NF-κB signaling pathway regulate endplate chondrocytes in rat vitro natural degeneration model

Abstract

To explore the relationship between nuclear factor (NF)-κB and vitro nature degeneration model of endplate chondrocyte in rats. Rats endplate chondrocytes were isolated and cultured in vitro.Rebulated vitro natural degeneration model and cells were divided into control group (P2 cells group), naturally passaged group (P5 cells group) and NF-κB signaling pathway inhibition group (added Bay11-7082 when the passaged to P5 cells). The changes of cellular morphology were observed by inverted phase contrast microscope, the phenotype of endplate chondrocyte were identified by toluidine blue staining, electromagnetic compatibility (EMC) were observed by alcian blue staining.Type Ⅱ collagen, sry related HMG box (SOX)-9, matrix metalloproteinase(MMP)13 and aggrecan genes were detected by real time-polymerase chain reaction (RT-PCR) to verify the degeneration mode.NF-κB transcriptional activity was assessed by examining cytosolic phosphorylated IκBα and nuclear phosphorylated p65 levels by Western blot. With chondrocytes passing, the cells lost the original morphology gradually.Alcian blue stains observed EMC decreased in naturally passaged group.The leave of Type Ⅱ collagen (P5/P2=0.182, P<0.01), aggrecan (P5/P2=0.287, P<0.01) and SOX-9 (P5/P2=0.488, P<0.01) were significantly reduced, MMP13 (P5/P2=1.324, P<0.05) significantly increased.Western blot analysis showed that in P5 cells nuclear phosphorylation p65 and cytosolic levels of phosphorylated IκBα increased and Bay11-7082 treatment attenuated increase in nuclear phosphorylation p65 and blocked acidinduced increase in cytosolic levels of phosphorylated IκBα.Moreover, the leave of Type Ⅱ collagen (Bay11-7082/P5=4.173, P<0.01), aggrecan (Bay11-7082/P5=2.732, P<0.05) and SOX-9 (Bay11-7082/P5=1.567, P<0.05) significantlyincreased, MMP13 (Bay11-7082/P5=0.611, P<0.05) significantly reduced in treatment group. Inhibitor NF-κB signaling pathway plays an important role in the vitro degeneration of endplate cartilage.Reasonable regulation of NF-κB signaling pathway may be a new way to prevent Intervertebral disc degeneration.