Li Juncong, Hu Chao, Yao Yongming, Yang Hongming
Burns Institute, the First Hospital Affiliated to the PLA General Hospital, Beijing 100048, China.
Zhonghua Shao Shang Za Zhi. 2016 May;32(5):266-71. doi: 10.3760/cma.j.issn.1009-2587.2016.05.003.
To observe the effects of ulinastatin on immune function of splenic CD4(+) T lymphocytes and CD4(+) CD25(+) regulatory T lymphocytes (Tregs) and content of high mobility group box 1 (HMGB1) in peripheral blood of severely burned rats, and to analyze the possible mechanisms.
Ninety-six male SD rats were divided into sham injury group, burn group, and ulinastatin group according to the random number table, with 32 rats in each group. Rats in sham injury group were sham injured on the back by immersing in 37 ℃ warm water for 12 s. Rats in burn group and ulinastatin group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back by immersing in 94 ℃ hot water for 12 s. Immediately after injury, rats in each group were intraperitoneally injected with saline (40 mL/kg), meanwhile rats in ulinastatin group were intraperitoneally injected with ulinastatin (4×10(4) U/kg), once per 12 h, till post injury hour 72. Eight rats of each group were respectively selected on post injury day (PID) 1, 3, 5, and 7 to collect abdominal aortic blood samples. Serum content of HMGB1 was detected by enzyme-linked immunosorbent assay (ELISA). And then, rats of the 3 groups were sacrificed immediately to collect spleens and separate CD4(+) CD25(+) Tregs and CD4(+) T lymphocytes. Flow cytometer was used to detect positive expression rates of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and forkhead-winged helix transcription factor p3 (Foxp3) in CD4(+) CD25(+) Tregs. Content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs, and content of interleukin 2 (IL-2), IL-4, and γ interferon (IFN-γ) in culture supernatant of CD4(+) T lymphocytes was detected by ELISA. The proliferative activity of CD4(+) T lymphocytes was determined by microplate reader. The sample number of above-mentioned experiments was 8 at each time point in each group. Data were processed with analysis of variance of factorial design and LSD test.
(1) Compared with that in sham injury group, serum content of HMGB1 of rats in burn group was significantly increased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, serum content of HMGB1 of rats in ulinastatin group was significantly decreased from PID 1 to 7 (with P values below 0.01). (2) Compared with those in sham injury group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in burn group were significantly increased from PID 1 to 7 (with P values below 0.01), peaking on PID 3 [(65±10)%, (76±10)%, and (28.2±4.4) pg/mL respectively]. These 3 indexes of rats in sham injury group on PID 3 were (45±7)%, (46±7)%, and (11.2±2.3) pg/mL respectively. Compared with those in burn group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in ulinastatin group were significantly decreased from PID 1 to 7 (P<0.05 or P<0.01), reaching the nadir on PID 7 [(43±6)%], PID 1 [(50±8)%], and PID 7 [(12.4±3.4) pg/mL] respectively. These 3 indexes of rats in burn group on PID 7, 1, and 7 were (58±8)%, (71±9)%, and (19.7±2.8) pg/mL respectively. (3) Compared with those in sham injury group, the content of IL-2 and IFN-γ in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased in burn group from PID 1 to 7, with P values below 0.01. Compared with that in burn group, the content of IL-2 and IFN-γ in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased in ulinastatin group from PID 1 to 7, P<0.05 or P<0.01. (4) Compared with that in sham injury group, the proliferative activity of CD4(+) T lymphocytes of rats in burn group was significantly decreased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, the proliferative activity of CD4(+) T lymphocytes of rats in ulinastatin group was significantly increased from PID 1 to 7 (with P values below 0.01).
Ulinastatin can weaken the immunosuppressive function mediated by splenic CD4(+) CD25(+) Tregs in severely burned rats, and improve proliferative function and secretory function of splenic CD4(+) T lymphocytes, which may be attributed to the inhibiting effect of ulinastatin on the release of HMGB1 in large amount.
观察乌司他丁对严重烧伤大鼠脾脏CD4(+) T淋巴细胞及CD4(+) CD25(+)调节性T淋巴细胞（Tregs）免疫功能和外周血高迁移率族蛋白B1（HMGB1）含量的影响，并分析其可能机制。
将96只雄性SD大鼠按随机数字表法分为假伤组、烧伤组和乌司他丁组，每组32只。假伤组大鼠背部浸入37℃温水中12 s造成假伤。烧伤组和乌司他丁组大鼠背部浸入94℃热水中12 s造成30%总体表面积的全层烫伤（以下简称烧伤）。伤后各组大鼠立即腹腔注射生理盐水（40 mL/kg），同时乌司他丁组大鼠腹腔注射乌司他丁（4×10(4) U/kg），每12 h注射1次，至伤后72 h。分别于伤后1、3、5、7 d每组选取8只大鼠采集腹主动脉血样本。采用酶联免疫吸附测定法（ELISA）检测血清HMGB1含量。然后，立即处死3组大鼠，取脾脏并分离CD4(+) CD25(+) Tregs和CD4(+) T淋巴细胞。采用流式细胞仪检测CD4(+) CD25(+) Tregs中细胞毒性T淋巴细胞相关抗原4（CTLA-4）和叉头翼状螺旋转录因子p3（Foxp3）的阳性表达率。采用ELISA检测CD4(+) CD25(+) Tregs培养上清液中IL-10含量以及CD4(+) T淋巴细胞培养上清液中白细胞介素2（IL-2）、IL-4和γ干扰素（IFN-γ）含量。采用酶标仪测定CD4(+) T淋巴细胞的增殖活性。每组上述实验各时间点样本数均为8只。数据采用析因设计方差分析和LSD检验进行处理。
（1）与假伤组相比，烧伤组大鼠血清HMGB1含量在伤后1～7 d显著升高（P值均<0.01）。与烧伤组相比，乌司他丁组大鼠血清HMGB1含量在伤后1～7 d显著降低（P值<0.05或P值<0.01）。（2）与假伤组相比，烧伤组大鼠CD4(+) CD25(+) Tregs中CTLA-4和Foxp3阳性表达率及CD4(+) CD25(+) Tregs培养上清液中IL-10含量在伤后1～7 d显著升高（P值均<0.01），于伤后3 d达到峰值[分别为(65±10)%、(76±10)%和(28.2±4.4) pg/mL]。假伤组大鼠伤后3 d这3项指标分别为(45±7)%、(46±7)%和(11.2±2.3) pg/mL。与烧伤组相比，乌司他丁组大鼠CD4(+) CD25(+) Tregs中CTLA-4和Foxp3阳性表达率及CD4(+) CD25(+) Tregs培养上清液中IL-10含量在伤后1～7 d显著降低（P<0.05或P<0.01），分别于伤后7 d[(43±6)%]、伤后1 d[(50±8)%]和伤后7 d[(12.4±3.4) pg/mL]降至最低点。烧伤组大鼠伤后7 d、1 d和7 d这3项指标分别为(58±8)%、(71±9)%和(19.7±2.8) pg/mL。（3）与假伤组相比，烧伤组大鼠CD4(+) T淋巴细胞培养上清液中IL-2和IFN-γ含量在伤后1～7 d显著降低，而CD4(+) T淋巴细胞培养上清液中IL-4含量在伤后1～7 d显著升高，P值均<0.01。与烧伤组相比，乌司他丁组大鼠CD4(+) T淋巴细胞培养上清液中IL-2和IFN-γ含量在伤后1～7 d显著升高，而CD4(+) T淋巴细胞培养上清液中IL-4含量在伤后1～7 d显著降低，P<0.05或P<0.01。（4）与假伤组相比，烧伤组大鼠CD4(+) T淋巴细胞增殖活性在伤后1～7 d显著降低（P值均<0.01）。与烧伤组相比，乌司他丁组大鼠CD4(+) T淋巴细胞增殖活性在伤后1～7 d显著升高（P值均<0.01）。
乌司他丁可削弱严重烧伤大鼠脾脏CD4(+) CD25(+) Tregs介导的免疫抑制功能，提高脾脏CD4(+) T淋巴细胞的增殖功能和分泌功能，这可能归因于乌司他丁对大量HMGB1释放的抑制作用。
