The Fourth Military Medical University, Changle Western Road, 710032, People's Republic of China; Institute for Hygiene of Ordnance Industry, Xi'an, 710065 Shaanxi, People's Republic of China.
Institute of Endemic Diseases, Key Laboratory of Trace Elements and Endemic Diseases, National Health and Family Planning Commission of the people's Rupublic of China, Xi'an Jiaotong University Health Science Center, People's Republic of China.
Eur J Pharmacol. 2018 Apr 5;824:40-47. doi: 10.1016/j.ejphar.2018.01.032. Epub 2018 Feb 6.
NO plays a key role in the pathological mechanisms of articular diseases. As cytoskeletal proteins are responsible for the polymerization, stabilization, and dynamics of the cytoskeleton network, we investigated whether cytoskeletal proteins are the intracellular pathological targets of NO. We aimed at clarifying whether the cytoskeleton perturbations involved in apoptosis are induced in rabbit articular chondrocytes by NO, which can be liberated by sodium nitroprusside (SNP) treatment. The first passage rabbit articular chondrocytes were cultured as monolayer for the experiments, and the effects of NO were tested in the presence of JNK-specific inhibitor, SP600125. SNP treatment of cultured chondrocytes caused significant apoptosis in a concentration-dependent manner (time and dose), as evaluated by TUNEL assay and Annexin V flow cytometry, while the apoptosis was reduced by the SP600125 addition 30 min before SNP treatment. Besides, SP600125 decreased significantly the protein expression of total caspase-3 and the intracellular gene expression of caspase-3, measured by Western blot analysis and PCR. SP600125 also increased the cytoskeletal protein expressions. These results suggested that JNK pathway plays a critical role in the NO-induced chondrocyte apoptosis, and SP600125 treatment blocks the dissolution of the cytoskeletal proteins via activation of caspase-3 pathways.
一氧化氮（NO）在关节疾病的病理机制中起着关键作用。由于细胞骨架蛋白负责细胞骨架网络的聚合、稳定和动态变化，我们研究了细胞骨架蛋白是否是 NO 的细胞内病理靶标。我们旨在阐明由硝普钠（SNP）处理释放的 NO 是否诱导兔关节软骨细胞中的凋亡相关细胞骨架扰动。第一阶段的兔关节软骨细胞作为单层培养进行实验，并在存在 JNK 特异性抑制剂 SP600125 的情况下测试 NO 的作用。SNP 处理培养的软骨细胞以浓度依赖性方式（时间和剂量）引起明显的细胞凋亡，如 TUNEL 测定和 Annexin V 流式细胞术评估所示，而在 SNP 处理前 30 分钟添加 SP600125 可减少细胞凋亡。此外，SP600125 通过 Western blot 分析和 PCR 显著降低总 caspase-3 的蛋白表达和 caspase-3 的细胞内基因表达。SP600125 还增加了细胞骨架蛋白的表达。这些结果表明 JNK 途径在 NO 诱导的软骨细胞凋亡中起着关键作用，并且 SP600125 治疗通过激活 caspase-3 途径阻止细胞骨架蛋白的溶解。
