Zhao Chang-Qing, Zhang Yue-Hui, Jiang Sheng-Dan, Jiang Lei-Sheng, Dai Li-Yang
Department of Orthopedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Age (Dordr). 2010 Jun;32(2):161-77. doi: 10.1007/s11357-009-9121-4. Epub 2009 Dec 4.
Intervertebral disc cell apoptosis occurs through either death receptor or mitochondrial pathway, but whether disc cell apoptosis is also mediated by the endoplasmic reticulum (ER) pathway remains unclear. The objective of this study was to investigate whether ER and mitochondria are co-involved in disc cell apoptosis and intervertebral disc degeneration (IVDD) in rats. Forty-eight rats were used for in vivo experiments. IVDD was characterized by X-ray and histomorphology examination, disc cell apoptosis was detected by TUNEL staining, and the co-involvement of ER and mitochondria in apoptosis was determined by immunohistochemical staining for GRP78, GADD153, caspase-12, and cytochrome C. Additional eight rats were used for annular cell isolation and culture. After sodium nitroprusside treatment, annular cell apoptosis was observed morphologically and quantified by flow cytometry; the expression of biomarkers of ER stress and mitochondrial dysfunction were analyzed by reverse transcriptase PCR (RT-PCR), fluorescence double labeling, and Western blot; and mitochondrial membrane potential was detected by 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbo cyanine iodide (JC-1) staining. Finally, NS3694 and Z-ATAD-FMK were employed to inhibit the formation of apoptosome complex and the activation of caspase-12, respectively, and apoptotic incidence and caspase-9 activity were assayed. We found that IVDD, induced by unbalanced dynamic and static forces in the rats, was accompanied by increased disc cell apoptosis and enhanced expression of GRP78, GADD153, caspase-12, and cytochrome C. Annular cell apoptosis induced by sodium nitroprusside was confirmed by morphologic observation and flow cytometry. With increased apoptosis, the expression of GRP78, GADD153, and caspase-12 upregulated, mitochondrial membrane potential decreased, and accumulation of cytochrome C in the cytosol manifested. Furthermore, NS3694 and Z-ATAD-FMK dramatically suppress annular cell apoptosis and caspase-9 activity. In conclusion, disc cell apoptosis mediated simultaneously by ER and mitochondria plays a potent role in IVDD.
椎间盘细胞凋亡可通过死亡受体途径或线粒体途径发生，但椎间盘细胞凋亡是否也由内质网（ER）途径介导仍不清楚。本研究的目的是探讨内质网和线粒体是否共同参与大鼠椎间盘细胞凋亡和椎间盘退变（IVDD）。48只大鼠用于体内实验。通过X射线和组织形态学检查对IVDD进行表征，通过TUNEL染色检测椎间盘细胞凋亡，并通过对GRP78、GADD153、caspase-12和细胞色素C进行免疫组织化学染色来确定内质网和线粒体在凋亡中的共同参与情况。另外8只大鼠用于分离和培养环状细胞。在硝普钠处理后，通过形态学观察环状细胞凋亡，并通过流式细胞术进行定量；通过逆转录聚合酶链反应（RT-PCR）、荧光双标记和蛋白质免疫印迹分析内质网应激和线粒体功能障碍生物标志物的表达；并通过5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑羰花青碘化物（JC-1）染色检测线粒体膜电位。最后，分别使用NS3694和Z-ATAD-FMK抑制凋亡小体复合物的形成和caspase-12的激活，并检测凋亡发生率和caspase-9活性。我们发现，大鼠体内不平衡的动态和静态力诱导的IVDD伴随着椎间盘细胞凋亡增加以及GRP78、GADD153、caspase-12和细胞色素C表达增强。硝普钠诱导的环状细胞凋亡通过形态学观察和流式细胞术得到证实。随着凋亡增加，GRP78、GADD153和caspase-12的表达上调，线粒体膜电位降低，细胞色素C在细胞质中的积累显现出来。此外，NS3694和Z-ATAD-FMK显著抑制环状细胞凋亡和caspase-9活性。总之，内质网和线粒体同时介导的椎间盘细胞凋亡在IVDD中起重要作用。
