高迁移率族蛋白B 1诱导小鼠腹腔巨噬细胞凋亡的受体机制

The receptor mechanism of high mobility group box-1 protein induced apoptosis in peritoneal macrophages in mice

目的了解高迁移率族蛋白B1(HMGB1)对小鼠腹腔巨噬细胞凋亡的影响及其受体机制。方法分离培养小鼠腹腔巨噬细胞,在巨噬细胞中加入不同的刺激物,分为HMGB1组:加入10μg/ml的HMGB1;HMGB1+抗晚期糖基化终末产物受体(RAGE)组:先加入RAGE多克隆抗体5μg/ml孵育2h后,再加入HMGB1;HMGB1+重组鼠(rm)RAGE/Fc组:将10μg/ml的HMGB1与10μg/ml的rmRAGE/Fc混合作用2h后,再加入巨噬细胞;对照组:加入磷酸盐缓冲液。采用流式细胞仪检测细胞表面RAGE的表达强度。激光共聚焦显微镜观察细胞凋亡情况,流式细胞仪检测细胞凋亡率。结果HMGB1组RAGE阳性细胞率(54±12)%明显高于对照组[(13±5)%,P<0.01],其荧光强度(126±10)也显著高于对照组(34±8,P<0.01)。HMGB1+rmRAGE/Fc组、HMGB1+抗RAGE组凋亡细胞明显多于对照组,而HMGB1组晚期凋亡及坏死细胞明显多于其他3组。HMGB1组细胞凋亡率(39.5±2.3)%高于HMGB1+rmRAGE/Fc组[(17.3±3.6)%]、HMGB1+抗RAGE组[(14.8±4.8)%]及对照组[(5.4±2.3)%,P<0.01]。结论HMGB1可诱导RAGE表达上调,RAGE是HMGB1诱导巨噬细胞凋亡的主要受体之一。

Objective To investigate the influence of high mobility group box-1 protein （HMGB1） on apoptosis of peritoneal macrophages in mice and its receptor mechanism. Methods The peritoneal macrophages were isolated from female BALB/c mice and divided into 4 groups according to different stimuli： i. e, HMGB1 group （with treatement of 10 μg/ml HMGB1 for 24 hours）, HMGB1 and anti-receptor advanced glycation end products （RAGE） antibody group（ with treatment of 5μg/ml anti-RAGE antibody for 2 hours followed by HMGB1 stimulation） , Recombinant mouse RAGE/Fc chimera （rmRAGE/Fc） and HMGB1 group （ 10 μg/ml of rmRAGE/Fc and 10 μg/ml HMGB1 were pre-mixed for 2 hours, then the peritoneal macrophages were treated with the mixture） , control group（ with treatment of phosphate buffer）. Expression of RAGE on the surface of macrophages, and the apoptotic rate of the cells were determined by flow cytometry. Laser scanning confocal microscopy was used to observe the apoptosis of the cells. Results The percentage of macrophages with positive RAGE expression in HMGB1 group [ （54 ± 12% ） ] was markedly increased compared to the controls [ （13 ± 5 ）% , P 〈 0.01 ] , and fluorescence density of RAGE expression was also significantly different between two groups （ 126 ± 10 vs 34 ± 8, P 〈 0.01 ）. The occurence of apoptosis in HMGB1 and rmRAGE/Fc group, as well as in HMGB1 plus anti-RAGE group were much higher than that in control group , and the number of macrophages with apoptosis and necrosis at late stage was obviously increased in HMGB1 group. The apoptic rate in HMGB1 group was （39.5 ± 2.3）% , which was significantly higher than those in HMGB1 and rmRAGE/Fc group（ 17.3 ± 3.6）% , and HMGB1 and anti RAGE group（ 14.8 ± 4.8） % , （ P 〈 0.01 ） , which were significantly higher than those in control groups （5.4 ± 2.3） % , （ P 〈 0.01 ）. Conclusion RAGE is one of the major receptor to induce apoptosis of macrophages, and the up-regulation of its expression is induced by HMGB1.