Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Orthop Res. 2021 Jul;39(7):1496-1504. doi: 10.1002/jor.24828. Epub 2020 Oct 14.
This study aimed to assess the negative effect of oxidized low-density lipoprotein (oxLDL) on annulus fibrosus (AF) cells and decipher the mechanism of action of the process. After treating AF cells with various concentrations (0, 25, 50, 100, and 200 μg/mL) of oxLDL for 24 and 48 hours, their viability was evaluated using cell counting kit-8 and live/dead staining. The percentage of AF cell death was determined with Annexin V/propidium iodide apoptosis staining. The expression of proteins related to the mitochondrial apoptosis pathway was determined using Western blot. Additionally, mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) were assessed with JC-1 staining and dichlorodihydrofluorescein diacetate ormitoSOX probes, respectively. Mitochondrial morphology was observed with a transmission electron microscope. After treatment with oxLDL, AF cell viability decreased, pro-apoptosis proteins (such as Bax, cleaved caspase-9, and cleaved caspase-3) increased, and anti-apoptosis proteins (Bcl-2) declined. Excessive ROS and diminished MMP were also detected during this process, as were enhanced mitochondrial fission and augmented Drp1 expression. Furthermore, knocking down the expression of Drp1 rescued oxLDL-induced AF cell death. Collectively, these results suggest that oxLDL induces AF cell death through a mitochondria-related pathway. Enhanced mitochondrial fission was involved in oxLDL-induced AF cell death. Targeting Drp1, a target for regulating the process of mitochondrial fission, may be a feasible strategy for preventing intervertebral disc degeneration in hyperlipidemia.
本研究旨在评估氧化型低密度脂蛋白（oxLDL）对纤维环（AF）细胞的负面影响，并阐明其作用机制。用不同浓度（0、25、50、100 和 200μg/ml）的 oxLDL 处理 AF 细胞 24 和 48 小时后，用细胞计数试剂盒-8 和死活染色法评估细胞活力。用 Annexin V/碘化丙啶凋亡染色法测定 AF 细胞死亡的百分比。用 Western blot 法测定与线粒体凋亡途径相关的蛋白表达。此外，用 JC-1 染色和二氯二氢荧光素二乙酸酯或 mitoSOX 探针分别评估线粒体膜电位（MMP）和细胞内活性氧（ROS）。用透射电子显微镜观察线粒体形态。oxLDL 处理后，AF 细胞活力下降，促凋亡蛋白（如 Bax、cleaved caspase-9 和 cleaved caspase-3）增加，抗凋亡蛋白（Bcl-2）减少。在此过程中还检测到过多的 ROS 和降低的 MMP，以及增强的线粒体裂变和增加的 Drp1 表达。此外，敲低 Drp1 的表达可挽救 oxLDL 诱导的 AF 细胞死亡。综上所述，这些结果表明 oxLDL 通过与线粒体相关的途径诱导 AF 细胞死亡。增强的线粒体裂变参与了 oxLDL 诱导的 AF 细胞死亡。靶向 Drp1，一种调节线粒体裂变过程的靶点，可能是预防高脂血症中椎间盘退变的一种可行策略。
