A total of 24 healthy Sprague-Dawley (SD) rats (3 weeks old) with an average body weight of 362±35 g were obtained from the Experimental Animal Center of Wuhan University (Wuhan, China). Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA), fetal bovine serum (FBS) was obtained from GE Healthcare Life Sciences (Logan, UT, USA). CMCS (purity >99%) was supplied by the Institute of Chemistry and Environmental Science of Wuhan University (Wuhan, China). Primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). Anti-p38 and anti-p-p38 antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-β-actin antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). A Caspase-3 Activity Assay kit (cat. no. C1115) was purchased from the Beyotime Institute of Biotechnology (Haimen, China). A Cell Counting Kit-8 (CCK-8; cat. no. CK04) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). SB203580 (cat. no. S3807), type-2 collagenase (cat. no. C6885) and SNP (cat. no. 1614501) were obtained from Sigma-Aldrich (St. Louis, MO, USA). An Annexin V-Fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection kit (cat. no. 556547) was obtained from BD Biosciences (Franklin Lakes, NJ, USA). Other commonly used reagents were of high purity and commercially available.

Rat articular chondrocytes were isolated and cultured, as previously described (20). The utilization of rat articular cartilage was approved by the Animal Ethics Committee of Wuhan University. Rats were anesthetized with a single intraperitoneal injection of 1% sodium pentobarbital (40 mg/kg; purchased from Sigma-Aldrich; cat. no. 1507002). The articular cartilage tissue was removed from the knee joints of the 3-week-old rats using a scalpel. The extracted cartilage was cut into 1×1×1 mm³ sections. Animals were sacrificed by an overdose of sodium pentobarbital. Subsequently, chondrocytes were isolated from the extracted cartilage by enzymatic digestion with 0.25% trypsin (Sigma-Aldrich) in phosphate-buffered saline (PBS) for 1 h, and 0.2% type-2 collagenase (381 U/mg; Sigma-Aldrich) in DMEM for 4 h. Following collection of individual cells by brief centrifugation, the chondrocytes were resuspended in DMEM supplemented with 10% FBS, 100 U/ml streptomycin and 100 U/ml penicillin (Hyclone, Logan, UT, USA). The culture medium was replaced every other day, and the second and third passage of chondrocytes were used in the subsequent experiments.

To establish the apoptotic model of cultured chondrocytes, SNP was used, which generates NO (21). Briefly, the chondrocytes (5×10⁴ cells/cm²) were seeded and cultured at 37°C for 24 h, following which the cells were randomly divided into five groups, in which the medium was replaced with complete medium (DMEM with 10% FBS and antibiotics) containing 0, 0.5, 1, 2 or 3 mM SNP, cultured at 37°C for different durations (3, 6, 12 and 24 h). CMCS was added into the culture medium at different concentrations (50, 100 and 200 µg/ml). Subsequently, in order to determine the activation of the p38/MAPK signaling pathway, the p38/MAPK specific inhibitor, SB203580 was used (22).

The chondrocytes were cultured in 96-well microplates at a density of 2×10⁴ cells per well with DMEM containing 0.1% FBS at 37°C for 24 h, and then exposed to different concentrations of SNP (0.5, 1, 2 and 3 mM), CMCS (50, 100 and 200 µg/ml) or SB203580 (10 µM) for 24 h, and for different durations (0, 3, 6, 12 or 24 h). For the quantitative analysis of the cell viability, 10 µl CCK-8 solution was added to each well and incubated at 37°C for 1 h. The optical densities were measured at 450 nm using an ELx800 Absorbance Microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Cell viability was determined as a percentage of the number of control (untreated) cells. Viability in the control group was designated as 100%, and cell viability in the treatment groups was determined as follows:

Viability (% of control) = [(Ae − Ab) / (Ac − Ab)] × 100%. Ae, Ab and Ac represent the A450 of the experimental, blank and control groups, respectively. All experiments were performed in triplicate in three independent experiments.

The apoptotic rates of the chondrocytes was measured using flow cytometry (FCM), according to the manufacturer's protocol of the Annexin V-FITC/PI Apoptosis Detection kit. In brief, the chondrocytes were treated with SNP (0.5, 1, 2 and 3 mM), CMCS (50, 100 and 200 µg/ml) or SB203580 (10 µM). Following culture of the chondrocytes for the indicated durations (0, 3, 6, 12 or 24 h), trypsinization was performed with 0.25% trypsin without ethylene diamine tetraacetic acid for analysis of apoptosis using the Annexin V-FITC/PI Apoptosis Detection kit. Cell apoptosis was analyzed using a BD FACSVerse™ flow cytometer (Becton Dickinson, Heidelberg, Germany) at 488 nm. The data were analyzed using CellQuest™ software (version 4.01; Becton Dickinson).

Caspase-3 activity was determined using a colorimetric assay kit (cat. no. C1115; Beyotime Institute of Biotechnology). The chondrocytes were first cultured in a mixture of DMEM supplemented with 5% FBS, SNP and CMCS. Following incubation at 37°C for 24 h, the cells were treated, according to the manufacturer's protocol. The cell lysates were prepared, and assays were performed in 96-well plates (at a density of 2×10⁴ cells/well) by incubating 10 µl cell lysate per sample in 80 µl reaction buffer (1% NP-40, 20 mM Tris-HCl, 137 mM NAD and 10% glycerol) containing 10 µl caspase-3 substrate (Ac-DEVD-pNA; Beyotime Institute of Biotechnology). The cell lysates were incubated at 37°C for 4 h. The absorbance at 405 nm was read using an ELx800 Absorbance Microplate reader (BioTek Instruments, Inc.). Caspase-3 activities were determined as the percentage of enzyme activity, compared with the control. All experiments were performed in triplicate.

The mRNA of the treated chondrocytes was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. An ABI Prism 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to detect the mRNA expression levels of the target genes. The sequences of the primers used, for B cell lymphoma (Bcl)-2, Bcl-2-associated X protein (Bax) and GAPDH, are shown in Table I.

For RT reaction, 5 µl mRNA was reverse transcribed into cDNA and amplified using specific reverse primers of the target genes using a RevertAid™ First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.). The PCR reaction mixture included 1 µl forward and reverse primers (10 µM), 1 µl SYBR Green I fluorescent dye and 1 µl cDNA. The PCR conditions were as follows: Initial step at 95°C for 2 min, followed by 40 cycles at 95°C for 20 sec and 60°C for 40 sec. The relative expression levels of target genes were analyzed using the comparative cycle threshold method (23). Data were standardized by GAPDH. The results were obtained from three independent experiments.

The chondrocytes were treated with SNP, with or without CMCS, following which the chondrocytes were lysed in lysis buffer at 4°C for 30 min. The lysates were centrifuged at 13,000 g or 15 min at 4°C. The supernatants were collected and stored at −80°C. Protein concentrations were determined using a bicinchoninic acid method (Pierce Biotechnology; Thermo Fisher Scientific, Inc.). Total protein (50 µg) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology) and electroblotted onto polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). After being blocked with 5% non-fat milk in Tris-buffered saline, the membrane was incubated overnight at 4°C with primary antibodies against p38 (rabbit polyclonal IgG, cat. no. 9212; Cell Signaling Technology, Inc.), p-p38 (rabbit polyclonal IgG, cat. no. 9211; Cell Signaling Technology, Inc.) and β-actin (rabbit polyclonal IgG, cat. no. sc-10731; Santa Cruz Biotechnology, Inc.) at dilutions of 1:200 for 2 h at room temperature. Subsequently, the membrane was incubated with a peroxidase-conjugated secondary antibody (goat anti-rabbit horseradish peroxidase-conjugated polyclonal IgG, cat. no. sc-45101; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Signals were detected by enhanced chemiluminescence. Optical band density was quantified using a Geliance 200 Imaging system (PerkinElmer, Waltham, MA, USA) and Gene Snap software (version 6.08.04; Syngene, Cambridge, UK). The values for p38 and p-p38 were normalized to that of β-actin.

The data are presented as the mean ± standard deviation. Statistical significance was analyzed by one-way analysis of variance using SPSS, version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine the protective effects of CMCS on chondrocytes, a model of apoptosis was established in the present study by exposure of chondrocytes to SNP, an inorganic compound with the formula Na₂[Fe(CN)₅-NO]·2H₂O. As shown in Fig. 1A, SNP induced a dose-dependent cytotoxic effect on the chondrocytes, and treatment with 3 mM SNP for 24 h induced a decrease in cell viability by >90%. In addition, treatment with 3 mM SNP for different durations induced a time-dependent cytotoxic effect (Fig. 1B). Therefore, the results indicated that SNP induced dose- and time-dependent cytotoxic effects on the chondrocytes. Based on these data, chondrocytes treated with 3 mM SNP for 24 h were used as the in vitro apoptosis model for subsequent experiments. To investigate the protective effects of CMCS on chondrocytes, the chondrocytes were pretreated with different concentrations of CMCS for 24 h prior to SNP induction, following which cell viability was measured. As shown in Fig. 1C, CMCS caused an increase in SNP-induced cytotoxicity, indicating that CMCS protected the chondrocytes from the SNP-induced decrease in viability, and may offer potential as a potent drug to treat OA.

To confirm the apoptosis-inducing effects of SNP, Annexin V-FITC/PI staining, followed by FCM was performed. As shown in Fig. 2, the numbers of normal cells in the SNP-induced groups were significantly lower, compared with the number in the control group, whereas the numbers of early apoptotic, late apoptotic and dead cells were significantly higher in number, compared with those in the control group. Increasing concentrations and durations increased the apoptotic rate, and the maximum pro-apoptotic effect was observed following treatment with 3 mM SNP for 24 h. These results are consistent with previous studies, demonstrating that SNP treatment leads to chondrocyte apoptosis (24,25). Following treatment with different concentrations of CMCS, the proportion of early apoptotic, late apoptotic and dead cells decreased, and there was a dose-dependent effect of CMCS treatment, with 200 µg/ml CMCS exerting the most marked inhibitory effect on SNP-induced apoptosis.

To further examine the mechanism underlying the effects of CMCS on signaling cascades, the present study analyzed the activation of p38/MAPK. As shown in Fig. 3, quantification of the Western blot bands showed that the protein levels of p-p38 were significantly higher in the SNP-treated groups, compared with the control group. Following treatment with CMCS or the p38 inhibitor, SB203580, the expression of p-p38 was decreased. As shown in Fig. 4, caspase-3 activity was significantly increased, to 63% of the control, in the SNP-induced chondrocytes, however, pretreatment with 50, 100 or 200 µg/ml CMCS attenuated the SNP-induced increase in caspase-3 activity by ~41, 54 and 56%, respectively.

In order to further investigate the mechanisms underlying the effects of CMCS on SNP-induced chondrocyte apoptosis, the mRNA expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 were detected using RT-qPCR analysis. As shown in Fig. 5A, the mRNA expression levels of Bcl-2 were markedly elevated in the CMCS-treated chondrocytes, compared with the 3 mM SNP-treated group without CMCS, at CMCS concentrations of 50 µg/ml (1.68±0.16-fold), 100 µg/ml (2.23±0.21-fold) and 200 µg/ml (4.12±0.23-fold). As shown in Fig. 5B, 3 mM SNP significantly increased the mRNA level of Bax (2.14±0.17-fold), compared with the control group. This increase was reversed by the addition of CMCS. These findings indicated that CMCS inhibited the mRNA expression of Bax and promoted the mRNA expression of Bcl-2 in the SNP-induced chondrocytes.

As p38/MAPK was found to be activated in response to SNP., the present study proceeded to address the role of p38/MAPK in the CMCS-induced inhibition of chondrocyte apoptosis, by treating the chondrocytes with SB203580, a specific inhibitor of p38 activation. The chondrocytes were pretreated with 10 µM SB203580 for 1 h and then stimulated with CMCS for 24 h. A subsequent CCK-8 assay showed that pretreatment with CMCS and/or SB203580 decreased the inhibitory effect of SNP on viability (Fig. 6A). In addition, FCM showed that pretreatment with CMCS or SB203580 prevented SNP-induced apoptosis (Fig. 6B). These findings suggested that CMCS prevented NO-induced apoptosis, possibly through the inhibition of p38/MAPK activation.