黄芪甲苷在体内对高迁移率族蛋白B1介导小鼠调节性T淋巴细胞免疫表型的作用

The effect and mechanism of Astragaloside IV on the immunophenotype of regulatory T cells mediated by high mobility group box 1 protein in vivo

目的进一步明确晚期炎症介质高迁移率族蛋白B1(HMGB1)在体内对小鼠调节性T细胞(Treg)免疫表型的影响,同时观察不同剂量黄芪甲苷(AST)对HMGB1介导的Treg细胞功能变化的作用。方法采用清洁级BALB/c小鼠,予实验当天早、晚各腹腔注射10μg重组HMGB1,并于注射后第2、4、6天腹腔注射生理盐水或黄芪甲苷。实验分组:将120只小鼠随机分为6组(每组20只)。(1)正常对照组:动物麻醉后活杀;(2)HMGB1攻击组:单纯注射重组HMGB1;(3)HMGB1+NS组:HMGB1+生理盐水治疗;(4)HMGB1+AST组Ⅰ:HMGB1+AST(10 mg/kg)治疗;(5)HMGB1+AST组Ⅱ:HMGB1+AST(20 mg/kg)治疗;(6)HMGB1+AST组Ⅲ:HMGB1+AST(30 mg/kg)治疗。以上6组再各分为4个亚组(每组5只),于实验第1、3、5、7天将动物处死,提取其脾脏Treg细胞。采用流式细胞术检测Treg细胞Foxp3蛋白表达水平,ELISA法检测Treg细胞孵育上清中IL-10及TGF-β表达水平。结果 HMGB1攻击组动物Foxp3表达水平在1~7d明显降低,与正常对照组比较差异有统计学意义(P<0.01),在攻击后第3天为最低值,细胞培养上清中IL-10及TGF-β表达水平呈现与Foxp3相同变化趋势;HMGB1+NS组动物Foxp3、IL-10及TGF-β表达水平与HMGB1攻击组比较差异无统计学意义(P>0.05);而HMGB1+AST组Ⅲ及HMGB1+AST组Ⅱ组Foxp3表达水平在给药后均较HMGB1+NS组明显升高(P<0.01~0.05),细胞培养上清中IL-10及TGF-β表达水平亦呈现与Foxp3相同变化趋势。结论 HMGB1在体内可抑制小鼠Treg细胞免疫抑制功能的发挥,而AST可针对性拮抗HMGB1对Treg细胞免疫功能的抑制效应,显示其对HMGB1介导的促炎效应具有治疗作用。

Objective To investigate the antagonistic effects of different doses of Astragaloside IV（ AST） on the immune function of Treg mediated by HMGB1 in vivo and its potential mechanism. Methods Animal models were constructed using i. p administration of recombinant HMGB1,in which normal saline or AST was administered i. p on Day2 nd,4 thand 6 th. One hundred and twenty mice were included,and divided into five groups（ 20 mice per group）,normal control group（ mice were sacrificed after anesthetized）,HMGB1 group,HMGB1 ＋ NS group（ HMGB1 ＋ normal saline）,HMGB1 ＋ AST Ⅰ group（ HMGB1 ＋ AST of 10 mg/kg）,HMGB1 ＋ AST Ⅱ group（ HMGB1 ＋ AST of 20 mg/kg）,and HMGB1 ＋ AST Ⅲ group（ HMGB1 ＋ AST of 30 mg/kg）. The latter four groups were further divided into four subgroups（ 8 rats each）,in which mice were sacrificed on the 1 st,3 rd,5 thand 7 thpost-stimulus day（ PSD）,respectively.Spleens were collected for harvest of Treg cells. Cells were cultured; phenotypes were analyzed by flow cytometry; and the contents of cytokines in supernatants were determined by ELISA. Results The expression levels of Foxp3 in Treg during PSD（ 1-7） were significantly reduced in HMGB1 group in comparison to Treg from control group（ P〈0. 01）,and it was lowest on the 3 rdPSD. The Foxp3 expressions were markedly increased in a dose-dependent manner in AST group as compared with those in HMGB1 group（ P〈0. 05）. The same results were found in the contents of IL-10 and TGF-β.Conclusion AST IV can rivalry the effects of HMGB1 on the immune function of Treg in vivo,indicating that AST Ⅳ has the therapeutic action on inflammation promoted by HMGB1.