Janus激酶-信号转导及转录活化因子通路对内毒素/脂多糖体外诱导大鼠腹腔巨噬细胞高迁移率族蛋白B1合成释放的调节作用

The role of Janus kinase-signal transducer and transcription activator pathway in the regulation of synthesis and release of lipopolysaccharide-induced high mobility group box-1 protein

目的探讨Janus激酶(JAK)-信号转导及转录活化因子(STAT)通路对内毒素/脂多糖(LPS)诱导大鼠腹腔巨噬细胞高迁移率族蛋白B1(HMGB1)合成释放的调节作用。方法取正常Wistar大鼠腹腔巨噬细胞,培养3d后用LPS刺激,分别于刺激前及刺激10、30、60、120min时观察JAK2、STAT1以及STAT3的活化情况,每时相点重复测定4次,以积分吸光度(IA)值表示;另取细胞分为正常组、LPS刺激组、JAK2抑制组、STAT1抑制组、STAT3抑制组,培养3d后用LPS刺激后4组细胞,刺激前2h,后3组分别加入AG490、氟达拉滨及雷帕霉素,观察各组HMGB1基因表达及蛋白释放情况,每组重复测定4次。结果LPS可诱导大鼠腹腔巨噬细胞JAK2、STAT1及STAT3在短时间(120min)内活化,其中STAT3活化最为迅速,10min即可达到峰值(7.47±0.56)。JAK2抑制组、STAT1抑制组、STAT3抑制组HMGB1基因表达均明显受抑制,其表达量均明显低于LPS刺激组(P<0.01),其中JAK2抑制组明显高于正常组(P<0.01),其余两个抑制组则与正常组相近(P>0·05);但3个抑制组HMGB1蛋白表达量与LPS刺激组基本相同,均明显高于正常组(P<0.01).结论JAK-STAT通路可在LPS刺激下早期活化,部分参与了诱导HMGB1合成的信号调控过程。

Objective To investigate the role of Janus kinase-signal transducer and transcription activator （JAK-STAT） pathway in the regulation of synthesis and release of lipopolysaccharide-induced high mobility group box-1 protein （ HMGB1 ）. Methods Peritoneal macrophages harvested from male Wistar rats were incubated for 3 days before the experiment. The activation of Janus kinase-2 （JAK2） , signal transducer and activator of transcription-1 （STAT1） and STAT3 was observed before and 10,30, 60 and 120 mins after LPS stimulation （4 determinations at each time point） and it was expressed as A value （absorption）. In addition, the cells were divided into normal control, LPS stimulation, JAK2 inhibition （with AG490 treatment 2 hours before LPS stimulation） , STAT1 inhibition （with fludarabine treatment 2 hours before LPS stimulation） and STAT3 inhibition （with rapamycin treatment 2 hours before LPS stimulation） groups. The cells in all groups except control group were stimulated with LPS 3 days after culture. The expression of HMGB1 gene and its protein release in each group were determined for 4 times and were expressed as A value. Results LPS could activate JAK2, STATl and STAT3 within 2 hours, especially the activation of STAT3 appeared more quickly, peaking at 10 minutes after LPS stimulation（7.47±0.56）. Pretreatment with the inhibitors of JAK-STAT pathway could markedly reduce the expression of HMGB1 mRNA （ P〈0.01 ） , but exerted no effect on HMGB1 release. Conclusion JAK-STAT pathway can be activated early during endotoxin challenge, and it may play a role in the regulation of HMGB1 synthesis.