ABSTRACT

Purpose: Reactive oxygen species (ROS) are related to compression stress-induced nucleus pulposus (NP) cell autophagy, but the specific mechanism is unknown in compression stress-induced intervertebral disc degeneration (IVDD). Here, we discuss the specific molecular mechanism and explore whether ROS scavengers could be employed as specific drugs to inhibit compression stress-induced IVDD.

Methods: Rat NP cells were exposed to 1.0 MPa compression and pretreatment with the ROS scavenger N-acetylcysteine (NAC) or the JNK-selective inhibitor SP600125 not. Intracellular ROS production was monitored by confocal microscopy. Autophagy was detected by observing the NP cell ultrastructural features using TEM and examining autophagic vacuoles by flow cytometry. The levels of autophagy-associated molecules, the JNK pathway and the PI3K/AKT/mTOR pathway were analyzed by western blotting.

Results: Compression-mediated autophagy in rat NP cells was implicated in ROS generation. The ROS scavenger NAC could protect compression-induced NP cell injures by inhibiting ROS production. And SP600125, a JNK inhibitor, attenuated compression-induced NP cell autophagy. Additionally, this is the first report showing that compression induces autophagy in rat NP cells by impeding the compression-induced ROS dependent PI3K/AKT/mTOR pathway and the ROS independent activation of JNK pathway. And the involvement of JNK pathway was in different mechanism of action that when inhibited leaded to increased cell death, increased generation of ROS but decreased autophagy.

Conclusions: These results show a new regulatory mechanism involving ROS-mediated autophagy in rat NP cells, which may provide ideas for drug development to improve compression stress-induced IVDD and help avoid eventual surgical treatment of IVD herniation.

摘要

目的：活性氧（ROS）与压迫应力诱导的髓核（NP）细胞自噬有关，但在压迫应力诱导的椎间盘退变（IVDD）中的具体机制尚不清楚。在这里，我们讨论了特定的分子机制，并探讨了 ROS 清除剂是否可以用作抑制压缩应激诱导的 IVDD 的特定药物。

方法：将大鼠 NP 细胞暴露于 1.0 MPa 的压力下，并用 ROS 清除剂 N-乙酰半胱氨酸 (NAC) 或 JNK 选择性抑制剂 SP600125 不进行预处理。通过共聚焦显微镜监测细胞内 ROS 的产生。通过使用 TEM 观察 NP 细胞超微结构特征并通过流式细胞术检查自噬泡来检测自噬。通过蛋白质印迹分析自噬相关分子、JNK通路和PI3K/AKT/mTOR通路的水平。

结果：大鼠 NP 细胞中压缩介导的自噬与 ROS 的产生有关。ROS 清除剂 NAC 可以通过抑制 ROS 产生来保护压缩诱导的 NP 细胞损伤。JNK 抑制剂 SP600125 可减弱压缩诱导的 NP 细胞自噬。此外，这是第一份显示压缩通过阻止压缩诱导的 ROS 依赖性 PI3K/AKT/mTOR 通路和 JNK 通路的 ROS 非依赖性激活来诱导大鼠 NP 细胞自噬的报告。JNK通路参与不同的作用机制，当被抑制时会导致细胞死亡增加，ROS的产生增加，但自噬减少。

结论：这些结果显示了一种新的调节机制，涉及大鼠 NP 细胞中 ROS 介导的自噬，这可能为药物开发以改善压缩应激诱导的 IVDD 并有助于避免最终手术治疗 IVD 疝提供思路。