乌司他丁对严重烧伤大鼠脾脏免疫功能的影响及机制

Effects of ulinastatin on immune function of spleen in severely burned rats and its mechanism

目的观察乌司他丁对严重烧伤大鼠脾脏CD4＋T淋巴细胞和CD4＋CD25＋调节性T淋巴细胞（Treg）免疫功能及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。方法将96只雄性SD大鼠按照随机数字表法分为假伤组、单纯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12S模拟致伤；单纯烧伤组、乌司他丁组大鼠背部浸入94℃热水中12S，造成30％TBSA Ⅲ度烫伤（以下称烧伤）。各组大鼠伤后即刻起腹腔注射生理盐水40mL／kg，乌司他丁组大鼠同时腹腔注射乌司他丁4×104U／kg，每隔12小时注射1次，持续至伤后72h。于伤后1、3、5、7d，每组各取8只大鼠采集腹主动脉血，采用ELISA法检测血清HMGB1含量，随即处死大鼠取脾脏组织，分选CD4＋CD25＋Treg和CD4＋T淋巴细胞。采用流式细胞仪检测CD4＋CD25＋Treg中细胞毒性T淋巴细胞相关抗原4（CTLA-4）、叉头翼状螺旋转录因子p3（Foxp3）阳性表达率，采用ELISA法检测CD4＋CD25＋Treg培养上清液中IL-10含量；采用ELISA法检测CD4＋T淋巴细胞培养上清液中IL-2、IL-4和γ干扰素含量，采用酶标仪测定CD4＋T淋巴细胞增殖活性，各组各时相点样本数为8。对数据行析因设计方差分析和LSD检验。结果（1）与假伤组比较，单纯烧伤组大鼠伤后1～7d血清HMGB1含量明显增加（P值均小于0．01）。与单纯烧伤组比较，乌司他丁组大鼠伤后1～7d血清HMGB1含量明显减少（P值均小于0．01）。（2）与假伤组比较，单纯烧伤组大鼠伤后1～7d CD4＋CD25＋Treg中CTLA4、Foxp3阳性表达率及细胞培养上清液中IL-10含量明显升高或增加（P值均小于0．01），于伤后3d达最大值，分别为（65±10）％、（76±10）％、（28．2±4．4）pg／mL，此时假伤组大鼠该3项指标分别为（45±7）％、（46±7）％、（11．2±2．3）pg／mL。与单纯烧伤组比较，乌司他丁组大鼠伤后1～7d CD4＋ CD25＋Treg中CTLA4、Foxp3阳性表达率及细胞培养上清液中IL-10含量明显降低或减少（P〈0．05或P〈0．01），分别于伤后7、1、7d达最低值[（43±6）％、（50±8）％、（12．4±3．4）pg／mL]，此时单纯烧伤组大鼠该3项指标分别为（58±8）％、（71±9）％、（19．7±2．8）pg／mL。（3）与假伤组比较，单纯烧伤组大鼠伤后1～7d CD4＋T淋巴细胞培养上清液中IL-2和γ干扰素含量明显减少、IL4含量明显增加（P值均小于0．01）。与单纯烧伤组比较，乌司他丁组大鼠伤后1～7dCD4＋T淋巴细胞培养上清液中IL-2和1干扰索含量明显增加、IL-4含量明显减少（P〈0．05或P〈0．01）。（4）与假伤组比较，单纯烧伤组大鼠伤后1～7dCD4＋T淋巴细胞增殖活性明显减弱（P值均小于0．01）。与单纯烧伤组比较，乌司他丁组大鼠伤后1～7dCD4＋T淋巴细胞增殖活性明显增强（P值均小于0．01）。结论乌司他丁可减弱严重烧伤大鼠脾脏CD4＋CD25＋Treg介导的免疫抑制功能，改善脾脏CD4＋T淋巴细胞的分泌和增殖功能，原因可能为其抑制了体内HMGB1的大量释放。

Objective 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. Methods 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 sa- line （40 mL/kg）, meanwhile rats in ulinastatin group were intraperitoneally injected with ulinastatin （4 × 104 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 sacri- ficed immediately to collect spleens and separate CD4＋ CD25＋ Tregs and CD4 ＋ T lymphocytes. Flow cytom- eter 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 cul- ture supernatant of CD4＋ CD25 ＋ Tregs, and content of interleukin 2 （ IL-2 ） , IL-4, and 3, 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 experi- ments was 8 at each time point in each group. Data were processed with analysis of variance of factorial de- sign and LSD test. Results （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 O. 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 PID3 [（65±10）%, （76±10）%, and （28.2 ±4.4） pg/mL respectively]. These3 indexes of rats in sham injury group on PID 3 were （45 ± 7 ） % , （46 ± 7 ） % , and （ 11.2 ± 2.3） pg/mL respective- ly. 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 sig- nificantly 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） Com- pared with those in sham injury group, the content of IL-2 and IFN-γ in culture supernatant of CD4＋ T lym- phocytes of rats was significantly decreased, while the content of IL-4 in culture supernatant of CD4＋ T lym- phocytes of rats was significantly increased in burn group from PID 1 to 7, with P values below 0.01. Com- pared 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 sig- nificantly 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 ）. Conclusions Ulinastatin can weaken the immunosuppressive function mediated by splenic CD4＋ CD25＋ Tregs in severely burned rats, and improve proliferative function and se- cretory function of splenic CD4 ＋ T lymphocytes, which may be attributed to the inhibiting effect of ulinasta- tin on the release of HMGB1 in large amount.