| Objective:An experimental autoimmune thyroiditis（EAT）model in mice was established to evaluate the proportion and function of regulatory T cells（Tregs）and Th17 cells in the splenocytes of the control group and the effect of different doses of total glucosides of paeony（TGP）in the treatment group.The aims of this study were to evaluate whether the autoimmune status of EAT mice improved after drug treatment.The effects of TGP on the immune balance of Tregs/Th17 cells in EAT mice were investigated,which laid a foundation for studying the mechanism of traditional C hinese medicine in the treatment of autoimmune thyroiditis.Methods:EAT model was established by immunising female Kunming mice with porcine thyroglobulin and Freund’s adjuvant.（2）Different doses of TGP（0.1,0.2 and 0.4 g/kg）and Tripterygium glycosides（10mg/kg）were administered intragastrically to the mice model.（3）Real-time PC R was performed to detect the expression of Foxp3 mRNA and ROR?t mRNA in the splenocytes of each group.（4）Flow cytometry was performed to detect the percentage of Tregs and Th17 cells in the mice splenocytes in each group.（5）Serum levels of TGF-beta1 and IL-17 in each group were measured by ELISA.（6）Serum levels of TGAb in each group were detected by chemiluminescence assay.Results:1.Foxp3 mRN A expression in the mice splenocytes in the TGP medium-dose group and the TGP high-dose group was significantly higher than that in the control model group(1.993×10-4±0.685×10-4 vs.1.166×10-4±0.496×10-4,P<0.05)(2.048×10-4±0.933×10-4 vs.1.166×10-4±0.496×10-4,P<0.05).Foxp3 mRN A expression in the mice splenocytes in the Tripterygium glycosides control group was also significantly higher than that in the control model group(1.971×10-4±0.615×10-4 vs.1.166×10-4±0.496×10-4,P<0.05).No difference was observed between the different dose groups of TGP and the control group of Tripterygium glycosides and among the different dose groups of TGP.2.No difference was observed between the different dose groups of TGP and the control model group in the expression of ROR?t mRNA,but there was a decreasing trend in the TGP medium-dose group and the TGP high-dose group.ROR?t mRN A expression in the mice splenocytes in the Tripterygium glycosides control group was significantly lower than that in the control model group(0.995×10-5±0.124×10-5 vs.1.553×10-5±0.602×10-5,P<0.01).ROR?t mRN A expression of in the splenocytes in the TGP low-dose and the high-dose group was significantly higher than that in the Tripterygiumglycosidescontrolgroup(1.611×10-5±0.342×10-5vs.0.995×10-5±0.124×10-5,P<0.01)(1.448×10-5±0.308×10-5vs.0.995×10-5±0.124×10-5,P<0.05).No difference was observed among the different dose groups of TGP.3.The percentages of CD4+CD25+Foxp3+T cells in the TGP low-dose and the high-dose group were significantly higher than that in the control model group（3.488±1.106%vs.1.881±0.695%,P<0.01）（3.413±1.618%vs.1.881±0.695%,P<0.05）.No difference was observed between the control model group and the control group of Tripterygium glycosides.No difference was observed between the different dose groups of TGP and the control group of Tripterygium glycosides and among the different dose groups of TGP.4.The percentages of T cells secreting IL-17 in the low-,medium-and high-dose groups of TGP were significantly lower than that in the control model group（1.213±0.629%vs.2.357±0.362%,P<0.001）,（1.038±0.478%vs.2.357±0.362%,P<0.001）,（1.075±0.385%vs.2.357±0.362%,P<0.001）.The percentage of T cells secreting IL-17 in the mouse splenocytes in the Tripterygium glycosides control group was significantly lower than in the control model group（1.125±0.385%vs.2.357±0.362%,P<0.001）.No difference was observed between the different dose groups of TGP and the control group of Tripterygium glycosides and among the different dose groups of TGP.5.Serum levels of TGF beta1 in the TGP medium-dose group and the TGP high-dose group were significantly higher than that in the control model group（5.506×105±1.645×105pg/ml vs.3.683×105±1.261×105pg/ml,P<0.01）.Serum level of TGF beta 1 in TGP medium-dose group was significantly higher than that in the Tripterygiumglycosidescontrolgroup（5.506×105±1.645×105pg/mlvs.4.257×105±0.843×105pg/ml,P<0.05）.No difference was observed between the control model group and the control group of Tripterygium glycosides and among the different dose groups of TGP.6.Serum levels of IL-17 in the TGP medium-dose group and the TGP high-dose group were significantly lower than that in the control model group（127.916±43.532pg/ml vs.202.456±72.712pg/ml,P<0.01）（137.435±37.007pg/ml vs.202.456±72.712 pg/ml,P<0.05）.Serum level of IL-17 in the Tripterygium glycosides control group was lower than that in the model control group（108.391±24.024 pg/ml vs.202.456±72.712 pg/ml,P<0.001）.No difference was observed between the different dose groups of TGP and the control group of Tripterygium glycosides and among the different dose groups of TGP.7.Serum levels of TGAb in the TGP medium-dose group and the TGP high-dose group were significantly lower than that in the control model group（18.294±6.789?g/dl vs.40.160±16.738?g/dl,P<0.05）（13.116±6.030?g/dl vs.40.160±16.738?g/dl,P<0.05）.Serum levels of TGAb in the Tripterygium glycosides control group were significantly lower than that in model control group（16.064±5.663?g/dl vs.40.160±16.738?g/dl,P<0.05）.No difference was observed between the different dose groups of TGP and the control group of Tripterygium glycosides and among the different dose groups of TGP.Conclusions:1.Compared with the normal group,there was a Treg/Th17 imbalance in EAT mice.This suggests that a Treg/Th17 imbalance exists in EAT mice and Th17 cells and the related cytokines play an important role in the onset and development of AIT.This finding helps us identify novel therapeutic targets based on the effects on the Treg/Th17immune balance.2.TGP could increase the number of Treg in spleen cells and the serum levels of TGF-?1 in EAT mice.TGP could significantly decrease the proportion of Th17 in spleen cells and the serum levels of IL-17 in EAT mice.TGP could reduce the serum levels of TGAb of EAT mice.Therefore,TGP can regulate the Treg/Th17 immune balance,which can be beneficial to the prognosis of AIT disease.The TGP medium-dose group and the TGP high-dose group had obvious curative effect. |
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