Cherng Yih-Giun, Chang Hua-Chia, Lin Yi-Ling, Kuo Ming-Liang, Chiu Wen-Ta, Chen Ruei-Ming
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, No. 250 Wu-Hsing St., Taipei 110, Taiwan, Republic of China.
J Orthop Res. 2008 Jul;26(7):1018-26. doi: 10.1002/jor.20578.
Nitric oxide (NO) can regulate chondrocyte activities. This study was aimed to evaluate the molecular mechanisms of NO donor sodium nitroprusside (SNP)-induced insults to human chondrocytes. Exposure of human chondrocytes to SNP increased cellular NO levels but decreased cell viability in concentration- and time-dependent manners. SNP time dependently induced DNA fragmentation and cell apoptosis. Treatment with 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide, an NO scavenger, significantly lowered SNP-induced cell injuries. Administration of SNP interrupted F-actin and microtubule cytoskeletons and stimulated phosphorylation of mitogen-activated protein kinase kinase kinase-1 (MEKK1) and c-Jun N-terminal kinase (JNK). Similar to SNP, cytochalasin D, an inhibitor of F-actin formation, disturbed F-actin polymerization and increased MEKK1 and JNK activations. Overexpression of a dominant negative mutant of MEKK1 (dnMEK1) in human chondrocytes significantly ameliorated SNP-induced cell apoptosis. Exposure to SNP promoted Bax translocation from the cytoplasm to mitochondria, but application of dnMEKK1 lowered the translocation. SNP time dependently decreased the mitochondrial membrane potential, complex I NADH dehydrogenase activity, and cellular ATP levels, but increased the release of cytochrome c from mitochondria to the cytoplasm. Activities of caspase-9, -3, and -6 were sequentially increased by SNP administration. This study shows that SNP can induce apoptosis of human chondrocytes through sequential events, including cytoskeletal remodeling, activation of MEKK1/JNK, Bax translocation, mitochondrial dysfunction, cytochrome c release, caspase activation, and DNA fragmentation.
一氧化氮（NO）可调节软骨细胞的活性。本研究旨在评估NO供体硝普钠（SNP）对人软骨细胞造成损伤的分子机制。将人软骨细胞暴露于SNP会使细胞内NO水平升高，但细胞活力会以浓度和时间依赖性方式降低。SNP可时间依赖性地诱导DNA片段化和细胞凋亡。使用NO清除剂2-苯基-4,4,5,5-四甲基-咪唑啉-1-氧基-3-氧化物进行处理，可显著降低SNP诱导的细胞损伤。给予SNP会破坏F-肌动蛋白和微管细胞骨架，并刺激丝裂原活化蛋白激酶激酶激酶-1（MEKK1）和c-Jun氨基末端激酶（JNK）的磷酸化。与SNP类似，F-肌动蛋白形成抑制剂细胞松弛素D会干扰F-肌动蛋白聚合，并增加MEKK1和JNK的激活。在人软骨细胞中过表达MEKK1的显性负突变体（dnMEK1）可显著改善SNP诱导的细胞凋亡。暴露于SNP会促进Bax从细胞质向线粒体的转位，但应用dnMEKK1可降低这种转位。SNP可时间依赖性地降低线粒体膜电位、复合体I烟酰胺腺嘌呤二核苷酸脱氢酶活性和细胞ATP水平，但会增加细胞色素c从线粒体释放到细胞质中的量。给予SNP会依次增加半胱天冬酶-9、-3和-6的活性。本研究表明，SNP可通过一系列事件诱导人软骨细胞凋亡，这些事件包括细胞骨架重塑、MEKK1/JNK激活、Bax转位、线粒体功能障碍、细胞色素c释放、半胱天冬酶激活和DNA片段化。
