SIRT1下调介导氧化应激损伤在高负荷压应力促进椎间盘退变中的作用及机制研究

High compressive stress is a crucial pathological factor of intervertebral disc degeneration, which can promote apoptosis- and senescence-like cellular fate of nucleus pulposus (NP) cells. However, the potential mechanism is not fully understood. Previous studies suggest that SIRT1 can alleviate NP cell apoptosis and senescence through inhibiting oxidative-stress damage. Our preliminary experiments show that the aggravated NP cell apoptosis and senescence are accompanied by the up-regulated SIRT1 expression and the intracellular reactive oxygen species (ROS) accumulation under high compressive stress. Therefore, we deduce that high compressive stress may promote the apoptosis- and senescence-like cellular fate of NP cells through down-regulating SIRT1 expression and thus aggravating oxidative-stress damage. To verify this hypothesis, this study will using technologies, including specific intervention and gene overexpression, to investigate the role and signal transduction of SIRT1 down-regulation in enhancing oxidative stress and than promoting the apoptosis- and senescence-like cellular fate of NP cells under high compressive stress in the disc organ culture and NP cell 3D culture. Meanwhile, we will observe that whether SIRT1 overexpression can alleviate oxidative stress and then alleviate apoptosis- and senescence-like cellular fate of NP cells. Additionally, we will observe that whether SIRT1 overexpression can retard intervertebral disc degeneration in vivo. This study will be helpful to understand the pathogenesis of disc degeneration and provide theoretical evidence for the prevention and therapy of disc degeneration.

高负荷压应力是椎间盘退变的重要外部病理因素，可促进髓核细胞发生凋亡和衰老样命运改变，但机制尚未阐明。既往国内外研究提示SIRT1可减轻氧化应激反应进而抑制细胞凋亡和衰老。我们前期研究发现高负荷压应力促进髓核细胞凋亡和衰老时，伴随SIRT1表达下调和胞内活性氧蓄积。因此，我们推测高负荷压应力是通过降低SIRT1表达增强氧化应激反应，继而导致髓核细胞凋亡和衰老样命运改变。为证实该假设，本项目拟在椎间盘器官培养和髓核细胞三维培养模型中，通过特异性阻断、基因过表达等技术，探讨高负荷压应力下SIRT1表达下调介导的氧化应激损伤在促进髓核细胞凋亡和衰老中的作用及机制；并观察高负荷压应力下，SIRT1过表达能否减轻氧化应激反应进而抑制髓核细胞凋亡和衰老；另外，动物模型中探讨SIRT1过表达能否减缓动物体内椎间盘退变进程。本研究将有助于阐明椎间盘退变的病理机制，为探索新的防治策略提供理论依据。

高负荷压应力在加速椎间盘退变进程中发挥重要作用，国内外和我们的前期研究表明高负荷压应力下椎间盘髓核细胞衰老、凋亡率显著升高，但机制不完全清楚。文献报道SIRT1表达下调可促进多种细胞发生衰老及凋亡。本研究展开前我们预实验发现高负荷压应力促进髓核细胞衰老、凋亡的同时，可诱导髓核细胞SIRT1表达下调和氧化应激损伤加剧，因此，我们推测高负荷压应力可通过下调SIRT1表达继而加剧氧化应激损伤来促进髓核细胞衰老及凋亡样命运改变。为证实这一推测，一方面，本项目探讨了SIRT1表达下调在高负荷压应力促进髓核细胞衰老及凋亡中的作用；另一方面，本研究通过条件性增强SIRT1表达等技术深入研究其具体分子机制。我们的研究结果表明：高负压压应力下(20%压缩性变)，髓核细胞SIRT1基因和蛋白表达明显降低，同时髓核细胞氧化应激损伤加剧(活性氧含量增加，线粒体功能失常)。同时，髓核细胞衰老、凋亡增强（细胞衰老及凋亡率增加、P53/21活性增强、caspase-3活性增强）；然而，当过表达髓核细胞SIRT1后，高负荷压应力下髓核细胞氧化应激损伤状态缓解，P53/21介导的应激下细胞早衰以及NF-κβ介导的细胞凋亡减弱；最后，在鼠尾间盘轴向加压诱导退变模型中，通过显微注射慢病毒上调髓核组织SIRT1表达后，髓核退变进程得到显著缓解，进一步验证了SIRT1对高负荷压应力相关髓核退变的保护性作用。通过本研究，有助于进一步认识压应力相关椎间盘退行性疾病的病理机制，为椎间盘退变的防治策略提供新的干预靶点及其理论依据。