烧伤后高迁移率族蛋白B1对调节性T细胞免疫功能影响的实验与临床研究
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摘要
目的:
实验一:(1)采用严重烫伤延迟复苏动物模型,明确高迁移率族蛋白B1(HMGB1)的变化及其与调节性T细胞(Treg)免疫功能的关系。(2)通过严重烫伤延迟复苏动物模型,阐明HMGB1对Treg分化成熟的受体作用机制。(3)探讨严重烫伤延迟复苏后HMGB1介导的Treg对树突状细胞(DC)免疫功能的影响及调节作用。(4)探讨严重烫伤延迟复苏后HMGB1介导的Treg对T淋巴细胞免疫功能的影响及调节途径。(5)观察丙酮酸乙酯(EP)对重度烫伤延迟复苏动物免疫功能及脏器损害的可能保护效应。
实验二:(1)对临床严重烧伤患者进行动态监测,明确严重烧伤后病人外周血HMGB1、Treg分化成熟以及T淋巴细胞免疫功能的变化规律,探讨HMGB1、Treg及T淋巴细胞免疫功能改变在患者烧伤后发生脓毒症及不良预后中所起的作用及临床意义。(2)探讨严重烧伤后HMGB1、Treg及T淋巴细胞免疫指标间的相互关系及可能的作用机制。
方法:
1.采用重度烫伤延迟复苏动物模型,即雄性清洁级Wistar大鼠,浸于沸水中造成30%体表面积Ⅲ度烫伤。伤后延迟6小时抗休克复苏,在伤后6小时从腹腔给予林格液(40 ml/kg)抗休克治疗,此外在烫伤后12、24、36、48小时分别给予4 ml/次林格液腹腔内注射。实验分组:136只大鼠随机分为5组,(1)正常对照组(n=8):麻醉后活杀;(2)假烫伤组(n=32);(3)烫伤组(n=32);(4)EP治疗组(n=32):EP加入林格液中随补液给药(EP为28 mM);(5)晚期糖基化终末产物受体(RAGE)抗体(1 mg/ml)治疗组(n=32):烫伤后6、24小时从阴茎背静脉给予RAGE抗体(1 mg/kg)治疗。以上后4组实验动物再分为4个亚组,分别于烫伤后1、3、5、7天处死动物,无菌留取血和脾脏标本。分离血清,-20℃贮存。脾脏一部分无菌、无酶液氮冻存,另一部分用于提取Treg、DC和T淋巴细胞。Treg、DC的分离纯化采用MiniMACS免疫磁性分离系统进行。利用B细胞和单核细胞具有易粘附于尼龙纤维表面的特性,分离T淋巴细胞。采用流式细胞术检测细胞免疫表型和细胞表面受体,MTT法检测脾T淋巴细胞增殖,酶联免疫吸附试验(ELISA)检测血清、细胞孵育上清及组织匀浆液的细胞因子和T淋巴细胞活化的核因子(NF)-kB。采用荧光定量PCR检测目的基因表达水平。
2.对106例烧伤总面积大于或等于30%的患者进行动态观察,对所采集临床病历资料进行分析。实验分组:(1)按烧伤总面积将患者分为三组:Ⅰ组41例(烧伤总面积30%～49%),Ⅱ组34例(烧伤总面积50%～69%),Ⅲ组31例(烧伤总面积70%～99%);(2)根据烧伤脓毒症的诊断标准,将患者进一步分为脓毒症组(59例)与非脓毒症组(47例);(3)根据脓毒症患者的预后情况,将其分为死亡组(17例)与存活组(42例)。同时设正常对照组(25位健康献血员)。分别于烧伤后1、3、7、14、21天采集患者外周静脉血,分离外周血T淋巴细胞,采用MiniMACS免疫磁性分离系统对外周血Treg进行分离纯化,同时分离血清,-20℃贮存。采用流式细胞术检测细胞免疫表型,MTT法检测T淋巴细胞增殖活性,ELISA检测血清HMGB1及细胞孵育上清的细胞因子水平,采用荧光定量PCR检测目的基因表达水平。
结果:
实验一:(1)烫伤延迟复苏导致动物脾脏HMGB1基因、蛋白表达和血清HMGB1水平在伤后1～7天显著升高,脾组织中HMGB1含量第1天达峰值,血清HMGB1水平于第3天达峰值。EP干预后烫伤动物脾脏HMGB1基因、蛋白表达和血清HMGB1水平1～7天均明显降低。RAGE抗体干预对烫伤动物脾脏和血清HMGB1无明显影响。(2)严重烫伤后脾脏Treg表面分子CTLA-4的表达1～5天明显增强,表面标记物Foxp3的表达1～7天明显增高,与假烫伤动物比较差异显著,表明严重烫伤可促使Treg成熟。EP及RAGE抗体干预组动物脾脏Treg表面CTLA-4和Foxp3的表达明显降低,与烫伤组比较均有统计学差异,提示EP处理和RAGE抗体干预可抑制Treg成熟。(3)严重烫伤后1～7天动物脾脏Treg表面RAGE的表达明显增强。EP干预后脾脏Treg表面RAGE的表达于1～7天显著降低,但仍明显高于假烫伤组。RAGE抗体干预后Treg表面RAGE表达1～7天显著降低。(4)烫伤延迟复苏后动物脾脏IL-10 mRNA表达以及Treg孵育液中IL-10含量明显增高。EP或RAGE抗体干预后,烫伤动物脾脏IL-10 mRNA表达和Treg孵育液中IL-10含量在伤后1～7天明显降低。(5)严重烫伤后1～7天脾脏DC表面CD80的表达与假烫伤组比较无统计学差异,CD86表达明显增强,MHCⅡ的表达仅在伤后第1天明显增高,DC摄取葡聚糖的能力与假烫伤组比较1～7天明显减低,表明DC向成熟发展,但表型表达不完全。EP或RAGE抗体干预后烫伤动物脾脏DC表面CD80、MHCⅡ的表达1～7天均明显升高,CD86的表达仍保持较高,DC摄取葡聚糖的能力在1～7天仍较低,表明DC成熟,表型表达趋于正常。(6)烫伤延迟复苏后1～7天脾T淋巴细胞增殖反应受抑制,IL-2mRNA表达1、3天无改变,5、7天明显降低,分泌IL-2的量1～7天显著下降,IL-2RαmRNA、IL-2Rα蛋白表达1～5天明显降低。EP或RAGE抗体干预能明显减轻1～7天烫伤对脾T淋巴细胞增殖的抑制,并且IL-2 mRNA表达、IL-2的分泌和IL-2RαmRNA、IL-2Rα表达明显上升。(7)严重烫伤后动物脾T淋巴细胞分泌IL-4的量比假烫伤组明显增加,而分泌IFN-γ的量显著降低,提示严重烫伤后T淋巴细胞向Th2漂移。用EP或RAGE抗体干预后,烫伤动物脾T淋巴细胞分泌IL-4的量明显下降,分泌IFN-γ水平则明显升高,提示EP或RAGE抗体干预可使烫伤后T淋巴细胞向Th1漂移。(8)烫伤组与假烫伤组比较,大鼠脾脏T淋巴细胞NF-kB活性伤后1～7天明显降低。应用EP或RAGE抗体干预均可明显提高1～7天烫伤动物脾T淋巴细胞NF-kB活性。(9)烫伤组与假烫伤组比较,大鼠血中丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)及心肌激酶(CK-MB)伤后1～7天明显升高,血中肌酐(Cr)、尿素氮(BUN)1～5天明显升高。EP或RAGE抗体干预可明显降低烫伤后动物血清ALT、AST、Cr、BUN及CK-MB含量。(10)脾组织中HMGB1含量与Treg表面CTLA-4、Foxp3、RAGE、IL-10的表达呈正相关。脾组织中HMGB1含量与脾T淋巴细胞增值反应、表面IL-2Rα表达、孵育上清中IL-2、IFN-γ含量、NF-kB活性呈负相关,与血清sIL-2R含量、孵育上清中IL-4含量呈正相关。血清HMGB1含量与血清生化指标ALT、AST、Cr、BUN及CK-MB水平均呈正相关。
实验二:(1)与正常对照组比较,严重烧伤患者不同烧伤面积组T淋巴细胞HMGB1基因表达及血浆HMGB1水平均明显升高,Ⅰ组与Ⅲ组比较存在明显差异;严重烧伤患者伤后各时间点HMGB1基因表达及血浆HMGB1水平均明显升高,脓毒症组HMGB1基因表达及血浆HMGB1水平在伤后7～21天显著高于非脓毒症组;脓毒症患者死亡组HMGB1基因表达及血浆HMGB1水平在伤后3～21天显著高于存活组。(2)与正常对照组比较,严重烧伤患者各烧伤面积组Treg表面分子CTLA-4及标记物FOXP3表达均明显增强,各组间比较存在明显差异;严重烧伤患者伤后不同时间点CTLA-4及FOXP3表达均明显升高,脓毒症组CTLA-4及FOXP3表达在伤后3～21天显著高于非脓毒症组;脓毒症患者死亡组CTLA-4及FOXP3表达在伤后3～21天显著高于存活组。(3)与正常对照组比较,严重烧伤患者不同烧伤面积组Treg IL-10基因/蛋白表达及TGF-β1水平均显著升高,各组间比较存在明显差异;严重烧伤患者伤后各时间点Treg IL-10基因/蛋白表达及TGF-β1水平均明显升高,脓毒症组Treg IL-10基因/蛋白表达及TGF-β1水平在伤后3～21天显著高于非脓毒症组;脓毒症患者死亡组Treg IL-10基因/蛋白表达及TGF-β1水平在伤后3～21天显著高于存活组。(4)与正常对照组比较,严重烧伤患者各烧伤面积组外周血T淋巴细胞增殖反应活性及IL-2基因/蛋白表达水平均明显降低,Ⅰ组与Ⅲ组比较存在明显差异;严重烧伤患者伤后T淋巴细胞增殖反应活性及IL-2基因/蛋白表达水平均明显降低,脓毒症组T淋巴细胞增殖反应活性及IL-2基因/蛋白表达水平在伤后3～21天显著低于非脓毒症组;脓毒症患者死亡组T淋巴细胞增殖反应活性及IL-2基因/蛋白表达水平在伤后3～21天显著低于存活组。(5)与正常对照组比较,严重烧伤患者各烧伤面积组T淋巴细胞分泌IL-4水平明显升高,而分泌IFN-γ水平显著降低;与Ⅰ组比较,Ⅲ组IL-4分泌水平明显升高,而IFN-γ分泌水平显著降低;严重烧伤患者伤后各时间点T淋巴细胞分泌IL-4水平明显升高,而分泌IFN-γ水平显著降低;脓毒症组T淋巴细胞分泌IL-4/IFN-γ水平在伤后3～21天显著高/低于非脓毒症组;脓毒症患者死亡组T淋巴细胞分泌IL-4/IFN-γ水平在伤后3～21天显著高/低于存活组。(6)血浆HMGB1含量与Treg及T淋巴细胞各实验组相关免疫指标均无明显相关性。Treg各实验组免疫指标(部分或全部)与T淋巴细胞分泌IL-4水平呈正相关,与T淋巴细胞增值反应活性、IL-2及IFN-γ分泌水平呈负相关。
结论:
实验一:(1)HMGB1在严重烫伤后具有升高较晚、持续时间较长的特征。HMGB1可能参与了烫伤后脓毒症所致多器官损害的发病过程。(2)HMGB1的持续升高可刺激Treg向成熟发展,从而介导T淋巴细胞增殖反应低下,并向Th2漂移,免疫功能抑制。(3)烫伤后大量的HMGB1可能主要通过受体RAGE介导Treg表型表达、Treg功能成熟。(4)HMGB1介导的Treg功能成熟可影响烫伤后DC细胞向成熟发育,但其表型表达异常,功能出现障碍。(5)HMGB1介导的Treg功能成熟还可通过下调T淋巴细胞NF-kB活性而减少了IL-2基因表达和蛋白合成,进而影响T淋巴细胞的增殖反应和免疫功能。(6)EP可能主要通过抑制HMGB1的合成释放,改善烫伤延迟复苏动物细胞免疫功能紊乱,保护动物的主要脏器功能。
实验二:(1)HMGB1在严重烧伤后具有增高较晚、持续时间较长的特征,其含量与烧伤严重程度、并发脓毒症及患者预后相关,HMGB1参与了严重烧伤后机体免疫紊乱、发生脓毒症并致患者死亡的病理过程。(2)严重烧伤后Treg功能向成熟发展,使其免疫抑制功能充分发挥,从而可能导致机体的免疫功能紊乱。其表面分子表达及细胞因子分泌在不同烧伤面积、是否并发脓毒症及是否存活等不同组间存在显著差异,Treg可通过分泌抑制性细胞因子参与了严重烧伤后机体免疫失衡、诱发脓毒症甚至最终造成患者死亡的病理过程。(3)严重烧伤后T淋巴细胞免疫功能受到抑制,并引起T淋巴细胞向Th2漂移。烧伤程度越重、脓毒症发生率和患者死亡率越高,机体免疫抑制状态也越明显。(4)HMGB1含量与Treg及T淋巴细胞相关免疫指标均无明显相关,而Treg免疫指标与T淋巴细胞免疫功能指标间存在显著相关性(正相关或负相关)。说明Treg可能对严重烧伤后T淋巴细胞免疫功能低下,并出现Th1向Th2极化现象具有重要影响。
Objective:
Part 1. The present study was performed: (1) to investigate in vivo the changes in high mobility group box 1 protein (HMGB1) and its effect on the maturation of regulatory T cell (Treg) after severe thermal injury; (2) to identify if RAGE is the possible receptor on surface of Treg to bind HMGB1; (3) to study in vivo the effect of HMGB1 stimulated Treg on splenic dendritic cell (DC) and its potential mechanisms after severe thermal injury; (4) to investigate the effect of the HMGB1 stimulated Treg on splenic T lymphocyte-mediated immunity and its potential mechanism; and (5) to observe the protective effect of ethyl pyruvate (EP) on immune function and organ function in burn rats with delayed resuscitation.
Part 2. The clinical study was performed to investigate the systemic release and kinetics of HMGB1, and to observe the potential roles of the maturation of regulatory T cell and T lymphocyte-mediated immunity in severely burned patients. Meanwhile, the significance of changes in plasma HMGB1 levels, Treg and T lymphocyte immunity and their relationship with sepsis as well as outcome of the patients were investigated in patients after major burns.
Methods:
1. A widely used technique for induction of full-thickness scald injury was used in the present study. For rats with burn injury, and the dorsal and lateral surfaces of animals were shaved under anesthesia. After being secured in a protective template with an opening corresponding to 30% of the total body surface area, and the exposed skin of the back was immersed in 99°C water for 12 s. Sham-injured rats were subjected to the same procedure except the temperature of the bath was of room temperature. 40 ml/kg lactated Ringer's solution was administered i.p 6 hours after the injury for delayed resuscitation, followed by i.p injection of 4 ml at 12,24,36 and 48 h after burn injury, respectively.
One hundred and thirty six male Wistar rats were randomly divided into five groups as follows: normal control group (8 rats), sham burn group (32 rats), burn group (32 rats), burn with ethyl pyruvate (EP) treatment group (32 rats), and burn with anti-RAGE (advanced glycation end-products) antibody treatment group (32 rats), and the later four groups were further divided into four subgroups of 8 rats each, which were sacrificed on postburn days (PBD) 1, 3, 5 and 7 respectively. EP was added to lactated Ringer's solution (EP 28 mM) in EP treatment group.. Anti-RAGE antibody lmg/kg was given via dorsal penile vein at 6 h and 24 h respectively after burn injury in anti-RAGE antibody treatment group. Animals of all groups were sacrificed at designated time points, and blood as well as spleen samples were harvested aseptically to determine organ damage related variables and levels of various cytokines. Spleen was divided into two portions as following: one portion was used to detect gene and protein expression levels of HMGB1 and IL-10, and the other portion was used to procure Treg and DC by MACS microbeads and T cell by using column of nylon wool. Cells were cultured, and phenotypes were analyzed by flow cytometry and the contents of cytokines released into supernatants were also determined. All cytokines in blood, supernatant and tissue, as well as activated NF-kB of T cell were determined by ELISA kits for rats. Gene expression was measured by real-time quantitative PCR taken GAPDH as the internal standard.
2. One hundred and six patients with total burn surface area larger than 30% were included in the present study, and they were divided into three burn size groups: 30%-49% total body surface area (TBSA) burn (group I, n=41), 50%-69% TBSA burn (group II, n=34), and >70% TBSA burn (groupIII, n=31). According to wheather there was development of sepsis or not, patients were divided into sepsis group (n=59) and none-sepsis group (n=47); then the patients with sepsis were further divided into non-survival group (n=17) and survival group (n=42). Healthy volunteers served as normal controls (n=25). The periphery blood samples were collected on PBD 1,3,7,14, and 21. The blood samples were divided into two portions as following: one portion was used to procure T cell and Treg by MACS microbeads, and the other portion was used to detect levels of HMGB1 in serum. Cells were cultured, and phenotypes were analyzed by flow cytometry and the contents of cytokines released into supernatants were also determined. All cytokines in blood, supernatant and tissue were determined by ELISA kits for human. Gene expression was assessed by real-time quantitative PCR taken GAPDH as the internal standard.
Results:
Part 1. (1) The significantly elevated expression levels of splenic HMGB1 gene and protein and levels of serum HMGB1 were detected on PBD 1-7, and the elevation was significantly inhibited by the treatment of EP, but not by anti-RAGE antibody. (2) The expression levels of CTLA-4 on PBD 1-5 and levels of Foxp3 in Treg on PBD 1 -7 were significantly enhanced in burn rats in comparison to Treg from sham-injured rats. Treatment with EP or anti-RAGE antibody to inhibit HMGB1 could significantly decrease the expression levels of CTLA-4 and Foxp3 of Treg. (3) The expression of RAGE on the surface of Treg from burned rats was found to be markedly elevated on PBD 1-7 compared with sham-injured rats, and it was not influenced by treatment of EP, but was blocked by treatment of anti-RAGE antibody. (4) The expression levels of splenic IL-10 mRNA and levels of IL-10 produced by Treg in supernatant were found to be significantly enhanced on PBD 1-7, and they were significantly inhibited by treatment of EP, but not by anti-RAGE antibody. (5) It was noted that DC expressed similar levels of CD80, strongly enhanced levels of CD86 and slightly enhanced levels of MHC class II on PBD 1-7 compared with DC from sham-injured rats. The capacity of DC to engulf dextran was decreased markedly after burn injury. Treatment with EP or anti-RAGE antibody to inhibit HMGB1 could significantly raise the expression levels of CD80, MHC class II of DC, but not the capacity of DC to engulf dextran. (6) The T cell proliferative activity in response to ConA in burn-injured rats was significantly suppressed on PBD 1-7 as compared with sham-injured rats, and gene/protein expressions of IL-2 and expressions of IL-2Ra of T cells in burn-injured rats were simultaneously suppressed after burn injury to different extent. EP or anti-RAGE antibody treatment could restore T cell proliferative activity response to Con A, gene/protein expressions of IL-2, and expression of IL-2Ra after burn injury. (7) Levels of IL-4 produced by T cell as a response to Con A increased markedly after burn injury, whereas levels of IFN-γlowered markedly, indicating that Th cells might shift into Th2 cells. Intervention with EP or anti-RAGE antibody could significantly inhibit the release of IL-4 and enhance the production of IFN-γby T cell response to Con A after thermal injury, indicating that EP or anti-RAGE antibody intervention might influence the polarization of T cells in animals subjected to thermal injury and induced Th cells to drift to Th1 cells. (8) The NF-kB activation of splenic T cell was downregulated significantly on PBD 1-7. Treatment with EP or anti-RAGE antibody could completely restored the NF-kB activity of splenic T cell after burn injury. (9) The serum ALT, AST, Cr, BUN and CK-MB levels were significantly elevated after burns, and treatment with EP or anti-RAGE antibody could inhibit these increase to different extent. (10) The levels of splenic HMGB1 were positively correlated with the levels of CTLA-4, Foxp3, RAGE, IL-10, sIL-2R, IL-4, and were negatively correlated with the levels of IL-2Rα, IL-2, IFN-γand T cell proliferative activity. Significant positive correlations were also found between the levels of serum HMGB1 and levels of serum ALT, AST, Cr, BUN and CK-MB.
Part 2. (1) The gene/protein levels of blood HMGB1 were significantly elevated on PBD 1-21 in patients with various burn sizes compared with normal controls, and there were obvious differences between group I and group III. The blood HMGB1 mRNA and plasma HMGB1 levels were significantly higher in septic patients than those without sepsis on PBD 7-21. Among septic patients, the HMGB1 levels in the survival group were markedly lower than those with fatal outcome on PBD 3-21. (2) Increased expressions of CTLA-4 and Foxp3 on the surface of Treg from burned patients were found on PBD 1-21 compared with normal control group, and there were obvious differences among patients with various burn sizes. The expressions of CTLA-4 and Foxp3 were significantly higher in patients with serious burns at all time points, and they were even higher septic patients than those without sepsis on PBD 3-21. Among septic patients, the expressions of CTLA-4 and Foxp3 in the survival group were obviously lower than those with fatal outcome on PBD 3-21. (3) Elevated gene/protein expression of IL-10 and TGF-β1 in Treg from burned patients were detected on PBD 1-21 in comparison to normal controls, and there was obvious difference among patients with different extent of burn injury. The gene/protein expression of IL-10 and TGF-β1 in Treg were significantly higher in septic patients than those without sepsis on PBD 3-21. Among septic patients, expression levels of IL-10 and TGF-β1 in the survival group were obviously lower than those with fatal outcome on PBD 3-21. (4) The T cell proliferative activity in response to PHA and gene/protein expression of IL-2 of T cell in burn-injured patients were significantly suppressed on PBD 1-21 compared with normal control group, and there were obvious differences between group I and group III. The T cell proliferative activity in response to PHA and gene/protein expression of IL-2 of T cell were significantly lower in septic patients than those without sepsis on PBD 3-21. Among septic patients, the T cell proliferative activity in response to PHA and gene/protein expression of IL-2 in the survival group were markedly higher than those with fatal outcome on PBD 3-21. (5) Levels of IL-4 produced by T cell response to PHA increased markedly after burn injury, whereas levels of IFN-γdecreased markedly as compared with normal control group, and there were obvious differences between group I and groupIII, indicating that Th cells might have shifted to Th2 cells. The same results were found in non-septic patients and the survival group as compared with those with sepsis and the non-survival group on PBD 3-21. (6) No significant correlations were found between serum HMGB1 levels and the immune indexes of Treg and T cells. In addition, the immune indexes of Treg were positively correlated with levels of IL-4 produced by T cells, but were negatively correlated with levels of IL-2, IFN-γand T cell proliferative activity.
Conclusion:
Part 1. Serum and splenic HMGB1 were markedly up-regulated for a prolonged period but delayed after severe thermal injury. HMGB1 might play an important role in the development of excessive inflammatory response and subsequent multiple organ dysfunction syndrome. The excessive release of HMGB1 might stimulate splenic Treg to mature (which might mainly induced by binding RAGE on the surface of Treg), and further stimulate splenic DC to mature abnormally, thereby inducing suppression of proliferative activity of T lymphocytes and drifting of Th1 to Th2 after burn injury. NF-κB signaling might be involved in the normal Treg mediating suppression of T lymphocyte associated with excessive release of HMGB1. EP has a therapeutic potential for suppressing HMGB1-induced immune dysfunction and ameliorating multiple organ injury.
Part 2. (1) Plasma HMGB1 was markedly elevated for a delayed but prolonged period after severe thermal injury in patients. Extensive burn injury could result in increased serum HMGB1 levels, and this phemenon was found to be associated with the development of sepsis and fatal outcome of burned patients.. (2) Severe burn injury per se could lead to activation and maturation of Treg cells, thus invoking its immunodepressive activity to full extent, resulting in immunosuppression. Similar to HMGB1, different degree of elevated levels of cytokines produced by Treg and activation markers on Treg surface might also take a role involved in the pathogenesis of sepsis and mortality in burn patients. (3) Suppression of T lymphocyte immune function and drifting of Th1 to Th2 were induced by severe burn injury. The immunosuppressive state of T lymphocytes was related to the extent of burn injury, development of sepsis and poor outcome in burned patients. (4) Plasma HMGB1 levels showed no significant correlations with the immune indexes of Treg and T lympholeukocyte, but the immune indexes of Treg were significantly correlated with T lymphocyte immune function, suggesting that Treg might have a potential effect on suppression of the proliferative activity, cytokine release of T cells and drifting Th1 to Th2 following major burns.
实验一:(1)采用严重烫伤延迟复苏动物模型,明确高迁移率族蛋白B1(HMGB1)的变化及其与调节性T细胞(Treg)免疫功能的关系。(2)通过严重烫伤延迟复苏动物模型,阐明HMGB1对Treg分化成熟的受体作用机制。(3)探讨严重烫伤延迟复苏后HMGB1介导的Treg对树突状细胞(DC)免疫功能的影响及调节作用。(4)探讨严重烫伤延迟复苏后HMGB1介导的Treg对T淋巴细胞免疫功能的影响及调节途径。(5)观察丙酮酸乙酯(EP)对重度烫伤延迟复苏动物免疫功能及脏器损害的可能保护效应。
实验二:(1)对临床严重烧伤患者进行动态监测,明确严重烧伤后病人外周血HMGB1、Treg分化成熟以及T淋巴细胞免疫功能的变化规律,探讨HMGB1、Treg及T淋巴细胞免疫功能改变在患者烧伤后发生脓毒症及不良预后中所起的作用及临床意义。(2)探讨严重烧伤后HMGB1、Treg及T淋巴细胞免疫指标间的相互关系及可能的作用机制。
方法:
1.采用重度烫伤延迟复苏动物模型,即雄性清洁级Wistar大鼠,浸于沸水中造成30%体表面积Ⅲ度烫伤。伤后延迟6小时抗休克复苏,在伤后6小时从腹腔给予林格液(40 ml/kg)抗休克治疗,此外在烫伤后12、24、36、48小时分别给予4 ml/次林格液腹腔内注射。实验分组:136只大鼠随机分为5组,(1)正常对照组(n=8):麻醉后活杀;(2)假烫伤组(n=32);(3)烫伤组(n=32);(4)EP治疗组(n=32):EP加入林格液中随补液给药(EP为28 mM);(5)晚期糖基化终末产物受体(RAGE)抗体(1 mg/ml)治疗组(n=32):烫伤后6、24小时从阴茎背静脉给予RAGE抗体(1 mg/kg)治疗。以上后4组实验动物再分为4个亚组,分别于烫伤后1、3、5、7天处死动物,无菌留取血和脾脏标本。分离血清,-20℃贮存。脾脏一部分无菌、无酶液氮冻存,另一部分用于提取Treg、DC和T淋巴细胞。Treg、DC的分离纯化采用MiniMACS免疫磁性分离系统进行。利用B细胞和单核细胞具有易粘附于尼龙纤维表面的特性,分离T淋巴细胞。采用流式细胞术检测细胞免疫表型和细胞表面受体,MTT法检测脾T淋巴细胞增殖,酶联免疫吸附试验(ELISA)检测血清、细胞孵育上清及组织匀浆液的细胞因子和T淋巴细胞活化的核因子(NF)-kB。采用荧光定量PCR检测目的基因表达水平。
2.对106例烧伤总面积大于或等于30%的患者进行动态观察,对所采集临床病历资料进行分析。实验分组:(1)按烧伤总面积将患者分为三组:Ⅰ组41例(烧伤总面积30%～49%),Ⅱ组34例(烧伤总面积50%～69%),Ⅲ组31例(烧伤总面积70%～99%);(2)根据烧伤脓毒症的诊断标准,将患者进一步分为脓毒症组(59例)与非脓毒症组(47例);(3)根据脓毒症患者的预后情况,将其分为死亡组(17例)与存活组(42例)。同时设正常对照组(25位健康献血员)。分别于烧伤后1、3、7、14、21天采集患者外周静脉血,分离外周血T淋巴细胞,采用MiniMACS免疫磁性分离系统对外周血Treg进行分离纯化,同时分离血清,-20℃贮存。采用流式细胞术检测细胞免疫表型,MTT法检测T淋巴细胞增殖活性,ELISA检测血清HMGB1及细胞孵育上清的细胞因子水平,采用荧光定量PCR检测目的基因表达水平。
结果:
实验一:(1)烫伤延迟复苏导致动物脾脏HMGB1基因、蛋白表达和血清HMGB1水平在伤后1～7天显著升高,脾组织中HMGB1含量第1天达峰值,血清HMGB1水平于第3天达峰值。EP干预后烫伤动物脾脏HMGB1基因、蛋白表达和血清HMGB1水平1～7天均明显降低。RAGE抗体干预对烫伤动物脾脏和血清HMGB1无明显影响。(2)严重烫伤后脾脏Treg表面分子CTLA-4的表达1～5天明显增强,表面标记物Foxp3的表达1～7天明显增高,与假烫伤动物比较差异显著,表明严重烫伤可促使Treg成熟。EP及RAGE抗体干预组动物脾脏Treg表面CTLA-4和Foxp3的表达明显降低,与烫伤组比较均有统计学差异,提示EP处理和RAGE抗体干预可抑制Treg成熟。(3)严重烫伤后1～7天动物脾脏Treg表面RAGE的表达明显增强。EP干预后脾脏Treg表面RAGE的表达于1～7天显著降低,但仍明显高于假烫伤组。RAGE抗体干预后Treg表面RAGE表达1～7天显著降低。(4)烫伤延迟复苏后动物脾脏IL-10 mRNA表达以及Treg孵育液中IL-10含量明显增高。EP或RAGE抗体干预后,烫伤动物脾脏IL-10 mRNA表达和Treg孵育液中IL-10含量在伤后1～7天明显降低。(5)严重烫伤后1～7天脾脏DC表面CD80的表达与假烫伤组比较无统计学差异,CD86表达明显增强,MHCⅡ的表达仅在伤后第1天明显增高,DC摄取葡聚糖的能力与假烫伤组比较1～7天明显减低,表明DC向成熟发展,但表型表达不完全。EP或RAGE抗体干预后烫伤动物脾脏DC表面CD80、MHCⅡ的表达1～7天均明显升高,CD86的表达仍保持较高,DC摄取葡聚糖的能力在1～7天仍较低,表明DC成熟,表型表达趋于正常。(6)烫伤延迟复苏后1～7天脾T淋巴细胞增殖反应受抑制,IL-2mRNA表达1、3天无改变,5、7天明显降低,分泌IL-2的量1～7天显著下降,IL-2RαmRNA、IL-2Rα蛋白表达1～5天明显降低。EP或RAGE抗体干预能明显减轻1～7天烫伤对脾T淋巴细胞增殖的抑制,并且IL-2 mRNA表达、IL-2的分泌和IL-2RαmRNA、IL-2Rα表达明显上升。(7)严重烫伤后动物脾T淋巴细胞分泌IL-4的量比假烫伤组明显增加,而分泌IFN-γ的量显著降低,提示严重烫伤后T淋巴细胞向Th2漂移。用EP或RAGE抗体干预后,烫伤动物脾T淋巴细胞分泌IL-4的量明显下降,分泌IFN-γ水平则明显升高,提示EP或RAGE抗体干预可使烫伤后T淋巴细胞向Th1漂移。(8)烫伤组与假烫伤组比较,大鼠脾脏T淋巴细胞NF-kB活性伤后1～7天明显降低。应用EP或RAGE抗体干预均可明显提高1～7天烫伤动物脾T淋巴细胞NF-kB活性。(9)烫伤组与假烫伤组比较,大鼠血中丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)及心肌激酶(CK-MB)伤后1～7天明显升高,血中肌酐(Cr)、尿素氮(BUN)1～5天明显升高。EP或RAGE抗体干预可明显降低烫伤后动物血清ALT、AST、Cr、BUN及CK-MB含量。(10)脾组织中HMGB1含量与Treg表面CTLA-4、Foxp3、RAGE、IL-10的表达呈正相关。脾组织中HMGB1含量与脾T淋巴细胞增值反应、表面IL-2Rα表达、孵育上清中IL-2、IFN-γ含量、NF-kB活性呈负相关,与血清sIL-2R含量、孵育上清中IL-4含量呈正相关。血清HMGB1含量与血清生化指标ALT、AST、Cr、BUN及CK-MB水平均呈正相关。
实验二:(1)与正常对照组比较,严重烧伤患者不同烧伤面积组T淋巴细胞HMGB1基因表达及血浆HMGB1水平均明显升高,Ⅰ组与Ⅲ组比较存在明显差异;严重烧伤患者伤后各时间点HMGB1基因表达及血浆HMGB1水平均明显升高,脓毒症组HMGB1基因表达及血浆HMGB1水平在伤后7～21天显著高于非脓毒症组;脓毒症患者死亡组HMGB1基因表达及血浆HMGB1水平在伤后3～21天显著高于存活组。(2)与正常对照组比较,严重烧伤患者各烧伤面积组Treg表面分子CTLA-4及标记物FOXP3表达均明显增强,各组间比较存在明显差异;严重烧伤患者伤后不同时间点CTLA-4及FOXP3表达均明显升高,脓毒症组CTLA-4及FOXP3表达在伤后3～21天显著高于非脓毒症组;脓毒症患者死亡组CTLA-4及FOXP3表达在伤后3～21天显著高于存活组。(3)与正常对照组比较,严重烧伤患者不同烧伤面积组Treg IL-10基因/蛋白表达及TGF-β1水平均显著升高,各组间比较存在明显差异;严重烧伤患者伤后各时间点Treg IL-10基因/蛋白表达及TGF-β1水平均明显升高,脓毒症组Treg IL-10基因/蛋白表达及TGF-β1水平在伤后3～21天显著高于非脓毒症组;脓毒症患者死亡组Treg IL-10基因/蛋白表达及TGF-β1水平在伤后3～21天显著高于存活组。(4)与正常对照组比较,严重烧伤患者各烧伤面积组外周血T淋巴细胞增殖反应活性及IL-2基因/蛋白表达水平均明显降低,Ⅰ组与Ⅲ组比较存在明显差异;严重烧伤患者伤后T淋巴细胞增殖反应活性及IL-2基因/蛋白表达水平均明显降低,脓毒症组T淋巴细胞增殖反应活性及IL-2基因/蛋白表达水平在伤后3～21天显著低于非脓毒症组;脓毒症患者死亡组T淋巴细胞增殖反应活性及IL-2基因/蛋白表达水平在伤后3～21天显著低于存活组。(5)与正常对照组比较,严重烧伤患者各烧伤面积组T淋巴细胞分泌IL-4水平明显升高,而分泌IFN-γ水平显著降低;与Ⅰ组比较,Ⅲ组IL-4分泌水平明显升高,而IFN-γ分泌水平显著降低;严重烧伤患者伤后各时间点T淋巴细胞分泌IL-4水平明显升高,而分泌IFN-γ水平显著降低;脓毒症组T淋巴细胞分泌IL-4/IFN-γ水平在伤后3～21天显著高/低于非脓毒症组;脓毒症患者死亡组T淋巴细胞分泌IL-4/IFN-γ水平在伤后3～21天显著高/低于存活组。(6)血浆HMGB1含量与Treg及T淋巴细胞各实验组相关免疫指标均无明显相关性。Treg各实验组免疫指标(部分或全部)与T淋巴细胞分泌IL-4水平呈正相关,与T淋巴细胞增值反应活性、IL-2及IFN-γ分泌水平呈负相关。
结论:
实验一:(1)HMGB1在严重烫伤后具有升高较晚、持续时间较长的特征。HMGB1可能参与了烫伤后脓毒症所致多器官损害的发病过程。(2)HMGB1的持续升高可刺激Treg向成熟发展,从而介导T淋巴细胞增殖反应低下,并向Th2漂移,免疫功能抑制。(3)烫伤后大量的HMGB1可能主要通过受体RAGE介导Treg表型表达、Treg功能成熟。(4)HMGB1介导的Treg功能成熟可影响烫伤后DC细胞向成熟发育,但其表型表达异常,功能出现障碍。(5)HMGB1介导的Treg功能成熟还可通过下调T淋巴细胞NF-kB活性而减少了IL-2基因表达和蛋白合成,进而影响T淋巴细胞的增殖反应和免疫功能。(6)EP可能主要通过抑制HMGB1的合成释放,改善烫伤延迟复苏动物细胞免疫功能紊乱,保护动物的主要脏器功能。
实验二:(1)HMGB1在严重烧伤后具有增高较晚、持续时间较长的特征,其含量与烧伤严重程度、并发脓毒症及患者预后相关,HMGB1参与了严重烧伤后机体免疫紊乱、发生脓毒症并致患者死亡的病理过程。(2)严重烧伤后Treg功能向成熟发展,使其免疫抑制功能充分发挥,从而可能导致机体的免疫功能紊乱。其表面分子表达及细胞因子分泌在不同烧伤面积、是否并发脓毒症及是否存活等不同组间存在显著差异,Treg可通过分泌抑制性细胞因子参与了严重烧伤后机体免疫失衡、诱发脓毒症甚至最终造成患者死亡的病理过程。(3)严重烧伤后T淋巴细胞免疫功能受到抑制,并引起T淋巴细胞向Th2漂移。烧伤程度越重、脓毒症发生率和患者死亡率越高,机体免疫抑制状态也越明显。(4)HMGB1含量与Treg及T淋巴细胞相关免疫指标均无明显相关,而Treg免疫指标与T淋巴细胞免疫功能指标间存在显著相关性(正相关或负相关)。说明Treg可能对严重烧伤后T淋巴细胞免疫功能低下,并出现Th1向Th2极化现象具有重要影响。
Objective:
Part 1. The present study was performed: (1) to investigate in vivo the changes in high mobility group box 1 protein (HMGB1) and its effect on the maturation of regulatory T cell (Treg) after severe thermal injury; (2) to identify if RAGE is the possible receptor on surface of Treg to bind HMGB1; (3) to study in vivo the effect of HMGB1 stimulated Treg on splenic dendritic cell (DC) and its potential mechanisms after severe thermal injury; (4) to investigate the effect of the HMGB1 stimulated Treg on splenic T lymphocyte-mediated immunity and its potential mechanism; and (5) to observe the protective effect of ethyl pyruvate (EP) on immune function and organ function in burn rats with delayed resuscitation.
Part 2. The clinical study was performed to investigate the systemic release and kinetics of HMGB1, and to observe the potential roles of the maturation of regulatory T cell and T lymphocyte-mediated immunity in severely burned patients. Meanwhile, the significance of changes in plasma HMGB1 levels, Treg and T lymphocyte immunity and their relationship with sepsis as well as outcome of the patients were investigated in patients after major burns.
Methods:
1. A widely used technique for induction of full-thickness scald injury was used in the present study. For rats with burn injury, and the dorsal and lateral surfaces of animals were shaved under anesthesia. After being secured in a protective template with an opening corresponding to 30% of the total body surface area, and the exposed skin of the back was immersed in 99°C water for 12 s. Sham-injured rats were subjected to the same procedure except the temperature of the bath was of room temperature. 40 ml/kg lactated Ringer's solution was administered i.p 6 hours after the injury for delayed resuscitation, followed by i.p injection of 4 ml at 12,24,36 and 48 h after burn injury, respectively.
One hundred and thirty six male Wistar rats were randomly divided into five groups as follows: normal control group (8 rats), sham burn group (32 rats), burn group (32 rats), burn with ethyl pyruvate (EP) treatment group (32 rats), and burn with anti-RAGE (advanced glycation end-products) antibody treatment group (32 rats), and the later four groups were further divided into four subgroups of 8 rats each, which were sacrificed on postburn days (PBD) 1, 3, 5 and 7 respectively. EP was added to lactated Ringer's solution (EP 28 mM) in EP treatment group.. Anti-RAGE antibody lmg/kg was given via dorsal penile vein at 6 h and 24 h respectively after burn injury in anti-RAGE antibody treatment group. Animals of all groups were sacrificed at designated time points, and blood as well as spleen samples were harvested aseptically to determine organ damage related variables and levels of various cytokines. Spleen was divided into two portions as following: one portion was used to detect gene and protein expression levels of HMGB1 and IL-10, and the other portion was used to procure Treg and DC by MACS microbeads and T cell by using column of nylon wool. Cells were cultured, and phenotypes were analyzed by flow cytometry and the contents of cytokines released into supernatants were also determined. All cytokines in blood, supernatant and tissue, as well as activated NF-kB of T cell were determined by ELISA kits for rats. Gene expression was measured by real-time quantitative PCR taken GAPDH as the internal standard.
2. One hundred and six patients with total burn surface area larger than 30% were included in the present study, and they were divided into three burn size groups: 30%-49% total body surface area (TBSA) burn (group I, n=41), 50%-69% TBSA burn (group II, n=34), and >70% TBSA burn (groupIII, n=31). According to wheather there was development of sepsis or not, patients were divided into sepsis group (n=59) and none-sepsis group (n=47); then the patients with sepsis were further divided into non-survival group (n=17) and survival group (n=42). Healthy volunteers served as normal controls (n=25). The periphery blood samples were collected on PBD 1,3,7,14, and 21. The blood samples were divided into two portions as following: one portion was used to procure T cell and Treg by MACS microbeads, and the other portion was used to detect levels of HMGB1 in serum. Cells were cultured, and phenotypes were analyzed by flow cytometry and the contents of cytokines released into supernatants were also determined. All cytokines in blood, supernatant and tissue were determined by ELISA kits for human. Gene expression was assessed by real-time quantitative PCR taken GAPDH as the internal standard.
Results:
Part 1. (1) The significantly elevated expression levels of splenic HMGB1 gene and protein and levels of serum HMGB1 were detected on PBD 1-7, and the elevation was significantly inhibited by the treatment of EP, but not by anti-RAGE antibody. (2) The expression levels of CTLA-4 on PBD 1-5 and levels of Foxp3 in Treg on PBD 1 -7 were significantly enhanced in burn rats in comparison to Treg from sham-injured rats. Treatment with EP or anti-RAGE antibody to inhibit HMGB1 could significantly decrease the expression levels of CTLA-4 and Foxp3 of Treg. (3) The expression of RAGE on the surface of Treg from burned rats was found to be markedly elevated on PBD 1-7 compared with sham-injured rats, and it was not influenced by treatment of EP, but was blocked by treatment of anti-RAGE antibody. (4) The expression levels of splenic IL-10 mRNA and levels of IL-10 produced by Treg in supernatant were found to be significantly enhanced on PBD 1-7, and they were significantly inhibited by treatment of EP, but not by anti-RAGE antibody. (5) It was noted that DC expressed similar levels of CD80, strongly enhanced levels of CD86 and slightly enhanced levels of MHC class II on PBD 1-7 compared with DC from sham-injured rats. The capacity of DC to engulf dextran was decreased markedly after burn injury. Treatment with EP or anti-RAGE antibody to inhibit HMGB1 could significantly raise the expression levels of CD80, MHC class II of DC, but not the capacity of DC to engulf dextran. (6) The T cell proliferative activity in response to ConA in burn-injured rats was significantly suppressed on PBD 1-7 as compared with sham-injured rats, and gene/protein expressions of IL-2 and expressions of IL-2Ra of T cells in burn-injured rats were simultaneously suppressed after burn injury to different extent. EP or anti-RAGE antibody treatment could restore T cell proliferative activity response to Con A, gene/protein expressions of IL-2, and expression of IL-2Ra after burn injury. (7) Levels of IL-4 produced by T cell as a response to Con A increased markedly after burn injury, whereas levels of IFN-γlowered markedly, indicating that Th cells might shift into Th2 cells. Intervention with EP or anti-RAGE antibody could significantly inhibit the release of IL-4 and enhance the production of IFN-γby T cell response to Con A after thermal injury, indicating that EP or anti-RAGE antibody intervention might influence the polarization of T cells in animals subjected to thermal injury and induced Th cells to drift to Th1 cells. (8) The NF-kB activation of splenic T cell was downregulated significantly on PBD 1-7. Treatment with EP or anti-RAGE antibody could completely restored the NF-kB activity of splenic T cell after burn injury. (9) The serum ALT, AST, Cr, BUN and CK-MB levels were significantly elevated after burns, and treatment with EP or anti-RAGE antibody could inhibit these increase to different extent. (10) The levels of splenic HMGB1 were positively correlated with the levels of CTLA-4, Foxp3, RAGE, IL-10, sIL-2R, IL-4, and were negatively correlated with the levels of IL-2Rα, IL-2, IFN-γand T cell proliferative activity. Significant positive correlations were also found between the levels of serum HMGB1 and levels of serum ALT, AST, Cr, BUN and CK-MB.
Part 2. (1) The gene/protein levels of blood HMGB1 were significantly elevated on PBD 1-21 in patients with various burn sizes compared with normal controls, and there were obvious differences between group I and group III. The blood HMGB1 mRNA and plasma HMGB1 levels were significantly higher in septic patients than those without sepsis on PBD 7-21. Among septic patients, the HMGB1 levels in the survival group were markedly lower than those with fatal outcome on PBD 3-21. (2) Increased expressions of CTLA-4 and Foxp3 on the surface of Treg from burned patients were found on PBD 1-21 compared with normal control group, and there were obvious differences among patients with various burn sizes. The expressions of CTLA-4 and Foxp3 were significantly higher in patients with serious burns at all time points, and they were even higher septic patients than those without sepsis on PBD 3-21. Among septic patients, the expressions of CTLA-4 and Foxp3 in the survival group were obviously lower than those with fatal outcome on PBD 3-21. (3) Elevated gene/protein expression of IL-10 and TGF-β1 in Treg from burned patients were detected on PBD 1-21 in comparison to normal controls, and there was obvious difference among patients with different extent of burn injury. The gene/protein expression of IL-10 and TGF-β1 in Treg were significantly higher in septic patients than those without sepsis on PBD 3-21. Among septic patients, expression levels of IL-10 and TGF-β1 in the survival group were obviously lower than those with fatal outcome on PBD 3-21. (4) The T cell proliferative activity in response to PHA and gene/protein expression of IL-2 of T cell in burn-injured patients were significantly suppressed on PBD 1-21 compared with normal control group, and there were obvious differences between group I and group III. The T cell proliferative activity in response to PHA and gene/protein expression of IL-2 of T cell were significantly lower in septic patients than those without sepsis on PBD 3-21. Among septic patients, the T cell proliferative activity in response to PHA and gene/protein expression of IL-2 in the survival group were markedly higher than those with fatal outcome on PBD 3-21. (5) Levels of IL-4 produced by T cell response to PHA increased markedly after burn injury, whereas levels of IFN-γdecreased markedly as compared with normal control group, and there were obvious differences between group I and groupIII, indicating that Th cells might have shifted to Th2 cells. The same results were found in non-septic patients and the survival group as compared with those with sepsis and the non-survival group on PBD 3-21. (6) No significant correlations were found between serum HMGB1 levels and the immune indexes of Treg and T cells. In addition, the immune indexes of Treg were positively correlated with levels of IL-4 produced by T cells, but were negatively correlated with levels of IL-2, IFN-γand T cell proliferative activity.
Conclusion:
Part 1. Serum and splenic HMGB1 were markedly up-regulated for a prolonged period but delayed after severe thermal injury. HMGB1 might play an important role in the development of excessive inflammatory response and subsequent multiple organ dysfunction syndrome. The excessive release of HMGB1 might stimulate splenic Treg to mature (which might mainly induced by binding RAGE on the surface of Treg), and further stimulate splenic DC to mature abnormally, thereby inducing suppression of proliferative activity of T lymphocytes and drifting of Th1 to Th2 after burn injury. NF-κB signaling might be involved in the normal Treg mediating suppression of T lymphocyte associated with excessive release of HMGB1. EP has a therapeutic potential for suppressing HMGB1-induced immune dysfunction and ameliorating multiple organ injury.
Part 2. (1) Plasma HMGB1 was markedly elevated for a delayed but prolonged period after severe thermal injury in patients. Extensive burn injury could result in increased serum HMGB1 levels, and this phemenon was found to be associated with the development of sepsis and fatal outcome of burned patients.. (2) Severe burn injury per se could lead to activation and maturation of Treg cells, thus invoking its immunodepressive activity to full extent, resulting in immunosuppression. Similar to HMGB1, different degree of elevated levels of cytokines produced by Treg and activation markers on Treg surface might also take a role involved in the pathogenesis of sepsis and mortality in burn patients. (3) Suppression of T lymphocyte immune function and drifting of Th1 to Th2 were induced by severe burn injury. The immunosuppressive state of T lymphocytes was related to the extent of burn injury, development of sepsis and poor outcome in burned patients. (4) Plasma HMGB1 levels showed no significant correlations with the immune indexes of Treg and T lympholeukocyte, but the immune indexes of Treg were significantly correlated with T lymphocyte immune function, suggesting that Treg might have a potential effect on suppression of the proliferative activity, cytokine release of T cells and drifting Th1 to Th2 following major burns.
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