Zhao Lei, Lin Hui, Chen Songfeng, Chen Sheng, Cui Min, Shi Deyao, Wang Baichuan, Ma Kaige, Shao Zengwu
Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Department of Orthopaedic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China.
J Orthop Res. 2018 Apr;36(4):1269-1282. doi: 10.1002/jor.23751. Epub 2017 Dec 29.
This study aimed to systematically investigate whether programmed necrosis contributes to H O -induced nucleus pulposus (NP) cells death and to further explore the underlying mechanism involved. Rat NP cells were subjected to different concentrations of H O for various time periods. The cell viability was measured using a cell counting kit-8, and the death rate was detected by Hoechst 33258/propidium iodide (PI) staining. The programmed necrosis-related molecules receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), poly (ADP-ribose) polymerase (PARP), and apoptosis inducing factor (AIF) were determined by real-time polymerase chain reaction and Western blotting, respectively. The morphologic and ultrastructural changes were examined by phasecontrast microscopy and transmission electron microscopy (TEM). In addition, the necroptosis inhibitor Necrostatin-1 (Nec-1), the PARP inhibitor diphenyl-benzoquinone (DPQ) and small interfering RNA (siRNA) technology were used to indirectly evaluate programmed necrosis. Our results indicated that H O induced necrotic morphologic and ultrastructural changes and an elevated PI positive rate in NP cells; these effects were mediated by the upregulation of RIP1 and RIP3, hyperactivation of PARP, and translocation of AIF from mitochondria to nucleus. Additionally, NP cells necrosis was significantly attenuated by Nec-1, DPQ pretreatment and knockdown of RIP3 and AIF, while knockdown of RIP1 produced the opposite effects. In conclusion, these results suggested that under oxidative stress, RIP1/RIP3-mediated programmed necrosis, executed through the PARP-AIF pathway, played an important role in NP cell death. Protective strategies aiming to regulate programmed necrosis may exert a beneficial effect for NP cells survival, and ultimately retard intervertebral disc (IVD) degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1269-1282, 2018.
本研究旨在系统调查程序性坏死是否导致过氧化氢（H₂O₂）诱导的髓核（NP）细胞死亡，并进一步探究其潜在机制。将大鼠NP细胞暴露于不同浓度的H₂O₂中不同时间段。使用细胞计数试剂盒-8检测细胞活力，通过Hoechst 33258/碘化丙啶（PI）染色检测细胞死亡率。分别通过实时聚合酶链反应和蛋白质免疫印迹法测定程序性坏死相关分子受体相互作用蛋白1（RIP1）、受体相互作用蛋白3（RIP3）、聚（ADP-核糖）聚合酶（PARP）和凋亡诱导因子（AIF）。通过相差显微镜和透射电子显微镜（TEM）检查形态学和超微结构变化。此外，使用坏死性凋亡抑制剂Necrostatin-1（Nec-1）、PARP抑制剂二苯基苯醌（DPQ）和小干扰RNA（siRNA）技术间接评估程序性坏死。我们的结果表明，H₂O₂诱导NP细胞出现坏死性形态学和超微结构变化以及PI阳性率升高；这些效应是由RIP1和RIP3上调、PARP过度激活以及AIF从线粒体转位至细胞核介导的。此外，Nec-1、DPQ预处理以及敲低RIP3和AIF可显著减轻NP细胞坏死，而敲低RIP1则产生相反的效果。总之，这些结果表明，在氧化应激下，RIP1/RIP3介导的程序性坏死通过PARP-AIF途径发挥作用，在NP细胞死亡中起重要作用。旨在调节程序性坏死的保护策略可能对NP细胞存活产生有益影响，并最终延缓椎间盘（IVD）退变。© 2017骨科研究学会。由Wiley Periodicals, Inc.出版。《矫形外科研究杂志》36:1269 - 1282，2018年。
