| Background:Systemic Lupus Erythematosus(SLE)is a chronic,alternating seizure and remission systemic autoimmune disease involving multiple organs,characterized by congenital and adaptive immune abnormalities.It is characterized by B cell overactivation and T cell dysfunction,the production of multiple autoantibodies,complement activation,and the deposition of immune complexes,which further cause damage to tissues and organs.It is common in women of childbearing age and can affect multiple organ systems throughout the body.Among them,B cells play a central role in the pathogenesis of SLE.In addition to antibody secretion,B cells also mediate the presentation of autoantigens and regulate the inflammatory response of SLE by secreting cytokines and regulating other immune cells.In recent years,targeted therapy for B cells has also been a hot research topic in autoimmune diseases such as SLE.TNFAIP 3(tumor necrosis factor alpha induced protein 3),also known as A20,is a ubiquitin editing enzyme.It is considered to be a negative modulator of the NF-κB signaling pathway.Studies have shown that TNFAIP3 can inhibit the over-activation of NF-κB by regulating TLR and other pathways,thereby regulating the immune and inflammatory responses.A variety of autoimmune diseases,including SLE,such as rheumatoid arthritis(RA),type I diabetes,multiple sclerosis,and inflammatory bowel disease,are associated with the over-activation of NF-κB.Genome-wide association studies and several candidate gene studies have shown that TNFAIP3 is a susceptible gene for SLE and it plays an important role in regulating the autoimmune inflammatory response in SLE.Studies have pointed out that TNFAIP3 may prevent the occurrence of autoimmune reaction by limiting the survival of autoimmune B cells.The expressions of TLR7 and TLR9 in memory B cells were significantly higher than those in the initial B cells,suggesting that the growth and development of B cells might be closely related to these two receptors.Therefore,TNFAIP3 may affect the production of various inflammatory factors and autoantibodies as well as the disease progression of SLE by regulating the activity of TLR7/TLR9-NF-κB signaling pathway in B cells.Objective:In this experiment,we infected the spontaneous SLE animal model MRL/lpr mice with AAV,and increased the expression level of TNFAIP3 in B cells,to explore whether high expression of TNFAIP3 can alleviate the inflammatory response in SLE mice by affecting the maturation and differentiation of B cells and inhibiting the TLR7/TLR9-NF-κB/IRF7 signaling pathway in B cells,thereby deepening the understanding of the pathogenesis of SLE and providing an experimental basis for clinical research of SLE.Method:Five-to eight-week-old female MRL/lpr and C57BL/6 mice were randomly divided into the following four groups:a.C57BL/6 mouse normal saline group(A group;n=20)b.MRL/lpr mouse normal saline group(B group;n=20)c.MRL/lpr mouse null virus group(C group;n=20)d.MRL/lpr mouse TNFAIP3 overexpression group(D group;n=20)At 12 weeks of age,mice in groups C and D were injected with adeno-associated virus vector(1×1012vg/ml,150μl)through caudal vein.The other two groups were injected with the same dose of normal saline.During the virus transfection,attention was paid to observing the changes in the hair of mice in each group and the survival rate.When the mice were 17 weeks old,the B cell suspension was taken to observe the transfection of the virus under the fluorescence microscope.After confirming the successful transfection of the virus,the urine protein level was measured by Coomassie brilliant blue method,which was used to evaluate the kidney function of the mice.The levels of serum anti-DNA IgG,IL-6,NF-κB and TNF-α were detected by ELISA.The expression levels of TNFAIP3,TLR7 and TLR9 proteins in B lymphocytes were detected by western blot.The expression levels of surface receptors BAFF mRNA,Blimp-1 mRNA and IRF7 mRNA related to the maturation and differentiation of B cells as well as the expression levels of TNFAIP3 mRNA,TLR7 mRNA and TLR9 mRNA were detected by real-time quantitative PCR.Differences between groups were analyzed using one-way ANOVA,and the results were expressed as mean standard deviation(χ±s).Results:After treatment with adeno-associated virus,the TNFAIP3 gene was successfully overexpressed.After successful AAV transfection,the expression level of TNFAIP protein in group D was significantly higher than that in group C,and there was no significant difference compared with group A.There was also no significant difference in the expression level of TNFAIP3 protein between groups B and C.Western blot also showed that the expression levels of TLR7 and TLR9 proteins in group D were significantly lower than those in group C and there was no significant difference as compared with group A.The expression levels of TLR7 and TLR9 proteins in groups B and C were also not significantly different.PCR results showed that the expression levels of BAFFmRNA,Blimp-1 mRNA,TLR7 mRNA,TLR9 mRNA and IRF7 mRNA in group D were significantly reduced as compared with those in group C.There was no significant difference between the expression levels of BAFFmRNA,Blimp-1 mRNA,TLR7 mRNA,TLR9 mRNA and IRF7 mRNA in groups B and C.The expression level of TNFAIP3 mRNA in group D was significantly higher than that in group C and there was no significant difference as compared with group A;Elisa results showed that the levels of anti-DNA IgG,IL-6,NF-κB and TNF-α in the group D were significantly reduced compared with those in the group C.There was no significant difference between the groups A and B.The levels of anti-DNA IgG,IL-6,NF-κB and TNF-α in the groups B and C were also not significantly different.Secondly,the 24-hour urinary albumin level test showed that the level of mice in group D was significantly lower than that in group C.Compared with group A,there was no significant difference between groups B and C.In addition,from the general situation of mice,mice in groups B and C were emaciated,and their hair was messy,peeling,dry and lusterless.However,mice in groups A and D were in good condition,and the hair of mice in the same negative control group was still smooth and shiny,without significant peeling.During the experiment,no mouse in group A died,including eight in group B,seven in group C and three in group D.Conclusion:Overexpression of TNFAIP3 resulted in remission of inflammation and decreased mortality in lupus mice.After the TNFAIP3 gene was overexpressed in B cells of MRL/lpr mice,the protein expression levels of TLR7 and TLR9,IRF7 mRNA expression levels of BAFF,Blimp-1 and IRF 7,and the serum levels of anti-DNA IgG,IgM,IL-6,NF-κB and TNF-α were all significantly reduced.TNFAIP3 may inhibit the maturation of B cells and the activity of TLR7/TLR9-NF-κB signaling pathway in B cells,and inhibit the production of various inflammatory factors and autoantibodies,thereby relieving SLE. |
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