基于溶酶体损伤所致自噬流受阻探讨压力诱导椎间盘退变的机制

Recent studies have shown that autophagy is closely related to intervertebral disc degeneration, but the exact role of autophagy in intervertebral disc degeneration remains controversial. Previous studies on autophagy of intervertebral disc degeneration mainly focused on evaluating the level of autophagosome, but the change in autophagosome cannot reflect the level of autophagy. Autophagic activity must be evaluated by autophagic flux analysis. Excessive pressure is the primary cause of intervertebral disc degeneration. Our previous study found that pressure can induce autophagy by activating autophagy upstream pathways, but it also can inhibit autophagy downstream pathways by damaging lysosomal function, leading to autophagic flux blockade. Based on the above findings, the next step is necessary to clarify how the pressure damage lysosomes, and thus how to affect the self-help of autophagy in pressure-induced intervertebral disc degeneration. This project was designed to investigate the effects of pressure on the autophagic flux and lysosomal function of nucleus pulposus cells by self-developed controllable air compression cell culture apparatus, and further to explore the role of lysosomes in the autophagic flux blockade of nucleus pulposus cells induced by pressure. The use of lysosomal protectors in the treatment of controllable axial loading-induced rabbit lumbar disc degeneration model was to confirm its protective effect on pressure-induced intervertebral disc degeneration in vivo. The project will help clarify the role of autophagy in intervertebral disc degeneration, and it will open up new ideas for the prevention and treatment of intervertebral disc degeneration.

近年研究表明自噬与椎间盘退变密切相关，但自噬在椎间盘退变中的确切作用尚存在争议。既往对椎间盘退变的自噬研究主要集中于评价自噬体的多少，而自噬体数量的变化并不能反映自噬水平高低，必须通过自噬流分析才能准确评价自噬活性。过度压力是促进椎间盘退变的首要诱因，我们前期研究发现：压力尽管能通过激活自噬上游通路诱导自噬发生，但同时能通过损伤溶酶体功能抑制自噬的下游通路，进而导致自噬流受阻。基于以上发现，下一步有必要明确压力如何损伤溶酶体，并进而如何影响自噬对压力所致椎间盘退变的自救作用发挥？本项目拟借助自行研制的可控气压加压细胞培养装置，研究压力对髓核细胞自噬流及溶酶体功能的影响，进一步探讨溶酶体在压力诱导髓核细胞自噬流受阻中的作用。使用溶酶体保护剂治疗可控轴向压力致兔腰椎间盘退变模型，在体内证实其对压力诱导椎间盘退变的保护作用。本项目有助于明确自噬在椎间盘退变中的作用，为椎间盘退变的防治开辟新思路。

近年研究表明自噬与椎间盘退变密切相关，但自噬在椎间盘退变中的确切作用尚存在争议。既往对椎间盘退变的自噬研究主要集中于评价自噬体的多少，而自噬体数量的变化并不能反映自噬水平高低，必须通过自噬流分析才能准确评价自噬活性。过度压力是促进椎间盘退变的首要诱因，但压力诱导椎间盘退变的具体分子机制仍未阐明。此外，溶酶体及其介导的自噬是否在压力诱导的椎间盘退变中扮演关键角色，目前尚无报道。本研究借助自行研制的可控气压加压细胞培养装置，全面检测了压力对髓核细胞自噬流及溶酶体功能的影响，并进一步了评估溶酶体在压力诱导髓核细胞生物学行为改变中的作用。我们发现压力能通过损伤溶酶体结构和功能，主要包括引起溶酶体体积增大、pH值升高、酶活性及降解能力下降等，抑制自噬底物的降解，进而导致自噬流受阻，最终引起髓核细胞功能异常或死亡。此外，压力能引起髓核细胞溶酶体膜的通透性改变，导致溶酶体内的组织蛋白酶释放入胞浆而诱导细胞死亡。这些结果提示我们：溶酶体功能障碍及其介导的自噬流受阻参与了压力诱导的椎间盘退变。本项目有助于阐明压力诱导椎间盘退变的分子机制，为椎间盘退变的防治提供新的思路。