高迁移率组蛋白1中和抗体在失血性休克中的保护作用

HMGB1 neutralizing antibody attenuates hemorrhagic shock-induced cardiac injury and modulates apoptotic gene expression in murine heart

目的 探讨晚期炎症因子高迁移率组蛋白1（HMGB1）中和抗体在出血性休克导致小鼠心肌损伤中的保护作用及机制.方法 复制KM小鼠的失血性休克模型,将KM小鼠分为假手术组、对照组、失血性休克模型组、HMGB1中和抗体治疗组（各组n=8）.并比较测定血清肌酸激酶（CK）及乳酸脱氢酶（LDH）水平及白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF） -α及HMGB1的浓度.取小鼠的心肌组织固定,做HE染色及电镜观察组织病理变化的分析,免疫组织化学法测定Fas/FasL系统及HMGB1在心肌组织中的表达.逆转录多聚酶链反应实验检测 Bax蛋白、凋亡相关基因（Bcl-2）与Caspase-3蛋白的RNA水平的变化,免疫印迹实验观察心肌中HMGB1、Bax、Bcl-2与Caspase-3的蛋白水平的表达.结果 失血性休克/复苏造成明显的心肌细胞的损伤和凋亡,明显增加了血清中炎症因子的表达,升高了促凋亡因子的mRNA水平的升高,HMGB1中和抗体干预后显著减轻了失血性休克导致的心肌细胞的损伤和凋亡[α-HMGB1干预组与失血性休克组比较（4.5＆#177;1.3） 比（8.9＆#177;1.9）],减轻了炎症反应[α-HMGB1干预组与失血性休克组比较,IL-1β：（127.7＆#177;21.2）比（164.3＆#177;30.2）;IL-6：（226.7＆#177;33.4） 比（402.5＆#177;59.5）;TNF-α：（100.6＆#177;10.7） 比（146.5＆#177;15.4）],调节了凋亡相关因子的mRNA水平的变化[α-HMGB1干预组与失血性休克组比较,Bcl-2：（0.25＆#177;0.02 ）比（0.19＆#177;0.02;） Bax：（ 0.38＆#177;0.04） 比（0.50＆#177;0.03）;Caspase-3：（0.38＆#177;0.04）比（0.56＆#177;0.04）].结论 HMGB1中和抗体保护了失血性休克/复苏导致的小鼠心肌细胞的损伤及凋亡,其保护作用可能与其减轻炎症反应及凋亡相关基因的表达有关.

Objective To explore the possible protective effects and mechanisms of neutralizing antibody of HMGB1 on hemorrhagic shock-induced cardiac injury in mice. Methods The KM mice of hemorrhagic shock model were divided into sham （sham）, control＋IgG, hemorrhagic shock （HS） and HMGB1 neutralizing antibody treatment （HS＋α-HMGB1） groups （n=8 each）. After modeling, the animals were anesthetized and blood samples collected. The concentrations of creatine kinase （CK） and lactate dehydrogenase （LDH） were measured. And the serum levels of such inflammatory factors as interleukin-1β （IL-1β）, interleukin-6 （IL-6）, tumor necrosis factor-alpha （TNF-α） and HMGB1 were analyzed by enzyme-linked immunosorbent assay （ELISA）. The cardiac tissues were harvested, fixed; hemotoxylin-eosin stained and observed under transmission electron microscopy. The histopathological cardiac changes were examined after hemorrhagic shock and resuscitation （HS/R）. Immunohistochemical staining was performed to detect the cardiac expressions of HMGB1 and Fas/FasL after HS/R. Reverse transcription polymerase chain reaction （PCR） was performed to analyze the RNA levels of Bax, Bcl-2 and Caspase-3 in cardiac tissues. And the protein levels of HMGB1, Bax, Bcl-2 and Caspase-3 in cardiac tissues were tested by Western blot. Results Hemorrhagic shock and resuscitation resulted in significantly cardiac cell damages, enhanced inflammatory factors in sera and up-regulated the expressions of pro-apoptotic genes and proteins in murine hearts. The lowered protein level of Bcl-2 induced by HS/R was reversed by neutralizing HMGB1 antibody treatment. Neutralizing HMGB1 antibody administration obviously attenuated HS/R-induced cardiac damages and apoptosis （HS ＋α-HMGB1 group vs HS group, 4.5＆#177;1.3 vs 8.9＆#177;1.9）, inhibited inflammatory responses （HS＋α-HMGB1 vs HS, IL-1β： 127.7＆#177;21.2 vs 164.3＆#177;30.2; IL-6： 226.7＆#177;33.4 vs 402.5＆#177;59.5; TNF-α：100.6＆#177;10.7 vs 146.5＆#177;15.4）, and modulated apoptosis-associated genes （HS＋α-HMGB1 group vs HS group, Bcl-2： 0.25＆#177;0.02 vs 0.19＆#177;0.02; Bax： 0.38＆#177;0.04 vs 0.50＆#177;0.03; Caspase-3： 0.38＆#177;0.04 vs 0.56＆#177;0.04） and proteins expression （HS＋α-HMGB1 group vs HS group, Bcl-2： 0.47＆#177;0.04 vs 0.3＆#177;0.03; Bax： 0.11＆#177;0.02 vs 0.17＆#177;0.02; Caspase-3： 0.62＆#177;0.04 vs 0.8＆#177;0.04） in murine hearts after HS/R. Conclusion Neutralizing HMGB1 antibody may protect mice from HS/R-induced cardiac damages and apoptosis. Such an effect is probably due to its anti-inflammatory responses and anti-apoptosis related gene expression.