Effects of zinc finger protein A20 over-expression on inflammatory response and degeneration of human intervertebral disc nucleus pulposus cells stimulated with lipopolysaccharide

Abstract

Objective To observe the effects of zinc finger protein A20 over-expression on the expression 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 collected surgical specimens of intervertebral discs from patients undergoing orthopedic surgeries for lumbar intervertebral disc degeneration in our hospital between June, 2017 and July, 2018. The nucleus pulposus cells were isolated from the specimens and cultured, and the cells in passages 1 and 2 were divided into 4 groups, namely the control group, adenovirus-mediated A20 overexpression group, LPS (10 μg/mL) stimulation group, and LPS stimulation with A20 overexpression group. The cellular mRNA expression of A20 was examined using real-time PCR in all the 4 groups. Western blotting was used to detect the expression of A20 protein, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), NF-κB-binding proteins (p65 and phosphorylated p-p65) and the degeneration-related proteins (aggrecan and collagen Ⅱ). The levels of TNF-α and IL-1β were also detected in the supernatant of the cell cultures using ELISA. Results Compared with the control cells, the cells transfected with the adenoviral vector showed significantly increased mRNA and protein expression levels of A20 (P < 0.05) without obvious changes in the protein levels of TNF-α, IL-β, p-p65, aggrecan or collagen Ⅱ. LPS stimulation of the cells significantly increased the levels of TNF-α, IL-1β, and p-p65 proteins (P < 0.05) and lowered the levels 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 levels aggrecan and collagen Ⅱ (P < 0.05) and reduced levels of TNF-α, IL-1β, and p-p65 proteins (P < 0.05). Conclusion 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.

Keywords

• zinc finger protein a20
• lipopolysaccharide
• nucleus pulposus cells
• inflammation
• intervertebral disc degeneration