Total Glucosides Of Paeony (TGP) Induces Regulatory CD4~+CD25~+T Cells In Systemic Lupus Erythematosus

Part1TGP influences regulatory CD4+CD25+T cells and cytokines in SLE CD4+T cellsObjective To investigate the molecular mechanisms of total glucosides of Paeony (TGP) in the treatment of systemic lupus erythematosus (SLE) through studies on the effect of TGP on the number of CD4+CD25+Treg cells and the related cytokine mRNA and protein expression.Methods CD4+T cells was Isolated from peripheral blood of SLE and controls by immunomagnetic beads, and then treated with TGP for48h at concentration of0,62.5,312.5and1562.5μg/ml. The numbers of CD4+CD25+T cells was measured by flow cytometric analysis. The mRNA and protein levels of TGF-β1, IL-2and IFN-γ were detected by Real-time PCR and ELISA, respectivelyResults1. The percentage of CD4+CD25+T cells were significant increased in CD4+T cells stimulated with1562.5μg/ml of TGP in SLE group compared to blank control group (p<0.01). However, there were no significance difference among other groups in SLE and all health controls groups (p>0.05).2. IFN-γ and IL-2mRNA (p<0.01,p<0.05)and protein expression(p<0.05) was significantly increased in lupus CD4+T cells treated with1562.5μg/ml of TGP. However, no significant change was observed in lupus CD4+T cells treated with low and middle concentration of TGP (p>0.05), and in health control groups (p>0.05). There was no significantly changed in the mRNA of TGF-β1in lupus CD4+T cells treated with TGP (p>0.05).Conclusion TGP inhibits autoimmunity in SLE patients possibly by inducing Treg cell differentiation, which may in turn be due to its ability to activate IFN-γ and IL-2signaling. Part2The effect of TGP on the expression of Foxp3in SLE CD4+T cells and its epigenetic mechanismsObjective To investigate the effect of TGP on the expression of Foxp3in SLE CD4+T cells and its epigenetic mechanismsMethods CD4+T cells was Isolated from peripheral blood of SLE and controls by immunomagnetic beads, and then treated with TGP for48h at concentration of0,62.5,312.5and1562.5μg/ml. The mRNA and protein level of Foxp3were detected by Real-time PCR and Western blotting, respectively. The methylation status of Foxp3promoter was measured by bisulfite genomic sequencing (BSP).Results1. Foxp3mRNA and protein levels were significantly enhanced in lupus CD4+T cells treated with1562.5μg/ml TGP compared with control-treated cells (p<0.01), and the changes was most obviously in48hours. However, no significant change was observed in lupus CD4+T cells treated with low and middle concentration of TGP (p>0.05). In addition, we also found that Foxp3mRNA expression was not changed in healthy CD4+T cells treated by TGP with different concentrations (p>0.05).2. The mean methylation level of the eight CG pairs within Foxp3promoter (positions-18,-33,-40,-52,-88,-101,-113and-139) was significantly down-regulated in lupus CD4+T cells treated with high concentration of TGP compared with negative controls (P<0.01).In addition, we also found that promoter methylation level of Foxp3was lower in CD4+T cells from healthy controls, which cannot be decreased by TGP treatment.Conclusion TGP acts as an inducer of Treg cell response, via demethylation of the Foxp3promoter and activation of Foxp3expression.