Department of Orthopedics, Dalian Medical University , Dalian, Liaoning, China.
Department of Orthopedics, Clinical Medical College of Yangzhou University , Yangzhou, Jiangsu, China.
Connect Tissue Res. 2020 Nov;61(6):554-567. doi: 10.1080/03008207.2019.1631299. Epub 2019 Jul 11.
: To investigate the protective effect of naringin (Nar) on HO-induced apoptosis of nucleus pulposus-derived mesenchymal stem cells (NPMSC) and the potential mechanism in this process. : Rat NPMSC were cultured in MSC culture medium or culture medium with different concentrations of HO. Nar or the combination of Nar and LY294002 was added into the culture medium to investigate the effects of Nar. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The apoptosis rate was determined using Annexin V/PI dual staining and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays. Additionally, the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. ATP level in NPMSC was analyzed via ATP detection kit. Mitochondrial ultrastructure change was observed through transmission electron microscope (TEM). Levels of apoptosis-associated molecules (cleaved caspase-3, Bax and Bcl-2) were evaluated via RT-PCR and western blot, respectively. : The cells isolated from NP met the criteria for MSC. HO significantly promoted NPMSC apoptosis in a dose and time-dependent manner. Nar showed no cytotoxicity effect on NPMSC up to a concentration of 100 μM for 24 h. Nar exhibited protective effects against HO-induced NPMSC apoptosis including apoptosis rate, expressions of proapoptosis and antiapoptosis related genes and protein. Nar could also alleviate HO-induced mitochondrial dysfunction of increased mitochondrial ROS production, reduced MMP, decreased intracellular ATP and mitochondrial ultrastructure change. However, these protected effects were inhibited after LY294002 treatment. : Our results demonstrated that Nar efficiently attenuated HO-induced NPMSC apoptosis and mitochondrial dysfunction. The activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.
探讨柚皮苷（Nar）对 HO 诱导的髓核间充质干细胞（NPMSC）凋亡的保护作用及其潜在机制。
将大鼠 NPMSC 分别在 MSC 培养基或含有不同浓度 HO 的培养基中培养，将 Nar 或 Nar 与 LY294002 的混合物加入培养基中，以研究 Nar 的作用。通过细胞计数试剂盒-8（CCK-8）测定细胞活力。通过 Annexin V/PI 双重染色和末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）测定法测定凋亡率。此外，通过流式细胞术分析活性氧（ROS）和线粒体膜电位（MMP）水平。通过 ATP 检测试剂盒分析 NPMSC 中的 ATP 水平。通过透射电子显微镜（TEM）观察线粒体超微结构变化。通过 RT-PCR 和 Western blot 分别评估凋亡相关分子（cleaved caspase-3、Bax 和 Bcl-2）的水平。
从 NP 中分离的细胞符合 MSC 标准。HO 以剂量和时间依赖的方式显著促进 NPMSC 凋亡。Nar 在浓度高达 100 μM 且作用 24 h 时对 NPMSC 无细胞毒性作用。Nar 对 HO 诱导的 NPMSC 凋亡具有保护作用，包括凋亡率、促凋亡和抗凋亡相关基因和蛋白的表达。Nar 还可以减轻 HO 诱导的线粒体功能障碍，包括增加的线粒体 ROS 产生、降低的 MMP、减少的细胞内 ATP 和线粒体超微结构变化。然而，在用 LY294002 处理后，这些保护作用受到抑制。
我们的结果表明，Nar 有效减轻了 HO 诱导的 NPMSC 凋亡和线粒体功能障碍。ROS 介导的 PI3K/Akt 通路的激活可能是该过程的潜在机制。
