Establishment Of SLE Mouse Model And The Inhibitory Effect Of TJ-M2010-5 On Hyperactive B Cells In Vitro

[Objective] In this study,pristane was used as a inducer to establish a SLE mouse model,to explore the abnormal changes of B cells in SLE induced by Pristane,and to investigate the inhibitory effect of TJ-M2010-5 on hyperactive B cells in vitro and its mechanism.[Methods] 1.Vivo experiments: Female BALB/c mice weighing 18-20 g were selected and intraperitoneally injected with Pristane(25μl/kg)to establish a mouse SLE model.The My D88 level from spleen tissue was determined via western blotting after Pristane stimulation.The experimental mice were divided into two groups: the normal control group(group A): intraperitoneal injection with the same dose of normal saline as Pristane;the model group(group B): intraperitoneal injection of Pristane(25μl/kg).Collecting mouse sera 6 months after modeling and measuring Cr and BUN content.The renal function was determined by testing PRO of morning urine to.Detecting anti-ANA antibody expression levels to reflect SLE activity,and detecting the expression levels of cytokines,IL-10、TNF-α and IL-6 reflect systemic inflammation.Spleen tissues of mice were collected 6 months after modeling to detect the subgroup changes of spleen B cells by flow detection.Mouse kidney tissues were obtained in 6 months after modeling.H & E staining was used to evaluate the degree of kidney damage and immune cell infiltration.Immunofluorescence staining for total Ig G immune complex infiltration in kidney tissue 2.Vitro experiments: Spleen cells of female BALB/c mice aged 6-8 weeks were extracted.And primary B cells suspensions sorted by magnetic beads Primary B cells were cultured with 1640 medium and CD40L(40ng/ml).Primary B cells were treated with different concentrations of My D88 inhibitor TJ-M2010-5(10,20μmol/L)for 3 hours,and then stimulated with R848(50ng/ml)for 12 hours.Flow cytometry was used to detect B220+CD25+,B220+CD69+and B220+CD138+ cells.Primary B cells were treated with different concentrations of My D88 inhibitor TJ-M2010-5(10,20μmol/L)for 3 hours,and then stimulated with R848(500ng/ml)for 48 hours Collecting cell culture Supernatant and extracting protein and total RNA in 48 hours.RT-PCR was used to detect My D88,TLR7 and IL-6 transcription.IL-6,total Ig G and anti-ANA antibodies expression level were measured by ELISA from cell culture supernatants.Western blot was used to explore the expression of My D88,TLR7,P38 protein and P38 phosphorylated protein,and to view nuclear translocation of NF-kB in cells.Annexin V-FITC/PI apoptosis detection kit detects apoptosis and CFSE detects cell proliferation[Results] 1.Vivo experiments: SLE mouse model was established: After 6 months of Pristane stimulation,the protein levels of My D88 in mouse spleen tissue were highly expressed,which was significantly different from that of group A mice(P <0.01).The levels of serum Cr and BUN in group B mice increased and PRO in morning urine increased slightly(P <0.05).Serum ELISA detection of anti-ANA antibodies,TNF-α,IL-10 and IL-6 levels showed that the level of SLE disease activity and systemic inflammation in group B were higher than those in the normal control group(P <0.01).The proportion of CD25+CD19+B220+,CD69+CD19+B220+,CD27+CD19+B220+ and CD138hiCD3-B220 lo cells in spleen B cells of group B increased(P<0.05).Group B H&E pathological staining of the kidney indicates that there are hyaline thrombus in the glomerular capillaries,a large number of inflammatory cells infiltrating the renal interstitial,thickening of the glomerular basement membrane,unclear glomerular outline.And no obvious pathological changes in group A Above all suggest that Pristane can successfully induce SLE models in mice.2.Vitro experiments:The proportion of B220+CD25+,B220+CD69+,and B220+CD138+ cells were significantly increased by flow cytometry after stimulation of mouse primary B cells with R848.While TJ-M2010-5 pretreatment could reduce B220+CD25+,B220+CD69+ and B220+CD138+ cells and These effects were concentration gradient dependence.The results of real-time quantitative PCR showed that R848 stimulation promoted the transcription of My D88,TLR7 and IL-6 in B cells,and TJ-M2010-5 pretreatment could reduce the transcription of these proteins and factors.ELISA detected the secretion of IL-6 in the cell culture supernatant and the results were consistent with the above.At the same time,B cells secreted a large amount of total Ig G and anti-ANA antibodies after R848 stimulation.TJM2010-5 pretreatment could reduce the secretion of these antibodies.TJ-M2010-5 can reduce the high expression of My D88,TLR7 protein and the phosphorylation of P38,ERK.JNK.It also inhibits the nuclear translocation of NF-kB.In addition,R848 can inhibit B cell apoptosis and promote its proliferation,and TJ-M2010-5 pretreatment can promote the apoptosis of abnormally active B cells and inhibit its proliferation.[Conclusion] 1.My D88 molecule is involved in the pathophysiological process of mouse SLE induced by Pristane.2.TJ-M2010-5 can inhibit the activation,differentiation and proliferation of B cells induced by R848 in vitro,and promote the apoptosis of hyperactive B cells.3.This inhibitory effect is mainly due to the inhibition of TLR7/My D88/NF-κB and TLR7/My D88/MAPK signaling pathways,which is manifested by the decreased secretion of autoantibodies and inflammatory factors.