Increase in NO causes osteoarthritis and chondrocyte apoptosis and chondrocyte ERK plays a protective role in the process.

BACKGROUND: Nitric oxide (NO) and reactive oxygen species (ROS) play an important role in the pathology of human osteoarthritis (OA). Ankylosing spondylitis (AS) and atypical OA have similar clinical manifestations and often require differential diagnosis. The mechanism is however not totally clear yet. This study aims to investigate the effects of excessive NO-ROS in OA patients and the effects of extracellular signal-regulated kinases (ERK) pathway in NO-induced apoptosis of chondrocytes during OA progress. METHODS AND RESULTS: Serum samples from OA or AS as pathological control patients and healthy controls were collected for NO and related chemical measurements. The rabbit articular chondrocytes were cultured in vitro, and NO was applied by Sodium Nitroprusside (SNP) in culture medium to mimic OA condition in patients. The level of SNP-evoked chondrocyte apoptosis with or without PD98059 (ERK-specific inhibitor) was evaluated by TUNEL assay, Annexin V flow cytometry and Western blotting. The activity and mRNA expression of caspase-3 in chondrocytes were measured by assay kits and RT-PCR. The levels of NO and malondialdehyde (MDA) in serum were significantly higher in OA patients, while only MDA was significantly higher in AS patients. However, the level of superoxide dismutase (SOD) was lower in both OA and AS patients. SNP induced chondrocyte apoptosis was enhanced by PD98059 with increased protein expression and functional activity of caspase-3. CONCLUSIONS: The increase in nitric oxide occurs specifically in OA patients. ERK pathway may play a protective role on the NO-induced chondrocyte apoptosis, and inhibition of ERK pathway enhances the NO-induced apoptosis.

SP600125 blocks the proteolysis of cytoskeletal proteins in apoptosis induced by gas signaling molecule (NO) via decreasing the activation of caspase-3 in rabbit chondrocytes.

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.

[Role of JNK signaling pathway in chondrocyte apoptosis induced by nitric oxide].

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.

SNP-induced apoptosis may be mediated with caspase inhibitor by JNK signaling pathways in rabbit articular chondrocytes.

NO plays an important role in cartilage destruction by inducing apoptosis of chondrocytes. Here we investigated the role of c-Jun N-terminal kinase (JNK) signal transduction pathways in the apoptosis induced by NO donor sodium nitroprusside (SNP) in rabbit articular chondrocytes. We used Annexin V-FITC/PI flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) assay to detect apoptosis rate. The expressions of p38, NF-κB p65, caspase-3 and p53 genes at protein levels were measured by Western blotting assay. RT-PCR was performed to show the mRNA expression of caspase-3, and the activity of caspase-3 was also detected. To investigate the effect of JNK-specific inhibitor SP600125, chondrocytes were pretreated with SP600125 ahead of SNP treatment. Treatment with SNP accelerated apoptosis in a concentration dependent manner, while such acceleration was reduced by SP600125 pretreatment. Moreover, we found that SP600125 significantly decreased NO-induced NF-κB, p53, caspase-3 protein expressions and caspase-3 mRNA expression, as well as intracellular caspase-3 activity (P < 0.05). Collectively, these data suggest that JNK plays an important role through stimulating NF-κB, p53 and caspase-3 activation.

Selenium increases expression of HSP70 and antioxidant enzymes to lessen oxidative damage in Fincoal-type fluorosis.

Fincoal type fluorosis has only been reported from China, but its pathogenesis is unclear. Many people believe that fluorosis is associated with oxidative stress. Oxidative stress can be reduced at higher selenium (Se) level. Heat shock protein (HSP70) is the most conserved and induced against different stressors. The aim of this study is to detect the expression of HSP70 in fluorosis patients and explore the role of Se in fluorosis protection. The subjects were divided into four groups: "High Se + F group" (n = 50), "High F group" (n = 50), "High Se group" (n = 20) and "Control group" (n = 46). Expression of HSP70 was evaluated by Western blotting and real-time PCR techniques. The concentration of fluoride, content of Se in hair, activity of antioxidant enzymes (GSH-Px, SOD, CAT) and content of malondialdehyde (MDA) were determined. The relative amount of HSP70 gene transcription was significantly higher in "High Se + F group" than the other groups. The same results were found for expression of HSP70 protein to beta-actin ratio. There was a significant difference between "High Se + F group" and "High F group" regarding MDA content and glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activity. These results suggest that oxidative stress plays an important role in the pathogenesis of the Fincoal type fluorosis and it can be reduced at higher Se level.