Mechanism Study Of A20 Treatment For Systemic Lupus Erythematosus Mice Model

Background:Systemic lupus erythematosus (SLE) is an autoimmune disease, which affects multiple organs such as kidneys, blood vessels, skin, joints and brain. During the process of SLE, loss of tolerance to nucleic acids and their interacting proteins results in the production of pathogenic autoantibodies that cause inflammation and tissue damage. Although advances in the medical management of infections and renal failure have improved the survival of patients with lupus, the efficacy of available therapeutic strategies for lupus nephritis and SLE is limited. The current broad-spectrum immunosuppressive agents are not always adequate to control clinical symptoms or prevent disease flares. Although the introduction of biological therapy in other rheumatic diseases such as rheumatoid arthritis (RA) and psoriatic arthritis is successful, targeted therapies for systemic lupus erythematosus are disappointing failures. Therefore, development of new therapies for control of systemic inflammation is of significance in management of patients with SLE.A20, also known as tumor necrosis factor alpha induced protein 3 (TNFAIP3) is a zinc finger protein induced by tumor necrosis factor alpha. Genome-wide association studies confirmed that TNFAIP3 (A20) is a SLE susceptibility gene, and abnormal expression of A20 was involved in the pathogenesis of SLE. Previous studies have found that aberrant low expression of A20 in tumor necrosis factor alpha stimulated SLE monocytes mediates sustained NF-κB inflammatory response. Therefore, we infer that relatively insufficient A20 expression cause sustained NF-κB activation that results in systemic inflammatory response, which may be one of the pathogenesis of SLE. And pharmacological intervention to increase A20 expression may be new treatment strategies for SLE.Daphnetin is a natural product extracted from plants of the genus Daphne and belongs to the coumarin family, which induces high A20 expression and possesses properties of anti-inflammatory effects in the treatment for autoimmune arthritis. The research methods of overexpression target gene by recombinant adenovirus are the widely used in basic research and gene therapy. This study aimed to investigate whether enhanced A20 expression in mice SLE model can alleviate SLE inflammation by increasing A20 expression in SLE mice model using daphnetin and recombinant adenovirus respectively. Furthermore, we explore the potential mechanism underlying, which may deepen our understanding of the pathogenesis of SLE and provide new treatment strategies for the treatment of SLE.Part I Daphnetin inhibits inflammation in the NZB/W F1 systemic lupus erythematosus murine model via down-regulation of NF-κB activityObjective:To investigate the potential therapeutic effect of enhanced A20 expression by daphnetin on inflammation in the NZB/W F1 systemic lupus erythematosus (SLE) murine model.Mothods:Female 16-to 18-week-old NZB/WF1 and BABL/c mice were randomly assigned into three groups:NZB/W F1 mice with untreated SLE (SLE; n= 20), NZB/W F1 mice with SLE treated with daphnetin (Daphnetin; n= 20), and BABL/c mice (Sham; n=10). The blood samples were collected to analyze blood urea nitrogen (BUN) level by Urea Nitrogen Diacetylmonoxime Test Kit for the assessment of renal function; ELISA for serum levels of anti-nRNP IgG, anti-dsDNA IgG TNF-a and IL-6. pNFKB-p65 and A20 protein expression were detected by Western Blot. All values of the experiment were expressed as the mean± Std. The differences between groups were analyzed by the Mann-Whitney u test or one-way ANOVA when the data were normally distributed.Results:Daphnetin treatment significantly promoted A20 protein expression in NZB/WF1 mice compared with SLE group, A20 protein expression was significantly promoted in SLE group compared with sham group. SLE induced high serum levels of TNF-a and IL-6 in NZB/WF1 mice compared with sham group, the mice treated with daphnetin exhibited inhibited serum levels of TNF-a and IL-6 in NZB/WF1 mice as compared with SLE group mice. Serum levels of anti-nRNP IgG and anti-dsDNA IgG in SLE group mice were significantly higher than those in Sham group mice, serum levels of anti-nRNP IgG and anti-dsDNA IgG in daphnetin group mice were significantly lower than those in SLE group mice. Daphnetin treatment significantly reduced BUN level and renal damage in NZB/WF1 mice. pNFκB-p65 protein expression of SLE model group was substantially promoted compared with sham group, daphnetin treatment significantly inhibited the SLE-induced pNFKB-p65 protein expression in NZB/WF1 mice.Conclusion:Our studies indicated that enhanced A20 expression by daphnetin possesses protective effect on inflammation in the NZB/W F1 murine SLE model. Our data suggest that the therapeutic effect of daphnetin may be related to the down-regulation of NF-κB signaling mediated by up-regulated A20.PartⅡ A20 overexpression mediated by adeno virus alleviates pristine-induced lupus nephritisObjective:To investigate the potential therapeutic effect of A20 on renal inflammation in mouse pristine model of lupus.Methods:BALB/c mice at 6-8 weeks of age were injected intraperitoneally (i.p) with 0.5 mL pristine or PBS, the PBS-injected mice served as the healthy PBS control group, three months later the pristine-injected mice were randomly assigned and injected i.p with 1.0×109 plaque forming units (PFU) of Ad-A20, Ad-control or PBS (100 μL, n=6-8 per group). The levels of serum IL-1β, IL-6, IgG, anti-nRNP, anti-dsDNA and urinary albumin in individual mice were measured using ELISA kits. Paraffin-embedded renal tissue sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope, the pathologic changes in the kidney tissues were evaluated for the glomerular and tubulointerstitial activity scores of 8 fields of each mouse in a blinded manner. The frozen kidney sections were stained with IgG or C3, the sections were examined under a fluorescent microscope and the mean intensity of fluorescent antibody was evaluated using Image J software and scored for 0-3; A20, NLRP3, ASC, p-IκB, IκB, F4/80, CCL2, Caspase-1p20, p-NF-KBp65 and NF-KBp65 protein expression were detected by Western Blot. All values were expressed as the mean±Std. The differences between groups were analyzed by the Mann-Whitney u test or one-way ANOVA when the data were normally distributed.Results:A20 expression in peritoneal macrophages was slightly promoted in Pristane and Ad-control group mice as compared with PBS group mice, but it was significantly promoted in Ad-A20 group mice as compared with the mice of three other group. Serum levels of anti-dsDNA IgG and anti-nRNP IgG in Pristane and Ad-control group mice were significantly higher than those in PBS group mice, serum levels of anti-dsDNA IgG and anti-nRNP IgG in Ad-A20 group mice were significantly lower than those in Ad-control group mice but still higher than those in PBS group. Semi-quantitative analysis revealed that the glomerular and tubulointerstitial activity scores in the Ad-A20 group mice were only slightly higher than those in the PBS group mice, but were significantly lower than those in the Pristane and Ad-control group mice. Quantitative measurements of 24-h urinary albumin indicated that the level of urinary albumin in the Pristine group mice was indistinguishable and significantly higher than that in PBS group mice, and the level of urinary albumin in Ad-A20 group mice was lower than that in Ad-control group mice. CCL2 and F4/80 expression in kidneys was significantly higher in Pristane and Ad-control group mice than that in PBS group mice, and it was significantly decreased in Ad-A20 group as compared with Ad-control group mice. p-IKB and p-NF-KBp65 expression was significantly higher in Pristane and Ad-control group mice than that in PBS group mice, and it was significantly decreased in Ad-A20 group as compared with Ad-control group mice. NLRP3, ASC and Caspase-1p20 expression was significantly higher in Pristane and Ad-control group mice than that in PBS group mice, and it was significantly decreased in Ad-A20 group as compared with Ad-control group mice.Conclusion:Our data indicated that A20 overexpression mediated by adenovirus significantly mitigated pristine-induced systemic inflammation and renal injury in mice and suggest that the therapeutic effect of A20 may be associated with inhibition of the NLRP3 inflammasome and NF-κB activation in macrophages. Because of dual inhibition of A20 on the NLRP3 inflammasome and NF-κB activation, A20 may be a promising new candidate for SLE treatment.