Both endoplasmic reticulum and mitochondria are involved in disc cell apoptosis and intervertebral disc degeneration in rats
Both endoplasmic reticulum and mitochondria are involved in disc cell apoptosis and intervertebral disc degeneration in rats
复制标题
内质网和线粒体均参与大鼠椎间盘细胞凋亡和椎间盘退变
DOI：
10.1007/s11357-009-9121-4
复制
发表时间：
2010-06-01
期刊：
影响因子：
--
通讯作者：
Dai, Li-Yang
中科院分区：
文献类型：
--
作者：
Zhao, Chang-Qing;Zhang, Yue-Hui;Dai, Li-Yang
椎间盘细胞凋亡可通过死亡受体或线粒体途径发生，但椎间盘细胞凋亡是否也由内质网（ER）途径介导尚不清楚。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）。采用48只大鼠进行体内实验。采用x线和组织形态学检查，TUNEL染色检测椎间盘细胞凋亡，免疫组化GRP78、GADD153、caspase-12和细胞色素c检测内质网和线粒体共同参与细胞凋亡。另取8只大鼠进行环状细胞分离培养。硝普钠处理后，观察环状细胞凋亡形态学及流式细胞术定量；采用逆转录酶PCR （RT-PCR）、荧光双标记和Western blot分析内质网应激和线粒体功能障碍生物标志物的表达；采用5 ',6,6 ' -四氯-1,1 ',3,3 ' -四乙基苯并咪唑碳氰化物碘化染色法检测线粒体膜电位。最后用NS3694和Z-ATAD-FMK分别抑制凋亡复合物的形成和caspase-12的激活，并测定凋亡发生率和caspase-9活性。我们发现，在不平衡的动静力诱导下，IVDD大鼠的椎间盘细胞凋亡增加，GRP78、GADD153、caspase-12、细胞色素c的表达增加。通过形态学观察和流式细胞术证实硝普钠诱导的环状细胞凋亡。随着细胞凋亡的增加，GRP78、GADD153、caspase-12的表达上调，线粒体膜电位降低，胞浆中细胞色素C积累。此外，NS3694和Z-ATAD-FMK显著抑制环状细胞凋亡和caspase-9活性。综上所述，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。
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.