ERS-Beclin-1信号通路介导的自噬在压力致椎间盘退行性变中的作用及调控机制

Excessive or inappropriate compressive force, which is the main cause of degenerative disc disease, can lead to autophagy of nucleus pulposus (NP) cells of intervertebral discs (IVDs). Autophagy of NP cells is closely related with mitochondrial damage as well as endoplasmic reticulum stress (ERS). According to our previous studies, we demonstrated that mechanical pressure can trigger extensive dilation of endoplasmic reticulum, and up-regulate the expression of Beclin-1, moreover, the application of ERS inhibitor significantly reduced the autophagy level of NP cells initiated by the compression. All these results indicate that mechanical stress may cause autophagy of NP cells through ERS-Beclin-1 pathway. This study is designed to investigate the possible mechanism of compression-induced autophagy of NP cells through observing the influence of mechanical stress on the expression of ERS and Beclin-1 pathway related proteins, to reveal the potential coupling mechanism between ERS and Beclin-1 pathway by identifying the influence of mechanical stress on the expression of ERS downstream signal molecule such as PERK and IRE1α, to illustrate the specific function of autophagy in degenerative disc disease by studying the relationship between autophagy and cell death. Our subject will make great contribution to further studying the mechanism of IVD degeneration, providing a new therapeutic strategy and theoretical foundation in preventing IVD degenerative disease.

过度压力作为椎间盘退行性变的主要诱因，可诱发椎间盘髓核细胞发生自噬。细胞自噬与线粒体损伤、内质网应激（ERS）等密切相关。我们前期研究发现，压力可以引起细胞内质网广泛扩张、Beclin-1基因表达上调，且ERS抑制剂可以明显降低压力诱导的细胞自噬水平，提示压力可能通过ERS-Beclin-1信号通路引起髓核细胞自噬。本课题拟在成功构建细胞可控加压模型的基础上，观察压力对ERS相关蛋白及Beclin-1途径相关蛋白表达水平的影响，探讨压力诱导髓核细胞自噬的可能机制；观察压力对ERS下游相关信号分子蛋白PERK、IRE1α表达水平的影响，揭示ERS和Beclin-1信号通路之间可能的偶联机制；研究髓核细胞自噬水平与细胞死亡之间的关系，阐明细胞自噬在椎间盘退行性变中的具体作用。本课题将有助于进一步深入研究椎间盘退行性变的分子机制，为椎间盘退行性变的早期防治提供新的思路和理论依据。

过度压力是椎间盘退行性变的首要诱因，但压力导致椎间盘退行性变的机制尚不清楚。本课题组利用成功构建的细胞可控加压模型、大鼠尾椎可控加压模型，充分模拟人体内的压力条件，并行相关压力处理后，检测自噬、凋亡水平变化，阐明自噬、凋亡与压力条件下椎间盘退行性变的关系。前期研究已经证实自噬、凋亡与髓核细胞存活率密切相关，且推测内质网应激（ERS）可能参与调节自噬、凋亡发生过程。本研究着力探索压力条件下ERS与自噬、凋亡的关系。髓核细胞置于1MPa压力条件下培养后：透射电镜下观测内质网形态学变化、自噬体的形成，WB、PCR、流式细胞术检测自噬及凋亡水平变化。研究显示，ERS水平与自噬表达水平呈正相关，抑制ERS可降低压力条件下自噬水平，且ERS介导的自噬对髓核细胞起保护作用；同时,ERS与凋亡发生也密切相关，抑制ERS可导致凋亡水平升高，促进髓核细胞死亡进程。大鼠尾椎可控加压模型研究亦显示：随着压力强度的增大，ERS关键蛋白BIP与自噬相关蛋白LC3II、Beclin1表达水平均逐渐升高，也提示ERS与自噬发生存在紧密关联。进一步研究发现，ERS相关蛋白表达水平与髓核细胞细胞外基质如蛋白多糖、II型胶原的合成水平一致，抑制ERS可降低压力条件下髓核细胞蛋白多糖、II型胶原的合成，促进髓核细胞死亡。据此推测ERS介导自噬发生、对细胞起保护作用均可能与介导蛋白多糖、II型胶原合成相关。然而，压力条件下ERS介导的自噬和凋亡之间的具体转换关系，尚待进一步研究明确。