Liang Qian, Wang Xiao-ping, Chen Tong-sheng
Department of Pain Management, The First Affiliated Hospital of Jinan University, Guangzhou, 510630, China.
Apoptosis. 2014 Sep;19(9):1354-63. doi: 10.1007/s10495-014-1012-1.
This study aims to investigate the mechanism by which resveratrol (RV) prevents sodium nitroprusside (SNP)-induced chondrocyte apoptosis, which is a characteristic feature of osteoarthritis (OA). Rabbit articular chondrocytes were pre-incubated with 100 μM RV for 18 h before 1.5 mM SNP co-treatment for 6 h. Cell viability was evaluated by CCK-8. Annexin V/PI double staining and Hoechst 33258 staining were used to determine the fashion of SNP-induced chondrocytes death. Mitochondrial membrane potential (ΔΨm) was measured by using flow cytometry (FCM) with TMRM and Rhodamine 123 staining. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels were confirmed by FCM analysis with DCFH-DA and DAF-FM DA staining. Cytoskeleton proteins of chondrocytes co-stained with Actin-Trakcer Green and Tubulin-Trakcer Red were validated by confocal microscopy. SNP induced time- and dose-dependent chondrocytes apoptosis with decline of ΔΨm, activation of caspases as well as cytoskeletal remodeling. SNP induced a significant induction of both ROS and NO. RV remarkably prevented SNP-induced ROS production and apoptosis as well as cytoskeletal remodeling, but did not prevent SNP-induced NO production. Pretreatment with NO scavengers did not significantly prevent SNP-induced apoptosis and cytoskeletal remodeling. SNP induces NO-independent ROS production which dominates rabbit articular chondrocyte apoptosis, and RV protects chondrocytes against SNP-induced apoptosis via scavenging ROS instead of NO.
本研究旨在探究白藜芦醇（RV）预防硝普钠（SNP）诱导的软骨细胞凋亡的机制，而软骨细胞凋亡是骨关节炎（OA）的一个特征性表现。兔关节软骨细胞在与1.5 mM SNP共同处理6小时前，先用100 μM RV预孵育18小时。通过CCK-8评估细胞活力。采用Annexin V/PI双染和Hoechst 33258染色来确定SNP诱导的软骨细胞死亡方式。使用TMRM和罗丹明123染色，通过流式细胞术（FCM）测量线粒体膜电位（ΔΨm）。通过使用DCFH-DA和DAF-FM DA染色的FCM分析来确定细胞内活性氧（ROS）和一氧化氮（NO）水平。用肌动蛋白追踪绿和微管蛋白追踪红共同染色的软骨细胞细胞骨架蛋白通过共聚焦显微镜进行验证。SNP诱导软骨细胞出现时间和剂量依赖性凋亡，伴随着ΔΨm下降、半胱天冬酶激活以及细胞骨架重塑。SNP显著诱导ROS和NO生成。RV显著预防了SNP诱导的ROS生成、凋亡以及细胞骨架重塑，但未预防SNP诱导的NO生成。用NO清除剂预处理并不能显著预防SNP诱导的凋亡和细胞骨架重塑。SNP诱导不依赖NO的ROS生成，而这种ROS生成主导了兔关节软骨细胞凋亡，并且RV通过清除ROS而非NO来保护软骨细胞免受SNP诱导的凋亡。
