CAJ | 학술논문

目的观察不同剂量黄芪甲苷(astragalosideⅣ,ASTⅣ)对高迁移率族蛋白B1(high mobility group box-1 protein,HMGB1)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) HMGB1组:HMGB1(1μg/ml)+Treg;(3)HMGB1+ ASTⅣ治疗Ⅰ组:HMGB1(1 μg/ml)+ASTⅣ(50μg/ml)+Treg；(4) HMGB1+ ASTⅣ治疗Ⅱ组:HMGB1(1μg/ml)+ASTⅣ(100 μg/ml)+Treg.以上4组再各分为3个亚组(每组6孔),分别于HMGB1刺激后24,48,72 h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-β分泌水平. 结果 HMGB1刺激Treg后,Foxp3蛋白及基因表达水平在24 ～72 h均较对照组有所下降(P＜0.01),其中以作用72 h下调尤为明显,细胞培养上清中IL-10及TGF-β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24～72 h均较HMGB1组明显升高(P＜0.05),且HMGB1+ASTⅣ治疗Ⅱ组Foxp3蛋白及基因表达水平在给药后24 ～ 72 h明显高于HMGB1+ ASTⅣ治疗Ⅰ组(P＜0.05),细胞培养上清中IL-10及TGF-β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HMGB1对小鼠Treg免疫功能的影响,呈剂量依赖性,提示其对HMGB1介导的促炎效应具有显著抑制作用.
목적 관찰불동제량황기갑감(astragalosideⅣ,ASTⅣ)대고천이솔족단백B1(high mobility group box-1 protein,HMGB1)개도소서조절성T세포(regulatory T cells,Treg)면역공능적길항효응병탐토기약물작용궤제. 방법 채용청길급BALB/c소서,처사후분리비장CD4+ CD25+ Treg,재72공판상안수궤수자표법장Treg분위4조진행배양(매조18공):(1)정상대조조:단순Treg; (2) HMGB1조:HMGB1(1μg/ml)+Treg;(3)HMGB1+ ASTⅣ치료Ⅰ조:HMGB1(1 μg/ml)+ASTⅣ(50μg/ml)+Treg；(4) HMGB1+ ASTⅣ치료Ⅱ조:HMGB1(1μg/ml)+ASTⅣ(100 μg/ml)+Treg.이상4조재각분위3개아조(매조6공),분별우HMGB1자격후24,48,72 h수집Treg.채용류식세포의급형광정량PCR법검측Treg포내차두익상라선전록인자p3(Foxp3)단백화기인표체수평,ELISA법검측Treg세포부육상청액중IL-10급TGF-β분비수평. 결과 HMGB1자격Treg후,Foxp3단백급기인표체수평재24 ～72 h균교대조조유소하강(P＜0.01),기중이작용72 h하조우위명현,세포배양상청중IL-10급TGF-β수평정현여Foxp3상동적변화추세；이ASTⅣ치료조Foxp3단백급기인표체수평재급약후24～72 h균교HMGB1조명현승고(P＜0.05),차HMGB1+ASTⅣ치료Ⅱ조Foxp3단백급기인표체수평재급약후24 ～ 72 h명현고우HMGB1+ ASTⅣ치료Ⅰ조(P＜0.05),세포배양상청중IL-10급TGF-β수평역정현여Foxp3상동적변화추세.결론 불동제량ASTⅣ재체외균가길항HMGB1대소서Treg면역공능적영향,정제량의뢰성,제시기대HMGB1개도적촉염효응구유현저억제작용.
Objective To investigate the antagonistic effect of different doses of astragaloside Ⅳ (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 Ⅳ exerts its influence.Methods CD4 + CD25 +Treg cells isolated from the spleens of clean-grade BALB/c mice were seeded on 72-well culture plate.Cells were divided into four groups (18 wells per group) according to the random number table,i.e.normal control group (cells were cultured merely),HMGB1 (1 μg/ml) group,HMGB1 (1 μg/ml) + AST Ⅳ (50 μg/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 cytometry and 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 IL-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 IL-10 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 HMGB1-mediated inflammatory response.