Effect Of TRIM21 On TLR9/NF-κB Pathway And Its Significance In SLE

Objectives:To determine whether the expressions of TLR9,TRIM21,NF-κB and IRF8 in peripheral blood monocytes and IFN-y,TNF-α,IL-6 and IL-17A in serum of SLE patients were different.In this study,TRIM21 was used as the research object to study the effect and regulatory mechanism of TRIM21 on NF-κB pathway at the cellular level,objective to explore the significance of TRIM21 in SLE,and to provide the basis for the treatment of SLE to find immune upstream targets.Methods:The research object were 35 patients with SLE in the Department of Rheumatology and Immunology,the Second Affiliated Hospital of Kunming Medical University.According to the SLE disease activity score,it was divided into 17 cases in remission group and 18 cases in active group,another 8 healthy volunteers were selected as normal control group.QPCR was used to detect the expression of TLR9,TRIM21,IRF8 and NF-κB in clinical blood PBMC,ELISA method detects the content of IFN-y,TNF-α,IL-6 and IL-17A in clinical blood serum,and analyze its correlation with clinical indicators.The recombinant plasmid vector for down-regulated expression of TRIM21 was constructed by means of gene recombination,THP-1 and Jurkat cells were infected with lentivirus package sh-TRIM21,and stable cell lines were screened,label the THP-1/Jurkat sh-TRIM21 cell group and THP-1/Jurkat NC cell group.Treat the cells with TLR9 agonists,detection of IκBα and p65 protein levels in cells,NF-κB mRNA,IRF8mRNA and the expression of IFN-y,TNF-α,IL-6 and IL-17A in the supernatant.And treat the cells with the inflammatory cytokine IFN-y/TNF-α/IL-6,collect cells at 8h and 24h,qPCR to detect the mRNA expression level of TRIM21 in cells.Statistical analysis of the data were performed by using SPSS 19.0 software,P<0.05 considered the difference was statistically significant,and Graphpad Prism 8 software was used for comparison between groups and statistical chart.Results:1.Clinical blood results(1)The expressions of TLR9,TRIM21 and NF-κB in peripheral blood PBMC of SLE patients in the remission and active phase groups were higher than those in the healthy control group,there were statistically significant.The expression of IRF8 was lower than that in the healthy control group,there were statistically significant.However,there was no significant difference in the expression of TLR9,TRIM21,IRF8 and NF-κB in peripheral blood PBMC of patients in remission and active phase.(2)The levels of IFN-y,IL-6 and IL-17A in peripheral blood serum of SLE patients in the remission and active phase groups were higher than those in the healthy control group,there were statistically significant,but the difference between the remission period group and the active period group was not statistically significant.For TNF-α expression,there was no statistically significant difference among the groups.(3)There was no correlation between the expression of TRIM21 mRNA in PBMC of healthy control group and the levels of IFN-y,TNF-α,IL-6 and IL-17A in serum.(4)TRIM21 mRNA in PBMC of SLE patients was positively correlated with WBC,and there was no correlation with IFN-y,TNF-α,IL-6 and IL-17A and clinical indicators,including HB,PLT,ALB,GLO,CR,IgG,IgA,IgM,C3,C4,SLEDAI.(5)There was no statistically significant difference in the expression of TRIM21 mRNA in peripheral blood PBMC in SLE patients with anti-TRIM21 antibody positive and anti-TRIM21 antibody negative.(6)There was no statistically significant difference in the expression of TRIM21 mRNA in peripheral blood PBMC in SLE patients with anti-dsDNA antibody positive and anti-dsDNA antibody negative.(7)SLE patients with positive anti-TRIM21 antibody,the expression of TRIM21 mRNA in peripheral blood PBMC is positively correlated with the levels of IFN-γand TNF-α in the serum,but has no correlation with the levels of IL-6 and IL-17A.SLE patients with negative anti-TRIM21 antibody,there was no correlation between the expression of TRIM21 mRNA and IFN-y,TNF-α,IL-6 and IL-17A levels.2.Cytological results(1)In THP-1 cells,when TLR9 agonists were not given,compared with NC group,sh-TRIM21 group had more degradation of IκBα,increased p65 in the nucleus,increased NF-κB mRNA transcription levels,and inflammatory factors IFN-y,TNF-α,IL-6 and IL-17A has a rising trend,and the expression of IRF8 mRNA increased,and the difference was statistically significant.After being stimulated with TLR9 agonist,compared with the NC group,the changes in IκBα and nuclear p65 in the sh-TRIM21 group were not obvious,and the NF-κB mRNA transcription level did not increase constitutively,instead declined,but the difference was not statistically significant.In Jurkat cells,the same results were obtained.(2)In THP-1 cells,the expression of TRIM21 in sh-TRIM21 and NC groups was significantly up-regulated when IFN-y and TNF-α were treated for 8h and 24h.In the sh-TRIM21 and NC groups treated with IL-6,the expression of TRIM21 did not change significantly at 8h,but could up-regulate the expression of TRIM21 at 24h.In Jurkat cells,the results of IFN-y treatment were the same as those in THP-1 cells.The expression of TRIM21 in the NC group treated with TNF-α was significantly up-regulated,the expression of TRIM21 in the sh-TRIM21 group did not change significantly at 8h,but at 24h was up-regulated.The expression of TRIM21 in the NC group treated with IL-6 was significantly up-regulated,the expression of TRIM21 in the sh-TRIM21 group was up-regulated at 8h,and there was no significant change in the expression of TRIM21 at 24h.Conclusions:1.TLR9,TRIM21,NF-κB,IRF8,IFN-y,IL-6 and IL-17A were involved in the pathogenesis of SLE.2.In healthy control group,there was no correlation between the expression of TRIM21 mRNA and the levels of IFN-y,TNF-α,IL-6,IL-17A.3.In SLE patients,TRIM21 mRNA in PBMC is positively correlated with WBC,but has no correlation with IFN-γ,TNF-α,IL-6,IL-17A and other clinical indicators.4.In SLE,the expression of TRIM21 is increased.Regardless of the presence of serum anti-TRIM21 antibodies and anti-ds-DNA antibodies,increased expression of TRIM21 is a characteristic of SLE patients.5.In SLE patients with positive anti-TRIM21 antibody,the expression of TRIM21 mRNA in peripheral blood PBMC is positively correlated with the levels of IFN-y and TNF-α,however it has no correlation with IL-6 and IL-17A.SLE patients with negative anti-TRIM21 antibody,the expression of TRIM21 mRNA have no correlation with serum levels of IFN-y,TNF-α,IL-6 and IL-17A.6.Down-regulation of TRIM21 can enhance the activation of NF-κB and regulate the downstream cytokines of the NF-κB pathway.Combined with TLR9 agonist stimulation,down-regulation of TRIM21 did not significantly regulate the NF-κB pathway.7.Inflammatory cytokines IFN-γ/TNF-α/IL-6 can induce the mRNA expression of TRIM21 in cells,but there is a difference between different inflammatory cytokines up-regulate the expression of TRIM21 in different cells.