The Effects Of Resveratrol On B Lymphocytes Of Pristane-induced Lupus Mouse Model In Vivo And In Vitro

BackgroundSystemic lupus erythematosus (SLE) is a prototype autoimmune disease characterized by hyperactive T and B cells, autoantibody production, immune complex deposition and multi-organ damage. To date, no therapy has been found to satisfactorily treat SLE. Multivariant analysis has begun to document abnormalities in B cell maturation that are primarily associated with lupus, or, alternatively related to disease duration, disease activity and concomitant medication. SIRT1deficiency results in the development of an autoimmune syndrome in mice, including a high titer of anti-nuclear antibody in serum, immunoglobulin deposition in the kidney, and immune complex glomerulonephritis. Resveratrol is an activator of SBRT1and possesses anti-inflammation and immune-regulatory properties. Our previous studies showed that resveratrol has protective effects on pristane-induced lupus mouse model and can inhibit activation and proliferation of CD4+T cells, induce their apoptosis, decrease Thl and the ratio of Th1/Th2. resveratrol can inhibit the activation and proliferation of SLE patients peripheral blood mononuclear cells (PBMC) and CD4+T cells. But There is no studies on the effects of resveratrol on B lymphocytes of lupus mouse model or SLE patients.ObjectiveTo investigate the effects of resveratrol on B lymphocytes of pristane-induced lupus mouse model in vivo; To study the effects of resveratrol on B lymphocytes activation, proliferation, apoptosis, differentiation and function in vitro, and to explore the possible mechanism; To provide a new evidence for the application of resveratrol on the treatment of SLE.Methods1. Female BALB/c mice at age9-10weeks were randomly divided into3groups:(1) Healthy Controls:BALB/c mice received a single intraperitoneal injection of0.5ml of0.9%sodium chloride.(2) Pristane-induced lupus controls:BALB/c mice received a single intraperitoneal injection of0.5ml of pristane.(3) Resveratrol group: Ten mice were feed with50mg/kg/d resverarol from the second day after pristane injection. The development was monitored until7months. Measurements of serum Ig subtypesand autobodies by ELISA and immunofluorescence assay of deposition of immunoglobulin (IgG, IgM) were performed.2. CD19+B lymphocytes separated from spleen mononuclear cells (SMC) of pristane-induced lupus mice using magnetic activated cell sorting (MACS) were activated by LPS or anti-CD40antibody, cultured with or without resveratrol, then detected the expression of CD69, CD80, CD86by flow cytometry; B lymphocytes were stained with CFSE, and activated by LPS, with or without resveratrol, detected their proliferation by flow cytometry; B lymphocytes were cultured with or without resveratrol, detected their apoptosis by flow cytometry; SMC or B lymphocytes were activated by LPS or anti-CD40antibody, with or without resveratrol, stained CD19, CD138antibodies, analyzed their differentiation by flow cytometry, or extract total RNA and detect Blimp-1mRNA expression by Real-time PCR; SMC were activated by LPS, with or without resveratrol, collect the culture supernatants and detect the relative concentration of Ig subtypes by ELISA; B lymphocytes were activated by LPS, with or without resveratrol, collect the culture supernatants and detect the level of cytokines IL-4, IL-6, IFN-gamma, IL-17A, IL-10, IL-12p70by CBA.Results1. IgG and IgM deposition in the kidney was significantly increased in the model group compared with the normal group (P=0.002; P=0.012; respectively); IgG deposition in the kidney was significantly decreased in the resveratrol group compared with the model group (P=0.022), and IgM deposition in the resveratrol group had a weakening trend (P=0.053).2. Levels of of serum IgGl, IgG2a, IgG3, IgM, IgA in normal group were significantly lower than that of model control group (P<0.001; P<0.001; P=0.015; P<0.001; P=0.035; respectively); Levels of of serum IgG1, IgG2a, IgG3were statistically decreased in resveratrol group compared with model group (P<0.001; P<0.001; P=0.044; respectively); Levels of ANA, anti-ds-DNA antibody, and anti-ssDNA antibody in normal group were significantly lower than that of model group (P=0.001; P=0.028; P=0.023; respectively), Levels of anti-RNP/Sm antibody in the model group had an increasing trend (P=0.063).3. Resveratrol significantly inhibited the LPS/anti-CD40antibody-activated CD69,CD80,CD86expression of B lymphocytes in vitro in a dose-dependent manner; Resveratrol significantly inhibited the LPS-induced preliferation of B lymphocytes in a dose-dependent manner (P<0.001), but there is no effect of resveratrol on apoptosis of B lymphocytes.4. Resveratrol significantly inhibited the LPS/anti-CD40antibody-induced CD138+plasma cell differentiation of B lymphocytes in a dose-dependent manner; Resveratrol (concentration of10μM,20μM) significantly inhibited the expression of Blimp-1mRNA of B lymphocytes (P=0.026; P=0.012; respectively).5. Resveratrol significantly inhibited the LPS-induced secretion of IgGl, IgG2a, IgG2b, IgG3, IgM, IgA of B lymphocytes in a dose-dependent manner (P=0.039; P=0.027; P=0.027; P=0.027; P=0.027; P=0.027; respectively); Resveratrol of concentration of10μM significantly increased the LPS-induced secretion of IL-10of B lymphocytes (P=0.04); but concentration of40μM of Resveratrol significantly decreased the LPS-induced secretion of IL-10of B lymphocytes (P=0.003); Resveratrol significantly inhibited the LPS-induced secretion of IL-6of B lymphocytes; Resveratrol significantly inhibited the LPS-induced secretion of IFN-y,IL-4,IL-12p70of B lymphocytes and decreased the ratio of IFN-y/IL-4.Conclusion:Pristane-induced lupus mouse model is associated with the B lymphocytes hyperactivity and production of autoantibodies; resveratrol can reduce the production of immunoglobulin IgG1, IgG2a and IgG3of lupus B lymphocytes and IgG and IgM deposition in kidney in vivo; resveratrol in vitro can inhibit the activation, proliferation, immunoglobulin and pro-inflammatory cytokines secretion of lupus B lymphocytes, and suppress their differentiation into plasma cells, also can regulate the secretion of cytokines correlated with Thl and Th2differentiation; resveratrol of small doses may promote their secretion of IL-10by regulating the function of regulatory B cells, therefore, resveratrol may have a immunoregulatory effect on lupus model B lymphocytes. Together, resveratrol may be a new potential drug for treatment of SLE.