 expression or treatment of chondrocytes with lysosomal function inhibitors increased the expression and mitochondrial translocation of BAX leading to Cytochrome c release. Lysosomal dysfunction-induced apoptosis in chondrocytes was not blocked by antioxidants MitoTempo or Diphenyleneiodonium (DPI) but was abrogated by inhibiting BAX.
Lysosomal dysfunction induce apoptosis in chondrocytes through BAX-mediated mitochondrial damage and release of Cytochrome c. Our data points to lysosomal function restoration and/or BAX inhibition in chondrocytes as a therapeutic approach for OA.
溶酶体是细胞的主要分解细胞器，调节大分子和细胞器的周转和程序性细胞死亡。在这里，我们研究了软骨中的溶酶体功能障碍及其在软骨细胞凋亡中的作用和相关机制。
通过 LysoSensor 染色测定骨关节炎 (OA) 和老年软骨中的溶酶体酸化。通过 siRNA 介导的溶酶体相关膜蛋白 2 (LAMP2) 耗竭或溶酶体抑制剂阻断软骨细胞中的溶酶体功能。通过 LDH 释放、Caspase-3/7 激活、TUNEL 和 PI 摄取测定软骨细胞凋亡。通过 JC-1 和 MitoSOX 染色分别测定线粒体膜电位 (MMP/ΔΨM) 和线粒体超氧水平的丧失。通过免疫染色测定线粒体与 BCL2 相关 X (BAX) 和细胞色素 c 的共定位。通过破坏内侧半月板 (DMM) 在小鼠中诱导 OA。
发现老化的小鼠和人类以及小鼠 OA 软骨中的溶酶体酸化明显降低，同时也显示出软骨细胞凋亡增加。溶酶体功能抑制导致单层和软骨外植体培养中的软骨细胞中氧化应激增加、功能失调的线粒体积累和凋亡。LAMP2 表达的耗竭或用溶酶体功能抑制剂处理软骨细胞增加了 BAX 的表达和线粒体易位，导致细胞色素 c 的释放。溶酶体功能障碍诱导的软骨细胞凋亡不能被抗氧化剂 MitoTempo 或 Diphenyleneiodonium (DPI) 阻断，但通过抑制 BAX 而被阻断。
溶酶体功能障碍通过 BAX 介导的线粒体损伤和细胞色素 c 的释放诱导软骨细胞凋亡。我们的数据表明，溶酶体功能恢复和/或 BAX 抑制在软骨细胞中作为 OA 的治疗方法。
