Effects Of Zinc Finger Protein A20 Over-expression On Inflammatory Response And Degeneration Of Human Intervertebral Disc Nucleus Pulposus Cells Stimulated With Lipopolysaccharide

Objective:To observe the effects of zinc finger protein A20 over-expression on the expressions of inflammatory cytokines and nuclear factor-κB(NF-κB)in human intervertebral disc nucleus pulposus cells and on cell degeneration in response to lipopolysaccharide(LPS)stimulation.Methods:We used degenerated lumbar intervertebral disc specimen resected from patients who received orthopaedic surgeries during June 2017 and July 2018 in the First Affiliated Hospital of Chongqing Medical University.From the primarily cultured degenerative nucleus pulposus cells of extracted samples,the P1,P2 generation cells are divided into 4 groups in a completely randomized way: control group,A20 overexpression adenovirus transfection group,LPS(10μg/mL)stimulation group,and LPS(10μg/mL)+A20 overexpression group.The mRNA expression level ofA20 is examined using real-time PCR.At the protein level,we used Western blot to test the expressions of A20 protein,TNF-α,IL-1,NF-κB binding protein(p65 and phosphorylated p-p65)and degeneration-related proteins like aggrecan and collagen Ⅱ.The expression of TNF-α and IL-1βin supernatant plasma was analyzed with ELISA.Results:Compared with the control cells,the cells transfected with the adenoviral vector showed significantly increased mRNA and protein expressions of A20(P<0.05)without obvious changes in the protein expressions of TNF-α,IL-β,p-p65,aggrecan or collagen Ⅱ.LPS stimulation of the cells significantly increased the expressions of TNF-α,IL-1β,and p-p65 proteins(P<0.05)and lowered the expressions of aggrecan and collagen Ⅱ proteins(P<0.05).Compared with the cells with LPS stimulation alone,the A20-overexpressing cells with LPS stimulation showed obviously increased expressions of aggrecan and collagen Ⅱ(P<0.05)and reduced expressions of TNF-α,IL-1β,and p-p65 proteins(P<0.05).Conclusions:The degeneration of human intervertebral disc tissue is closely related to inflammatory responses of the nucleus pulposus cells.The production and activation of the inflammatory cytokines in nucleus pulposus cells can be effectively inhibited by over-expression of Zinc finger protein A20,which may play an important role in the regulatory mechanism of nucleus pulposus cell degeneration.