黄芪糖蛋白对胶原诱导性关节炎小鼠Th17/Treg细胞免疫平衡的影响

Influences of AmGP on Th17/Treg immune balance in CIA mice

目的研究16k D黄芪糖蛋白对胶原诱导性关节炎(collagen-induced arthritis,CIA)模型小鼠Th17/Treg细胞免疫平衡的影响,探讨黄芪糖蛋白治疗胶原诱导性关节炎小鼠的免疫作用机理。方法采用雄性Balb/c小鼠建立牛Ⅱ型胶原诱导性关节炎小鼠模型,将造模成功的小鼠随机分为CIA模型组、氢化可的松阳性对照组、黄芪糖蛋白高、中、低剂量组,另设空白对照组;各治疗组腹腔注射药物14天,对照组与模型组注射等体积生理盐水。采用流式细胞术检测各组小鼠外周血CD3^+CD4^+IL-17A^+Th17细胞、CD4^+CD25^+Foxp3^+Treg细胞比例;Western blot法检测各组小鼠脾组织中维甲酸相关孤独受体γt(retinoid-related orphan receptorγt,RORγt)和叉头状转录因子3(forkhead box protein 3,Foxp3)的蛋白表达水平。结果流式检测结果显示,黄芪糖蛋白可显著降低模型组小鼠外周血中CD3^+CD4^+IL-17A^+Th17细胞比例(P<0.01),提高CD4^+CD25^+Foxp3^+Treg细胞的比例(P<0.01)。Western blot结果显示黄芪糖蛋白可显著降低模型组小鼠脾组织中RORγt的表达(P<0.01),提高Foxp3的表达(P<0.01)。结论黄芪糖蛋白提高Foxp3的表达水平,降低RORγt的表达水平,恢复Th17/Treg细胞之间的平衡,可能是黄芪糖蛋白治疗胶原诱导性关节炎小鼠的机制之一。

Objective To examine the impacts of 16 k D astragalus membranaceus glycoprotein（ Am GP） on the balance of Th17 /Treg in CIA mice; and to investigate the effects of Am GP on CIA mice and its＇ mechanism. Methods Bovine type Ⅱcollagen was used to establish CIA model. Mice were randomly divided into five groups： CIA model group,hydrocortisone（ HC） positive control group,Am GP groups with low,medium and high dose. In addition,a normal group was built. The HC and Am GP groups were treated by HC or Am GP with intraperitoneal injection,while normal saline was used in the other groups,and the treatment was lasted for 2 weeks. Flow cytometry was used to detect the proportions of CD3＋CD4＋IL-17A＋Th17 and CD4＋CD25＋Foxp3＋Treg cells in peripheral blood of the mice; The protein expressions of RORγt and Foxp3 were evaluated by western blot in mice spleen. Results It is showed that Am GP could significantly reduce the proportion of CD3＋CD4＋IL-17A＋Th17 in peripheral blood of CIA mice（ P 〈0. 01）. It could improve the proportion of CD4＋CD25＋Foxp3＋Treg cells（ P 〈0. 01）. Western blot results indicated that Am GP could significantly decrease the expression of RORγt（ P 〈0. 01）,while increase the expression of Foxp3 in spleen of CIA mice（ P 〈0. 01）. Conclusion Am GP recovers the balance of Th17 / Treg cells by improving the expressions of Foxp3,and reducing the expression of RORγt.This procedure may be one of the mechanisms for Am GP to cure the CIA mice.