B)?Example of a representative experiment. Open in a separate window Figure 4 MMP-13 protein levels in chondrocytes after MRC dysfunction. and mRNA expression was evaluated. An up regulation of MMP-1 and -3 mRNA levels was observed after the treatment with Oligomycin 5 and 100 g/ml (inhibitor of the complex Rabbit Polyclonal to STAT5A/B V) for 24 hours. MMP-13 mRNA expression was reduced after the incubation with AA 20 and 60 g/ml (inhibitor of complex III) and Oligomycin. Results were validated at protein level observing an increase in the intracellular levels of MMP-1 and -3 after Oligomycin 25 g/ml stimulation [(15.208.46 and 4.591.83 vs. basal=1, respectively (n=4; *(VWR, Bridgeport, NJ, USA), tissues were observed in the microscope. was used for mounting and visualization in the microscope with a Nikon camera (Nikon Instruments, Melville, NY). Safranine fast green was also used for proteoglycan detection. For this technique, FFPE tissues were cut in the microtom and washed to get rid of the paraffin. Fast green stained the background for 5 min. After washing for 10 sec in acetic acid and safranin 0.1% for 5 min tissues were dehydrated and mounted. Proteoglycan quantitation was done with Analisys software obtaining relative values. em Statistical analyses /em The data are expressed as mean SE. Individual donor assays were duplicated. The statistical software program SPSS (version 15.0, SPSS, Chicago, IL, USA) was used to perform analysis of variance (ANOVA) and Tukey tests. Differences were considered to be statistically significant at P0.05. Results Intracellular MMP-1, MMP-3 and MMP-13 expression after MRC dysfunction We evaluated the possible modulation at mRNA level of MMPs -1, -3 and -13 after the induction of 25-Hydroxy VD2-D6 the MRC dysfunction. According to the bibliography, we used Rotenone 10 and 50 g/ml to inhibit the MRC complex I, NPA 0.5 and 10 mM to inhibit the MRC complex II, Antimycin A (AA) 20 and 60 g/ml to inhibit the complex III, Sodium azide 2 and 25 mM to inhibit the complex IV and Oligomycin 5 and 100 g/ml to inhibit the activity of the complex V. After 24 hours of treatment, we analyzed the mRNA expression of MMPs -1, -3 and -13 as Figure?1 shows. Oligomycin 5 g/ml produced a tendency in the increase of MMP-1 and -3 expression (Figure?1A, ?A,1B)1B) to 68.1039.9 and 60.1329.7 vs. basal=1, respectively (n=9). On the other hand, the inhibition of the complex III with AA 20 g/ml, produced a decrease in the MMP-13 mRNA expression to 0.340.2 vs. basal=1 (Figure?1C). To confirm these results at protein level, we 25-Hydroxy VD2-D6 evaluated the intracellular protein expression of these MMPs by western blot (Figures?2, ?,33 and ?and4).4). We stimulated the cells at different concentrations of AA or Oligomycin according to the preliminary mRNA results. The positive control used was IL-1 5 ng/ml. The treatment of chondrocytes with the inhibitor of complex V (Oligomycin 2.5, 5, 10 and 25 g/ml) after 24 25-Hydroxy VD2-D6 hours produced an increase in the MMP-1 levels (Figure?2A). The levels increased significantly up to 12.203.24 and 15.208.46 vs. basal=1, Oligomycin 10 and 25 g/ml respectively, (n=4; * em P /em 0.05). Figure?2B represents an experiment of 4. As we expected, AA did not induce the MMP-1 modulation according to the mRNA results. In a similar way, MMP-3 was only induced by Oligomycin. Figure?3A shows these levels: at 24 h 5.652.08 and 4.591.83 vs. basal=1 for the concentrations of 10 and 25 g/ml, respectively (n=4; * em P /em 0.05). Figure?3B represents an experiment of 4. As we expected, AA did not induce the modulation of MMP-1. MMP-13 decreased after treatment with AA 40 g/ml and 25-Hydroxy VD2-D6 Oligomycin 25 g/ml (0.700.16 and 0.30.24.