黄芪甲苷对高迁移率族蛋白B1介导小鼠调节性T细胞免疫功能的拮抗作用  

Antagonistic effect of astragaloside on immune response of regulatory T cells mediated by highmobility group box-1 protein in rats

作　　者：李金凤[1] 姚咏明[3] 黄立锋[2] 张淑文[4] 张震宇[1] 盛志勇[3] 

机构地区：[1]首都医科大学附属北京朝阳医院妇产科,北京100020 [2]首都医科大学附属北京朝阳医院SICU,北京100020 [3]解放军总医院第一附属医院全军烧伤研究所 [4]首都医科大学附属北京友谊医院感染暨急救医学科

出　　处：《中华创伤杂志》2013年第10期919-923,共5页Chinese Journal of Trauma

基　　金：国家自然科学基金资助项目（81130035,81071545,81372042,30901561）;国家重点基础研究发展计划资助项目（2012CB518102）;全军“十二五”医学科学技术研究计划重大资助项目（AWSllJ008）

摘　　要：目的观察不同剂量黄芪甲苷（astragaloside IV，ASTIV）对高迁移率族蛋白B1（high mobility groupbox-1protein，HMGBl）介导小鼠调节性T细胞（regulatory T cells，Treg）免疫功能的拮抗效应并探讨其药物作用机制。方法采用清洁级BALB／c小鼠，处死后分离脾脏CD4^＋CD25^＋Treg，在72孔板上按随机数字表法将Treg分为4组进行培养（每组18孔）：（1）正常对照组：单纯Treg；（2）HMGBl组：HMGBl（1μg／m1）＋Treg；（3）HMGBl＋AST IV治疗I组：HMGBl（1μg／m1）＋ASTIV（50μg／m1）＋Treg；（4）HMGBl＋ASTIV治疗Ⅱ组：HMGBl（1μg／m1）＋ASTIV（100μg／m1）＋Treg。以上4组再各分为3个亚组（每组6孔），分别于HMGBl刺激后24，48，72h收集Treg。采用流式细胞仪及荧光定量PCR法检测Treg胞内叉头翼状螺旋转录因子p3（Foxp3）蛋白和基因表达水平，ELISA法检测Treg细胞孵育上清液中IL-10及TGF-B分泌水平。结果HMGBl刺激Treg后，Foxp3蛋白及基因表达水平在24-72h均较对照组有所下降（P〈0．01），其中以作用72h下调尤为明显，细胞培养上清中IL-10及TGF-B水平呈现与Foxp3相同的变化趋势；而ASTIV治疗组Foxp3蛋白及基因表达水平在给药后24～72h均较HMGBl组明显升高（P〈0．05），且HMGBl＋ASTIV治疗Ⅱ组Foxp3蛋白及基因表达水平在给药后24-72h明显高于HMGBl＋ASTIV治疗I组（P〈0．05），细胞培养上清中IL-10及TGF-B水平亦呈现与Foxp3相同的变化趋势。结论不同剂量ASTIV在体外均可拮抗HMGBl对小鼠Treg免疫功能的影响，呈剂量依赖性，提示其对HMGBl介导的促炎效应具有显著抑制作用。Objective To investigate the antagonistic effect of different doses of astragaloside 1V （AST IV） on immune function of regulatory T （Treg） cells mediated by high mobility group box-1 protein （HMGB1） in rats and the mechanism by which AST 1V exerts its influence. Methods CIM^＋ CD25 ^＋ Treg cells isolated from the spleens of clean-grade BALB/c mice were seeded on 72-well culture plate. Ceils were divided into four groups （ 18 wells per group） according to the random number table, i.e. nor- mal control group （cells were cultured merely） , HMGB1 （1 μg/ml） group, HMGB1 （1 μg/ml） ＋AST IV （50μg/ml） group, and HMGB1 （1 μg/ml） ＋ AST IV （100 μg/ml）group. Each group consisted of three subgroups （6 wells per group）, from which the Treg cells were collected at post-stimulation hours 24, 48 and 72 respectively. Foxp3 intracellular protein and mRAN expressions were detected by flow cytometryand quantitative fluorescent PCR. Contents of IL-10 and TGF-β released into the supernatants were determined by ELISA method. Results As compared with the control group, Foxp3 intracellular protein and mRAN expressions in the Treg cells were decreased at 24 hours to 72 hours after HMGB1 stimulation （ P 〈 0. 01 ）, with particular reduction at 72 hours. Additionally, changes of lL-10 and TGF-βin the supernatants presented the same trends with Foxp3. Foxp3 protein and mRAN expressions in AST IV group were markedly higher than that in HMGB1 group at 24-72 hours （ P 〈 0. 05 ） and the expressions in AST IV （100 μg/ml） group were much more significant than that in AST IV （50 μg/ml） group （P 〈 0. 05 ）. While contents of lL-lO and TGF-β in supernatants revealed the same trend with Fox3 as well. Conclusions AST IV antagonizes the impact of HMGB1 on the activity of the Treg cells in a dose-dependent manner in vitro. It is suggested that AST IV has a strong inhibitory effect on HMGBl-mediated inflammatory response.

关 键 词：脓毒症 高迁移率族蛋白质类 免疫耐受 调节性T细胞 

分 类 号：R285.5[医药卫生—中药学]