Purpose
The main purpose of this study was the investigation of the effect of Dexamethasone (DEXA) treatment on TNF-α stimulated intervertebral disc cells through the action mechanism of NF-κB in the cytoplasm and nucleus.
Materials and Methods
We separated cultured human intervertebral disc cells passed three times into four groups. A:
control group, B: TNF-α treatment group, C: DEXA treatment group, D: TNF-α and DEXA treatment simultaneously.
After extraction of cytoplasmic and nuclear protein from the 4 groups at time points including 10 minutes, 1 hour, and 2 hours, we measured the protein expression levels of p50, p65, p52, and p100 by western blot analysis. Also, we observed the expression of p50, p52, p65 in each group at the 1 hour time point by immunofluorescence analysis.
Results
Western blot analysis demonstrated that cytoplasmic levels of p50 and p65 at1 hour in groups B and D were decreased, groups C showed no significant change as compared to the control group. Nuclear levels of p50 at 1 hour in groups B (10.99 fold) and D (7.24 fold) were increased, and group D had decreased expression compared to group B. Nuclear levels of p50 expression at 2 hours in groups B (12.33 fold) was increased compared to the levels measured at 1 hour. Levels of nuclear p50 in group D at 2 hour time points showed no significant change as compared to the group D at 1 hour time points. In the nucleus, the level of p65 at 1 hour had the same pattern as the p50 expression, however, group B (4.13 fold) and D (4.13 fold) expression levels at 2 hours were decreased compared to the group B (7.49 fold) and D (6.79 fold) at 1 hour. In the cytoplasm, the expression of p100 in groups B and D were decreased after 1hour, and other groups had the same trend as that observed for the control group. Nuclear p100 expression levels were observed in, groups B, C, and D after 2 hours. The cytoplasmic levels of p52 at 10min, 1 hour, and 2 hours were same. The nuclear levels of p52 in group D at 1 hour had no expression and decreased at 2 hours (0.08 fold). The results of the immunofluorescence analysis and the western blot analysis at the 1 hour time point is quite consistent.
Conclusion
Transcriptional mediation of NF-κB was mainly focused on the classical pathway but several protein levels were influenced by the alternative pathway following stimulation with TNF-α in disc cells. The effect of DEXA on NF-κB transcription signaling was observed through the delayed expression of involved proteins and inhibited the translocation of p50, p52, p65 to prevent the expression of corresponding genes.
