BNIP3L/NIX介导的线粒体自噬在压力诱导椎间盘退行性变中的作用及机制

Interverterberal disc degeneration (IVDD) is the main reasons of low back pain. The decrease of nucleus pulposus (NP) cells is highly correlated with IVDD. Although we have known that autophagy and apoptosis are involved in the decrease of NP cells during compression-induced IVDD, the exact mechanism of IVDD is unclear. We have reported that the mitochondrial pathway is involved in compression-induced apoptosis of NP cells and autophagy is activated in compression-induced cell degeneration and is mediated by reactive oxygen species in NP cells exposed to compression. Recently, we found that mitophagy was associatied with compression-induced IVDD, the expression of BNIP3L/NIX genes were increased obviously when NP cells were exposed to compression. BNIP3L/NIX is closely related to the mitophagy. We will further study the mechanisms of compression-induced IVDD, reveal the function and mechanism of NP cell mitophagy in vivo and in vitro experiments. And the target proteins of BNIP3L/NIX mediated signaling pathway will be found to adjust NP cell mitophagy. The study will attempt to enhance the capacity of NP cell resistant to mechanical stress by adjusting the selective mitophagy. The target proteins may become the potential targets of IVD degeneration treatment. The study will provide brand new ideas and methods for the prevention and treatment of IVDD.

椎间盘退行性变与椎间盘内髓核细胞数目的减少密切相关。研究显示自噬、凋亡是过度压力诱导髓核细胞减少的重要原因，但椎间盘退行性变的确切机制目前尚未阐明。前期研究中我们发现过度压力可以通过线粒体途径诱导髓核细胞凋亡，及过度压力刺激可引起髓核细胞内产生大量活性氧，从而激活细胞自噬。且首次发现线粒体自噬参与压力诱导髓核细胞损伤，压力刺激可引起髓核细胞线粒体表面蛋白BNIP3L/NIX表达增多，而BNIP3L/NIX与线粒体自噬密切相关。因此本研究将通过体内外实验进一步明确BNIP3L/NIX介导的线粒体自噬在椎间盘退行性变中的作用机制，及其与髓核细胞凋亡关系，并通过干预该通路关键蛋白调节线粒体自噬预防细胞损伤，增强细胞抵抗压力刺激能力。本研究的完成，可以从机理方面阐明过度压力如何诱导髓核细胞线粒体自噬，并为调节压力诱导髓核细胞损伤提供调控途径及靶点，这些将为椎间盘退行性变防治提供全新的思路。

研究背景：腰背部疼痛是骨科常见的疾病，椎间盘退行性变是引起腰背部疼痛的重要原因，压力是引起椎间盘退行性变的重要诱因。压力引起椎间盘内髓核细胞的衰老是引起椎间盘盘退行性变的重要病理因素，本课题重点探讨是否线粒体自噬参与压力诱导椎间盘退行性变，及探索BNIP3/NIX, pink1/parkin介导的线粒体自噬与压力诱导的髓核细胞衰老关系。.研究内容：首先使用衰老相关蛋白p21和p53，及β半乳糖苷酶染色观察压力诱导髓核细胞衰老情况。其次通过免疫荧光，电镜及JC-1染色观察压力作用下线粒体膜通透性的改变，细胞内自噬相关蛋白，及线粒体自噬相关的BNIP3/NIX, pink1/parkin蛋白表达改变，同时使用线粒体膜通透性抑制剂环孢霉素A(cyclosporine A, CSA)及慢病毒转染抑制pink1表达，从而探讨线粒体自噬与压力诱导髓核细胞衰老关系，另外使用活性氧(reactive oxygen species, ROS)抑制剂NAC，观察影响压力诱导髓核细胞内产生的ROS对线粒体自噬的影响。.研究结果: 结果表明压力诱导髓核细胞衰老呈时间依赖性，同时发现压力诱导髓核细胞衰老过程中， pink1/parkin介导的线粒体自噬被活化, 而BNIP3/NIX在压力诱导椎间盘退行性变过程中表达相对较少。通过使用CSA或者沉默pink1信号通路抑制线粒体自噬，压力诱导的髓核细胞衰老可以明显被抑制，而且发现压力诱导髓核细胞损伤的同时，大量的ROS在髓核细胞内堆积，使用ROS抑制剂NAC后， pink1/parkin介导的线粒体自噬也被抑制，压力诱导的髓核细胞衰老也减轻。.研究结论:过度的线粒体自噬可能加速压力诱导髓核细胞衰老，通过调节pink1/parkin信号通路调节线粒体自噬及减少压力引起细胞内ROS产生将可能成为治疗椎间盘退行性变的有效的治疗方法。