ABSTRACT
Objective To find the relationship between nuclear factor kappB (NF-κB) activation and cell apoptosis.Methods Degenerative human nucleus pulposus cells were cultured in vitro.Useing CCK-8 to observe the proliferative effect of LPS on the nucleus pulposus cells in vitro, in the concentration of 100, 200, 500 and 1 000 μg/mL and choose the most apropriate concentration.The experiment was divided into blank control group, LPS(500 μg/mL)groups, and NF-κB inhibitor(BAY11-7082)plus LPS(500 μg/mL)group,Annexin V-FITC/PI flow cytometry and Hoechest33258 staining was used to analyze apoptosis.The expression of cleaved caspase-3,PARP,P65,P-P65 proteins were detected by Western blot respectively.Results When LPS was 500 μg/mL, the cell vitality was obviously declined.Compared with the blank control group, cell apoptosis rate of the LPS group is increasing (P<0.05), and the expression of P-P65,cleaved caspase-3, cleaved PARP were alsohigher (P< 0.05).Compared with the LPS group, cell apoptosis rate of the NF-κB inhibitor plus LPS group is significantly lower (P<0.05) and the expression of P-P65,cleaved caspase-3,cleaved PARP were also lower (P< 0.05).Conclusions NF-κB signaling pathway may be associated with the nucleus pulposus cell apoptosis in disc degeneration.
ABSTRACT
Objective To observe the expression of zinc finger protein A20(A20), NF-κB and related inflammatory factors before and after lipopolysaccharide (LPS) stimulates degeneration of rabbit intervertebral disc nucleus pulposus cells.Methods The normal and degenerative nucleus pulposus cells were isolated and cultured, then divided into normal group,degenerative group,LPS stimulation group and NF-κB inhibition group.HE staining observe the morphological changes of nucleus pulposus and annulus fibrosus,immunohistochemistry was used to detect the expression of A20,NF-κB/p65 and COL-Ⅱ.Real-time PCR was employed to analyze the expression of A20,IL-1β,TNF-α,NF-κB and COL-Ⅱ,Western blot was used to observe the A20 protein,p65 and COL-Ⅱexpression in the four groups, and TNF-α, IL-1β in cell supernatant was determined by ELISA.Results The number of nucleuspulposus cells significantly decreased, aggregation occured in the degenerative group.COL-Ⅱ was obvious lower and A20, p65 significantly higher than that in normal group by immunohistochemical staining.Compared with the normal group,A20,TNF-α,IL-1β,p65 expression was significantly increased and COL-Ⅱ decreased in the mRNA and protein levels in degenerative group.Above indexes changed more significant in LPS stimulation group than in degenerative group.The expression of A20, TNF-α, IL-1β, p65 in the NF-κB inhibitor group was lower than that in the LPS group, and the expression of type Ⅱ collagen increased(P<0.05).Conclusions Intervertebral disc inflammatory response is closely related to the development of intervertebral disc degeneration, A20 may play an important role.