ABSTRACT
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 30â¯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.
Subject(s)
Anthracenes/pharmacology , Apoptosis/drug effects , Caspase 3/metabolism , Chondrocytes/drug effects , Cytoskeletal Proteins/metabolism , Nitric Oxide/metabolism , Proteolysis/drug effects , Animals , Caspase 3/genetics , Chondrocytes/cytology , Chondrocytes/metabolism , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rabbits , Signal Transduction/drug effectsABSTRACT
OBJECTIVE: To study the role of c-jun N-terminal kinase (JNK) signaling pathway in chondrocyte apoptosis induced by nitric oxide (NO) using NO donor sodium nitroprusside (SNP) and JNK inhibitor SP600125. METHODS: Articular chondrocytes were separated from New Zealand rabbits aged 3 weeks by mechanical digestion and enzyme digestion and identified by toluidine blue staining, and then the chondrocytes were treated with SNP and SP600125 for 24 h. The cell apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL), and the expression levels of nuclear factor-kappa B (NF-κB) p65 and p53 were measured by western blot. RESULTS: Compared with those in control group, the early apoptotic rate of SNP-treated chondrocytes increased as the concentration of SNProse, exhibiting a concentration dependency (P < 0.05), and the expression levels of NF-κB p65 and p53 also increased (P < 0.05); JNK inhibitor SP600125 inhibited these increases (P < 0.05). CONCLUSION: JNK signaling pathway plays an important role in NO-induced chondrocyte apoptosis. JNK inhibitor SP600125 can reduce NO-induced apoptosis and expression of NF-κB p65 and p53 in articular chondrocytes of rabbits in a concentration-dependent manner.
