NF-κB inhibitor reduces the expression of zinc finger protein A20 in rabbit lumbar intervertebral disc nucleus pulposus cells induced by lipopolysaccharide
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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 in vitro,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 nucleus pulposus cells was significantly decreased, aggregation occurs in the degenerative group.COL-Ⅱ was obvious lower and A20, p65 was significantly higher than 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 change 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.