The Role Of Zinc-finger-protein A20 In The Pathogenesis Of Inflammatory Bowel Disease

Inflammatory bowel disease(IBD) includes ulcerative colitis(UC) and Crohn's disease(CD). The pathophysiological mechanism of the disease remains elusive. Clinical epidemiologic data derived from several studies in the pediatric population confirmed the increased incidence of inflammatory bowel disease in children. The annual incidence rates of 0.2-8.5 per100 000 for CD and 0.5-4.3 per 100 000 for UC have been reported. In addation to chronic abdomine pain and diarrhea, affected with IBD during childrenhood may also result in short stature and delayed puberty, which strongly affected the children's psychosocial and social functioning. Studies showed that health-related quality of life of IBD patients was lower than healthy children. Children represent a unique population with fewer environmental confounders such as smoking or contraceptive use. It is therefore possible that genetics and microbiology play a stronger role in the development of pediatric IBD. For this reason, pediatric inflammatory bowel disease was defined as a research priority.Inflammatory bowel disease and, in particular, Crohn's disease, is thought to result from a dysregulated interaction between the host immune system and normal luminal microflora. The monolayer of epithelial cells exposed to the gut luminal environment is endowed with the capacity for first-line defence against microbial pathogens. The intestinal epithelium is normally tolerant to commensal bacteria, and inappropriate functioning of the immune system to the commensal bacteria is linked to the pathogenesis of inflammatory bowel disease. Zinc finger protein A20 was originally identified as a TNF-inducible gene in human umbilical vein endothelial cells in 1990. Subsequent research demonstrated that A20 is also induced in many other cell types and by a wide range of other stimuli. It is demonstrated that zinc finger protein A20 is a key player in the negative feedback regulation of NF-κB signaling in response to multiple stimuli and has been described as central gatekeeper in inflammation and immunity.Excessive activation of NF-κB is central to the pathogenesis of inflammatory bowel disease. Nuclear factor (NF)-kB is a transcription factor that regulates the expression of an exceptionally large number of genes in response to infection, inflammation and other stressfμl situations requiring rapid reprogramming of gene expression. Inappropriate NF-κB activity has been linked with many autoimmune and inflammatory diseases. Mμltiple mechanisms normally ensure the proper termination of NF-kB activation. A20 can negatively regulate TNF-R, TLRs, NOD2, TCR and BCR induced NF-kB activity. The use of A20-deficient mice and RNA interference technologies has revealed the crucial role of A20 in a variety of pathogen-and cytokine-induced signaling pathways.Mice lacking A20 are born at normal mendelian ratios but die shortly after birth due to massive mμltiorgan inflammation, indicative of a key role for A20 in immune homeostasis of the host. Intestinal epithelial cells (IEC)-specific A20 knockout mice show increased susceptibility to DSS-induced colitis and develop more severe colitis symptoms, and are unable to cope with the initial DSS challenge and do not recover. In addation, a recent genome-wide association study for seven major common diseases, undertaken in the British population identified A20 as a Crohn's disease susceptibility gene. Yet to date, few studies have been done to explore the role of A20 in the pathogenesis of IBD. The aim of the study was to explore the role of zinc finger protein A20 in the pathogenesis of infalmmatory bowel disease.Part I Expression of zinc finger protein A20 in pediatric inflammatory bowel diseaseObjective To gain insight into the relationship between intestinal inflammation and the expression of A20 in IBD patients, we examined the expression of A20 and a series of inflammatory cytokines, such as NF-κB, IL-6, and IL-8, in children with IBD and controls. Methods Terminal ileal mucosal samples were obtained under endoscopy. Fifty-seven mucosal samples were divided into 4 groups:normal control group (n=16), IBD remission group (n=12), IBD active group (n=13) and non-IBD enteritis group (n=16). According to disease activity index scores, the IBD patients were divided into IBD remission group and IBD active group. Normal control group comes from the patients with functional bowel disorders or intestinal polypus. Non-IBD enteritis defined as endoscopy and histological examination showed inflammatory changes but could not diagnosed as IBD. Real-time PCR was adopted for detecting the mRNA levels of A20, IL-6 and IL-8. Meanwhile immunohistochemistry was performed to measure the expression of A20 and NF-κB.Results (1) The expression of A20 and NF-κB were very low in normal control group, but significantly up-regulated in IBD active group and non-IBD enteritis group (bothh<0.01); (2) Compared with normal control group, expression of NF-κB [(9.35±4.84)% vs (0.57±0.44)%, P<0.01], IL-6 (t=1.34, P> 0.05), IL-8 (t=1.38, P>0.05) were increased in IBD remission group, while the expression of A20 both in mRNA (t=1.03, P>0.05) and protein levels [(0.36±0.18)% vs (0.87±0.29)%, P<0.01] were decreased; (3) Compared with non-IBD enteritis group, although the expression of NF-κB [(24.17±11.27)% vs (55.29±21.84)%, P<0.01], IL-6 (t=2.22, P<0.05), IL-8 (t=2.97, P<0.01) were highly increased in IBD active group, the expression of A20 both in mRNA(t=2.26, P<0.05) and protein levels[(29.23±11.70)%vs (16.81±5.90)%, P<0.01] were significantly decreased; (4) The expression of IL-6, IL-8 were similar in IBD remission group and non-IBD enteritis group (both P>0.05), but the expression of A20 were much lower both in mRNA (t=4.42, P<0.01) and protein levels [(29.23±11.70)%vs (0.47±0.25)%, P<0.01] in IBD remission group.Conclusions The results demonstrate that there is an excessive inflammatory response but insufficient up-regulation of A20 expression in IBD patients. Low levels expression of A20 may play an important role in the pathogenesis of IBD.PartⅡThe role of zinc finger protein A20 in the regulation of intestinal epithelial-cell inflammation.Objective To explore the role of A20 in the regulation of intestinal epithelial-cell inflammation.Methods Through gene transfection, a stablely overexpression and knockdowned A20-expressed HT-29 cell lines were estabolished. And accordingly, the cells were divided into three groups:HT-29 Cell group, A20 overexpression group and A20 knockdowned group. Immunofluorescence and western-blot was adopted for detecting the role of A20 in regulating NF-κB translocation into nuclear, and ELISA was performed to explore A20 in regulating the release of inflammatory cytokines.Results (1) When intestinal-epithelial-cell was subjected to the stimulation of LPS, the expression of A20 was increased, and the expression of A20 was increasing with enhanced the amount of LPS stimulation; After 1 hour stimulation of LPS, the expression of nuclear NF-κB in A20 overexpression group was much lower than HT-29 cell group and A20 konckdowned group (both P<0.05); (2) Compared with HT-29 cell group, the expression of nuclear NF-κB was much higher in A20 knockdowned group after 1 hour stimulation of LPS (P<0.05); (3) Increasing the time of LPS stimulation, the expression of TNF-a, IL-1βwere increased. (4)There is significiant difference in the expression of TNF-a in HT-29 cell group, A20 konckdowned group and A20 overexpression group in 8 hours stimulation of LPS (F=42.247; DF=2; P<0.001); (5)The expression of TNF-a was different at different time points in 8 hours stimulation of LPS (F=31.33; DF=5; P<0.001), and the expression of TNF-a was increased with longer LPS stimulation time (F=111.435; DF=1; P<0.001); There is no significiant difference in the expression of IL-1βin HT-29 cell group, A20 konckdowned group and A20 overexpression group in 8 hours stimulation of LPS (F=2.456; DF=2; P=0.166); (7) The expression of IL-1βwas different at different time points in 8 hours stimulation of LPS (F=9.216; DF= 5; P<0.001), and the expression of IL-1βwas increased with longer LPS stimulation time (F=80.829; DF=1; P<0.001).Conclusions A20 plays a key role in the regulation of intestinal-epithelial-cell inflammation.