Necrostatin-1对创伤失血性休克大鼠肝脏高迁移率族蛋白B1表达的影响

The effect of necrostatin-I on high mobility group protein-B1 in liver of rats with hemorrhagic shock

目的探讨程序性坏死特异性抑制剂-1（necrostatin-1，Nec-1）对创伤失血性休克大鼠肝脏HMGB-1的影响及其机制。方法采用创伤失血性大鼠休克模型，将96只雄性SD大鼠按随机数字表法随机分为假手术组、DMSO组、Nec-1组，每组32只。假手术组仅进行麻醉、分离血管、结扎血管，并不进行创伤失血和再灌注。DMSO组建立创伤失心陆休克大鼠模型，再灌注前5min前股静脉给予DMSO溶剂。Nec-1组于再灌注5rain股静脉给予Nec-1（1ms／kg）。于再灌注后分别在2、8、16、24h各处死动物8只，取动物血清及肝脏组织。检测血清中丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）水平；HE染色观察肝脏病理变化；透射电镜观察再灌注后24h后细胞器水平的细胞坏死；酶联免疫分析法（ELISA）分析血清中HMGB-1含量；蛋白质免疫印迹法（western blotting）分别检测肝脏组织中胞质和总蛋白的HMGB—1含量。结果Nec-1组与DMSO组比较，血清AlJrr在8h（P〈0．05）、16h（P〈0．01）、24h（P〈0．01）表达较低，Nec-1组血清AST在8h（P〈0．01）、16h（P〈0．01）、24h（P〈0．01）表达较DMSO组低；与DMSO组比较，Nec-1组血清HMGB-1在8h（P〈0．05）、16h（P〈0．01）、24h（P〈0．01）有明显下降。光镜及电镜下DMSO组及Nec-1组可见肝小叶结构破坏，淤血，炎性细胞浸润及细胞器损伤，Nec-1组肝组织损伤明显减轻；与DMSO组比较，Nec-1组肝细胞中胞质蛋白HMGB-1在8h（P〈0．01）、16h（P〈0．01）、24h（P〈0．01）有明显下降，Nec—1组总蛋白HMGB—1在8h（P〈0．05）、24h（P〈0．05）有明显下降。结论Nec-1可以有效降低创伤失血性休克对肝脏的损伤，减少HMGB-1的释放，有效保护肝细胞。

Objective To investigate the effect and mechanism of necrostatin-1 （ Hec-1 ） on the level of HMGB-1 protein in liver of rats with hemorrhagic-traumatic shock. Methods A number of 96 male SD rats were divided into sham-operated group, dimethyl sulfoxide （DMSO） group and Nec-1 group （n = 32 in each） by randomized number method. Rat model of hemorrhagic-traumatic shock was made by fracture of femoral bone and tibia bone and exsanguination from femoral vein until 30 mmHg and maintained at 30 -40 mmHg for 90 min, then the shed blood was transfused back with Ringer＇s solution. The rats in shamoperated group were only under anesthesia for separating and ligating blood vessels, without exsanguination to induce hemorrhagic shock and without replenishment with blood. Rats in Nec-1 group were given 1 mg/kg Nec-1 through femoral vein 5 min before replenishment with blood and Ringer＇ s solution, while the rats in DMSO group were given equal volume of DMSO solution instead. Eight rats in each group were sacrificed separately at 2 h, 8 h, 16 h and 24 h after replenishment. The serum and liver tissues of rats in each group were collected to detect serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST）, and to observe the pathological changes in liver with hematoxylin-eosin （HE） staining. The level of HMGB-1 in serum was detected by using ELISA. The cytoplasm protein and total protein expressions of HMGB-1 were assessed by using western blot analysis. Results Compared with DMSO group, levels of serum ALT at 8 h （P〈0. 05）, 16 h （P〈0.01） and 24 h （P〈0.01） in Nec-1 group were significantly lower. Level of serum AST in Nec-1 group were lower compared with DMSO group at 8 h （ P 〈 0. 01 ） , 16 h （ P 〈 0. 01 ） and 24 h （P 〈 0. 01 ）, Compared with DMSO group, levels of serum HMGB-1 at 8 h （P 〈 0. 05）, 16 h （P 〈0. 01 ） and 24 h （P 〈 0. 01 ） in Nec-1 group were significantly lower. Under light microscopy and transmission electron microscope, hepatic lobule destroyed, the blood extravasated, the immunocyte infiltrated and cellular organelle destroyed were found. Compared with DMSO group, the level of HMGB-1 protein in cytoplasm protein in Nec-1 group were significantly decreased at 8 h （P 〈 0. 01 ）, 16 h （P 〈 0. 01 ） and 24 h （P 〈 0.01 ）. The level of HMGB-1 protein in total protein in Nec-1 group were significantly decreased 8 h （ P 〈 0. 05 ） and 24 h （ P 〈 0. 05 ）. Conclusions Nec-1 can remarkably protect the liver of rats with hemorrhagic-traumatic shock, decrease the level of HMGB-1, and protect the hepatocyte effectively.