Regulatory Effects Of Four Ginsenoside Monomers On Humoral Immunity Of Systemic Lupus Erythematosus

Background:Systemic lupus erythematosus（SLE）is a human autoimmune disease with multiple clinical manifestations.In most cases,the vital organs involved include the brain,heart,joints,skin and kidneys.The pathogenesis of SLE are complex,the existing research shows that immune dysfunction is one of the main reasons for its incidence.Ginsenosides（GS）,as the main active ingredient extracted from ginseng root,modern pharmacology has confirmed that GS could enhance immunity,so if GS can be used for the treatment of autoimmune diseases has been the focus of debate in the medical profession.In our previous study,we used a randomized controlled double-blind prospective clinical trial to confirm that GS could enhance the overall efficacy of prednisone in the treatment of SLE,relieved SLE disease progression and reduced SLEDAI score.In order to further confirm this effect,we designed this vitro experiment.In this study,we investigated the regulate effect of the four kinds of ginsenoside monomers on humoral immune of SLE.Objective:In this thesis,to investigate the effect of GS on the humoral immunity of SLE by in vitro experiments,we discuss the effects of GS on the proliferation and apoptosis of B cells and its regulate effect on B cell secretions,and the effects of GS on the distribution of B cells.Method:1.Human peripheral blood mononuclear cell suspension preparation:take healthy adult peripheral blood cells,the blood cells will be stratified after centrifugation,take the lymphocyte layer with a pipette,and then washed three times with PBS,add 1 ml of RMPI-1640 Medium to resuspend the cell to obtain peripheral blood mononuclear cell suspension（PBMC）.2.CD19⁺B cell sorting:PBMC was labeled with CD19 beads antibody,labeled PBMC was placed in the column（LS column）,and then LS column was placed in the magnetic field of the separator and collect labeled cells.3.Cell culture:CD19⁺B cells were cultured in RPMI-1640 medium containing 10%fetal bovine serum（FBS）and 1%double antibody（100U/ml penicillin and 100μg/ml streptomycin）and placed in a 5%CO2 humidified incubator at 37°C.4.Using a BrdU assay to test the effects of different concentrations（0.1,1,10 mg/L）of Rb1,Rh1,Rg1,Rg3+1μg/ml LPS on B cell proliferation,and compared with that stimulated with LPS.5.Using a ELISA assay to test the effects of different concentrations（0.1,1,10 mg/L）of Rb1,Rh1,Rg1,Rg3+1μg/ml LPS on secretory of IgG and IgM,and compared with that stimulated with LPS.6.Using a Western-Blot analysis to test the effects of 10 mg/L of Rb1,Rh1,Rg1,Rg3+1μg/ml LPS on apoptosis of B cells,and compared with that stimulated with LPS.7.Using a RT-PCR method to test the effects of 10 mg/L of Rb1,Rh1,Rg1,Rg3+1μg/ml LPS on expression of BAFF and BLyS and their receptors andβ₂M.8.Using a Flow cytometric analysis to test the effects of 10 mg/L of Rb1,Rh1,Rg1,Rg3+1μg/ml LPS on proportions B cell subsets:CD19⁺B cells,CD27⁺B cells and CD32⁺B cells.Results:1.The purity of CD19⁺B cells after magnetic separation（P<0.05）was higher（98.4%）compared with that before sorting（17.3%）.2.The results of BrdU showed that the proliferation rate of B cells in LPS group was higher than that in control group（P<0.05）.Compared with LPS group,the four kinds of ginsenoside monomers could inhibit B cell proliferation（P<0.05）,and the inhibitory effect was dose dependent.3.The results of ELISA showed that IgG and IgM were increased compared with control group（P<0.05）.Compared with LPS group,four kinds of ginsenoside monomers could inhibit the secretion of IgG and IgM（P<0.05）,and the inhibitory effect was dose dependent.4.The results of Western-Blot showed that the expression of Fas/Fas L and caspase-3in LPS group was lower than that in control group.Compared with LPS group,the four kinds of ginsenoside monomers could increase the expression of Fas/FasL and caspase-3.5.The results of RT-PCR showed that the mRNA expression of B lymphocyte stimulator（BLyS）and its receptor BCMA,TACI,and B cell activating factor（BAFF）and its receptor BAFF-R,andβ2-microglobulin（β₂M）in LPS group was higher than that in control group（P<0.05）.Compared with LPS group,the mRNA expression of BLyS and BAFF and its receptor andβ₂M were down-regulated by four kinds of ginsenoside monomers（P<0.05）.6.The results of flow cytometry showed that in LPS group,the percentage of CD27⁺B cells was increased（P<0.05）,the percentage of CD19⁺B cells was decreased（P<0.05）,and the expression of FcγRIIB（CD32）was decreased（P<0.05）compared with control group.Compared with LPS group,Rb1,Rh1 and Rg3 could increase the percentage of CD19⁺B cells（P<0.05）,decrease the percentage of CD27⁺B cells（P<0.05）,and increase the expression of FcγRIIB（P<0.05）.Conclusion:Ginsenoside monomers can regulate B cell proliferation,reduce the secretion of IgG and IgM,enhance expression of B cell apoptosis-related proteins and inhibit the expression of B cell-associated factors,and regulate the distribution of B cell subsets to regulate humoral immunity of SLE,inhibition effect of Rg1 is most obvious above these GS monomers.