The Effects Of Carbon Monoxide Releasing Molecule-3 On The Apoptosis Of Rat Temporomandibular Condylar Chondrocytes Induced By Interleukin-1?

ObjectiveTo investigate the effect of carbon monoxide releasing molecule-3 on the expression of type ? collagen and apoptosis-related factor Bax/Bcl-2 in a vitro model of rat temporomandibular condylar chondrocytes induced by interleukin-1? and try to explore the possible mechanism by which carbon monoxide releasing molecule-3 confers the protective effect.MethodsThe bilateral condylars of 3-4-week old Wistar rats were isolated under sterile conditions.The condylar cartilage(located at the head of the condyle which was milky white)was removed and alternately digested with 0.25%trypsin and type ?collagenase.The morphology of chondrocytes were observed under microscope.Cells of 2-4th generation were used for subsequent experiments.The second generation of cells were obtained to make a slide,toluidine blue staining solution was configured to dye the cells,then they were observed under microscope.Third generation of chondrocytes were stimulated with interleukin-1?(IL-1?)at different concentrations of 0,1,5,and 10 ng/ml for 24 hours,then they were collected for further analysis.The expression of type II collagen and apoptosis-related factor Bax/Bcl-2 were detected at the gene and protein level respectively.Optimal interleukin-1? concentration was chosen for the subsequent experiments.The cells were then pretreated with carbon monoxide releasing molecule-3(CORM-3)at dilferent concentration of 100,200,and 400 ?M for 24 hours,then treated with 10 ng/ml IL-1? for another 24 hours.The survival rate of each group was detected by CCK-8 method and the mRNA expression of Bax/Bcl-2 and type ? collagen in each group was detected by RT-qPCR.The most optimal concentration of carbon monoxide releasing molecule-3(CORM-3)for subsequent experiments was decided.The chondrocytes were pretreated with carbon monoxide releasing molecule-3(CORM-3)and degassed CORM-3 at concentration of 200 ?M for 24 hours,then treated with 10 ng/ml IL-1?.The cells were collected and appropriate amount of suspension was taken to applied the apoptosis kit,then the cell apoptosis were detected by flow cytometry.The mRNA expression of type ? collagen and Bax/Bcl-2 in each group were detected by RT-qPCR.The protein expression of Bax/Bcl-2 and type ? collagen were detected by western blotting.In order to explore the possible mechanism,the total protein was taken and western blotting was used to detect the expression of heme oxygenase-1 in each group.Results?The chondrocytes were observed to be spherical under an inverted microscope,and they were polygonal after adherence.After 7-8 days of culture,the cells reached a confluent state which resembled as a "paving stone".The generated cells were in good condition and evenly distributed,and the cell proliferation rate was increased.After the fourth generation,the cells showed irregular growth and some of them appeared to have tentacles extending out.The shape of cells changed from polygon to long spindle and the growth rate slowed down significantly.The stained cells were observed under the microscope,most of which had round and dark blue nucleus and light blue cytoplasm.There were abundant dark blue granular substances between the cells,which were proteoglycans in the extracellular matrix secreted by the cells.?Results from RT-qPCR showed that the mRNA expression of Bax was significantly enhanced by IL-1? stimulation dose-dependently compared with the control group Moreover,IL-1(3 dose-dependently suppressed the mRNA level of Bcl-2 and Col-2 significantly.In consistent with the results from RT-qPCR,in western blot analysis,IL-1? increased the protein expression of Bax significantly,and decreased the Bcl-2 and Col-2 expression significantly.Based on the data,10 ng/ml was chosen as the working concentration of IL-1? for the subsequent experiments.?CORM-3 pretreatment at different concentrations of 100,200,400?M concentrations increased the cell viability significantly in IL-]P-stimulated cells.Moreover,pre-treatment with CORM-3 decreased the mRNA expressions of Bax significantly and increased the expressions of Bcl-2 and Col-2 significantly in IL-1?-treated cells.Based on the data that CORM-3 at the concentration of 200 ?M had the most effective regulation on the expression of Bax,Bcl-2 and Col-2 in IL-1?-stimulated cells,200 ?M was therefore selected as the working concentration in the subsequent experiments.?The results of flow cytometry showed that the cell apoptosis in IL-1?P+200 ?M CORM-3 group was significantly lower than that in IL-1? group.The effect of CORM-3 on cell apoptosis was mediated by the release of CO since the degassed solution of CORM-3 failed to inhibit the cell apoptosis.And the apoptotic rate of degassed CORM-3 was not significantly different from that of IL-1?-stimulated group.The regulatory effect of CORM-3 on the expression of Bax,Bcl-2 and Col-2 in IL-1?-stimulated cells was abrogated when degassed CORM-3 was used.?The expression of HO-1 was significantly increased in the CORM-3 pretreatment group,however,there was no significant difference between degassed CORM-3 pretreatment group and IL-1? stimulation group.ConclusionsCarbon monoxide releasing molecule-3 inhibits the apoptosis of rat condylar chondrocytes induced by interleukin-1?,increases the expression of Bcl-2 and type ?collagen and suppresses the production of Bax.The possible mechanism might be that carbon monoxide release molecule-3 activates heme oxygenase-1 protein to exert its anti-apoptosis effect.