Chen Songfeng, Lv Xiao, Hu Binwu, Shao Zengwu, Wang Baichuan, Ma Kaige, Lin Hui, Cui Min
Department of Orthopaedic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou City, 450052, China.
Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Apoptosis. 2017 May;22(5):626-638. doi: 10.1007/s10495-017-1358-2.
The aim of this study was to systematically investigate the role of necroptosis in compression-induced rat nucleus pulposus (NP) cells death, as well as explore the underlying mechanisms involved. Rat NP cells underwent various periods of exposure to 1.0 MPa pressure. Cell viability and cell death were quantified by using cell counting kit-8 (CCK-8), and Calcein-AM/propidium iodine (PI) staining respectively. Necroptosis-associated target molecules receptor-interacing protein kinase 1 (RIPK1), phosphorylated RIPK1 (pRIPK1), receptor-interacing protein kinase 3 (RIPK3), phosphorylated RIPK3 (pRIPK3) and mixed lineage kinase domain-like (MLKL) were analyzed by Western-blot and RT-PCR. NP cells were also examined for morphological and ultrastructural changes, which can indicate necroptosis. To indirectly establish the presence of necroptosis, the RIPK1 specific inhibitor necrostatin-1 (Nec-1), RIPK3 inhibitor GSK'872, MLKL inhibitor necrosulfonamide (NSA) and small interfering RNA (siRNA) were utilized. The results established necroptosis was taking place in NP cells. The level of necroptosis increased in a time-dependent manner, and this effect was reduced by Nec-1 in vitro. Additionally, NP cells death were significantly attenuated following treatment with Nec-1, GSK'872 or NSA. SiRNA-induced knockdown of RIPK3 or MLKL increased cell survival rate, while knockdown of RIPK1 resulted in a decreased cell survival rate. In summary, RIPK1/RIPK3/MLKL-mediated necroptosis may play an important role in NP cells death induced by continuous mechanical stress. Treatment strategies which aim to regulate necroptosis may prove beneficial, by both reducing NP cells death and slowing IVD degeneration.
本研究的目的是系统地研究坏死性凋亡在压缩诱导的大鼠髓核（NP）细胞死亡中的作用，并探索其潜在机制。将大鼠NP细胞暴露于1.0MPa压力下不同时间段。分别使用细胞计数试剂盒-8（CCK-8）和钙黄绿素-AM/碘化丙啶（PI）染色来定量细胞活力和细胞死亡情况。通过蛋白质免疫印迹法（Western-blot）和逆转录-聚合酶链反应（RT-PCR）分析坏死性凋亡相关靶分子受体相互作用蛋白激酶1（RIPK1）、磷酸化RIPK1（pRIPK1）、受体相互作用蛋白激酶3（RIPK3）、磷酸化RIPK3（pRIPK3）和混合谱系激酶结构域样蛋白（MLKL）。还检查了NP细胞的形态和超微结构变化，这些变化可表明坏死性凋亡。为了间接确定坏死性凋亡的存在，使用了RIPK1特异性抑制剂坏死素-1（Nec-1）、RIPK3抑制剂GSK'872、MLKL抑制剂坏死磺酰胺（NSA）和小干扰RNA（siRNA）。结果证实NP细胞中发生了坏死性凋亡。坏死性凋亡水平呈时间依赖性增加，并且在体外这种作用被Nec-1降低。此外，用Nec-1、GSK'872或NSA处理后，NP细胞死亡明显减轻。SiRNA诱导的RIPK3或MLKL基因敲低提高了细胞存活率，而RIPK1基因敲低导致细胞存活率降低。总之，RIPK1/RIPK3/MLKL介导的坏死性凋亡可能在持续机械应力诱导的NP细胞死亡中起重要作用。旨在调节坏死性凋亡的治疗策略可能被证明是有益的，既可以减少NP细胞死亡，又可以减缓椎间盘退变。
