高迁移率族蛋白B1对烫伤大鼠调节性T细胞表型及T细胞功能极化的影响

Effects of high mobility group box-1(HMGB1) protein on phenotype of regulatory T cells and functional polarization of T cells in burned rats

目的探讨严重烫伤延迟复苏后脾脏高迁移率族蛋白B1(HMGB1)表达对调节性T细胞(Treg)表型及其介导T淋巴细胞功能性极化的影响。方法 104只Wistar大鼠随机分为正常对照组(8只)、假烫组(32只)、烫伤组(32只)、丙酮酸乙酯(EP)治疗组(32只)。后2组大鼠制作30%TBSA三度烫伤模型,伤后6h腹腔注射林格液或EP液延迟复苏抗休克。分别于伤后1、3、5、7d处死各组大鼠,免疫磁珠法分离大鼠脾脏CD4+CD25+Treg,检测脾组织HMGB1含量及T淋巴细胞上清液中白细胞介素4(IL-4)、干扰素γ(IFN-γ)水平,并采用流式细胞术检测Treg表面标记物叉头翼状螺旋转录因子(Foxp3)表达水平。结果与假烫组比较,烫伤组大鼠脾组织HMGB1水平在伤后1～7d显著升高(P<0.01),其中第1天时达峰值(46.73±8.27ng/mg),EP干预后HMGB1水平在1～7d显著低于烫伤组(P<0.01)。与假烫组比较,烫伤组大鼠伤后1d脾脏Treg表面Foxp3表达逐渐增强,于第3天达峰值(72.46%±11.02%),EP治疗组伤后1～7dFoxp3表达显著低于烫伤组(P<0.01或P<0.05)。与假烫组比较,烫伤组脾T淋巴细胞IL-4分泌量明显增高(P<0.01或P<0.05),IFN-γ分泌量明显降低(P<0.01),EP治疗组伤后1～5dIL-4分泌量明显低于烫伤组(P<0.05),伤后1～7dIFN-γ分泌量则明显高于烫伤组(P<0.05)。结论严重烧伤后HMGB1可促进Treg成熟,从而介导T细胞功能亚群从促炎反应优势向抗炎优势转化,诱导机体细胞免疫功能抑制。

Objective To explore the effects of high mobility group box-1（HMGB1） protein expression on the phenotype of regulatory T cells（Treg） and the functional polarization of splenic T cells in severe burn rats after delayed resuscitation.Methods One hundred and four Wistar rats were involved in present study and randomly divided into normal control group（n=8）,sham burn group（n=32）,burn group（n=32） and ethyl pyruvate（EP） treatment group（n=32）.Thirty percent TBSA full-thickness burn models were established with the animals in both burn and EP treatment group,and Ringer’s solution or EP solution was respectively injected intraperitoneally for delayed resuscitation.Animals in each of the later three groups were further divided into four subgroups（8 each）,sacrificed and simultaneously harvesting the spleen samples on day 1,3,5 and 7 after burn,respectively.Spleen CD4＋CD25＋ Treg were isolated by magnetic-activated cell sorting（MACS） system.The levels of HMGB1 in splenic tissue and IL-4,IFN-γ in T cells supernatant were determined by ELISA,and the expression of forkhead/winged helix transcription factor 3（Foxp3） as a surface marker of Treg was analyzed by flow cytometry.Results Compared with those in sham burn group,the HMGB1 level elevated significantly at day 1 to 7（P〈0.01）,reached the peak value（46.73±8.27ng/mg） on day 1,and declined obviously（P〈0.01） by EP treatment in burn group;The expression of Foxp3 on splenic Treg increased from day 1 and reached the peak（72.46%±11.02%） on day 3 in burn group,while after EP treatment,it decreased obviously on day 1 to 7（P〈0.01 or 0.05）.The IL-4 level increased（P〈0.01 or 0.05） and IFN-γ level decreased（P〈0.01） markedly in burn group compared with those in sham burn group.The IL-4 level was significantly lower（P〈0.05） on day 1 to 5,while the IFN-γ level was markedly higher on day 1 to 7（P〈0.05）,in EP group than in burn group.Conclusion The excessive release of HMGB1 after severe burn might stimulate the maturation of splenic CD4＋CD25＋ Treg,thereby mediate the transformation of T cell subsets from Th1 to Th2 and induce the inhibition of cellular immunity.