穿龙薯蓣皂苷元体外对CIA小鼠Treg细胞亚群及转录因子的影响

Effect of diosgenin on Treg cell and its transcription factor in collagen-induced arthritis mice in vitro

目的:探究穿龙薯蓣皂苷元体外对CIA小鼠Treg细胞及特异性转录因子的影响。方法:将DBA1/J小鼠用鸡Ⅱ型胶原乳剂进行免疫(0.1 ml/只),21 d等量再次刺激,建立CIA模型。选模型成功CIA鼠无菌取腹股沟淋巴结研磨成单细胞悬液,根据药物毒性确定药物处理浓度,且设为对照组、模型组、穿龙薯蓣皂苷元高、中、低剂量组。流式细胞术分析体外培养淋巴细胞中Treg细胞百分率,PCR、Western blot检测Treg特异性转录因子Foxp3 mRNA及蛋白状况。结果:选择CCK-8检测药物抑制率不低于70%为体外药物刺激高、中、低剂量,分别为25、12.5、6.25μmol/L。较对照组,模型组Treg细胞比率显著增高(P<0.05),Foxp3 mRNA水平提高(P<0.05),Foxp3蛋白表达下降(P<0.05)。较模型组,穿龙薯蓣皂苷元中剂量组Treg细胞增多,在统计学上有意义(P<0.05),高剂量组Foxp3 mRNA、蛋白表达均明显升高(P<0.05)。结论:体外实验中CIA小鼠Treg细胞的生长可被穿龙薯蓣皂苷元明显促进,这个作用许是通过调节Treg细胞特异性转录因子Foxp3而引起的。

Objective:To explore the effects of diosgenin on Treg cell and its specific transcription factors in vitro.Methods: Used chicken type Ⅱ collagen emulsion immuned DBA1/J mice(0.1 ml/mouse),with the same dose of stimulus again in 21 days,the CIA model was established.Then,excise the inguinal lymph nodes of the CIA mice under aseptic condition and made them into single cell suspension.Determined drug treatment concentration according to drug toxicity,the experiment were divided into control group,model group,high dose diosgenin group,medium dose diosgenin group,low dose diosgenin group.Following this,the percentage of Treg cells was determined by flow cytometry,and the mRNA and protein levels of Treg-specific transcription factor Foxp3 were detected by PCR and Western blot.Results: We selected the dose of CCK8 detection drug with inhibition rate of no less than 70% as the high,medium and low dose of drug stimulation in vitro: 25,12.5 and 6.25 mol/L respectively.Compared with the control group,Treg cell ratio was significantly increased(P<0.01),Foxp3 mRNA level was increased(P<0.05),and protein expression of Foxp3 was decreased(P<0.05) in the model group.Compared with the model group,Treg cells in the medium dose diosgenin group increased significantly(P<0.05),while Foxp3 mRNA and protein expression in the high dose diosgenin group increased significantly(P<0.05).Conclusion: The growth of Treg cells in CIA mice was significantly promoted by dioscogenin in vitro,which may be caused by the regulation of Treg cell-specific transcription factor Foxp3.