A20 inhibits lipopolysaccharide-induced inflammation in enterocytes

To examine the role of A20 in the regulation of intestinal epithelial cells (IECs) inflammation.

Using gene transfection, both stable overexpression and knockdown A20-expressed HT-29 cell lines were established. Accordingly, the cells were divided into the following groups: The control group, the A20 overexpression group, the A20 knockdown group and the respective controls. A20 was stimulated with lipopolysaccharide (LPS) in a dose- and time-dependent manner and was detected using western blotting and real-time polymerase chain reaction (PCR) analyses. Immunofluorescence and western blotting analyses were performed to investigate the role of A20 in the regulation of nuclear factor (NF)-κB activation and translocation into the nucleus. ELISA and real-time PCR were performed to examine A20 in regulating the release of the following inflammatory cytokines: Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-8.

The expression of A20 in IECs was inducible. When intestinal epithelial cells were subjected to the stimulation of LPS, the expression of A20 was increased, and the expression of A20 was induced in a dose- and time-dependent manner. The expression of A20 was very low in HT-29 cells without LPS stimulation but rapidly increased and was maintained at a high level 2-4 h after stimulation with LPS. These levels gradually declined with a change in time-course, and the expression of A20 increased with increasing LPS stimulation. Western blotting and immunofluorescence revealed that overexpression of A20 can inhibit NF-κB activation and its translocation to the nucleus. The overexpression of A20 can reduce the levels of proinflammatory cytokines involved in the pathophysiology of inflammatory bowel disease. There was no significant difference in the expression of IL-8 mRNA in the control group, A20 overexpression group or A20 knockdown group without LPS stimulation (P > 0.05); however, while after 2 h, 4 h and 8 h stimulation with LPS, the expression of IL-8 in the A20 overexpression group was lower than the control group and the A20 knockdown group (P < 0.05 or P < 0.01). The expression of TNF-α was different at different time points after 8 h of LPS stimulation (F = 31.33, DF = 5, P < 0.001), and the expression of TNF-α increased as the LPS stimulation time increased. Upon LPS stimulation, lower levels of TNF-α were detected in the A20 overexpression cell lines (P < 0.05). There were no significant differences in the induction of IL-6 and IL-1β among the control group, A20 overexpression group and A20 knockdown group (P > 0.05).

A20 plays an important role in limiting inflammation by inhibiting LPS-induced NF-κB responses in the gut luminal. A20 may be a potential therapeutic tool for inflammatory diseases.