Zhong Tian-yu, Tang Jing, Liu Ya-wei, Li Zhi-jie, Chen Deng-yu, Zhao Ming-zhe, Wang Wei, Liu Jing-hua, Jiang Yong
Key Laboratory of Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China. zhongtianyu @gmail.com
Nan Fang Yi Ke Da Xue Xue Bao. 2009 Aug;29(8):1517-20.
To examine the synergistic effect of recombinant human high mobility group box 1 (HMGB1) protein and lipopolysaccharides (LPS) on the release of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in human umbilic vein endothelial cells (HUVECs), and explore the role of mitogen-activated protein kinases (MAPK) signal transduction in cytokine release.
HUVECs were incubated with recombinant HMGB1 (0-75 ng/ml) for 24 h and the culture medium supernatant was harvested for detection of IL-8 and MCP-1 with LiquiChip system. At 0, 1, 3, 6, 12 and 24 h after stimulation with 15 ng/ml HMGB1 or 15 ng/ml HMGB1 plus 10 ng/ml LPS, the levels of IL-8 and MCP-1 in the HUVECs were examined. To test the effect of MAPK inhibitors, HUVCs were pretreated with the inhibitors SB203580 (20 mol/L), PD98059 (20 mol/L), and JNK inhibitor II (50 nmol/L) 1 h before HMGB1 and LPS stimulation.
The levels of IL-8 and MCP-1 were significantly increased in the HUVECs stimulated with HMGB1 protein at the concentrations of 3, 15 and 75 ng/ml in comparison with the control levels (P<0.01). Since 3-6 h after the stimulation with HMGB1, the levels of IL-8 and MCP-1 began to increase gradually, and steadily increased at 12 and 24 h, all significantly higher than those of the control group (P<0.01). Stimulation of the HUVECs with LPS (10 ng<ml) or HMGB1 (15 ng/ml) alone resulted in significantly increased levels of IL-8 and MCP-1 (P<0.01), which were further increased after costimulation with LPS and HMGB1, suggesting a synergistic effect between HMGB1 and LPS (P<0.01). This synergistic effect was significantly inhibited by pretreatment with MAPK signaling kinases inhibitors, especially the p38 MAP kinase inhibitor SB203580, and the cocktail of MAP kinase inhibitors almost totally blocked the expression of these chemokines in HUVECs treated with HMGB1 and LPS.
HMGB1 protein can activate HUVECs to produce the chemokines IL-8 and MCP-1 in a dose- and time-dependent manner. HMGB1 also acts synergistically with LPS to induce IL-8 and MCP-1 release, which might play an important role in the development of sepsis. MAPK signal transduction plays an important role in HMGB1 and LPS-induced IL-8 and MCP-1 release.
研究重组人高迁移率族蛋白B1（HMGB1）与脂多糖（LPS）对人脐静脉内皮细胞（HUVECs）释放白细胞介素-8（IL-8）和单核细胞趋化蛋白1（MCP-1）的协同作用，并探讨丝裂原活化蛋白激酶（MAPK）信号转导在细胞因子释放中的作用。
将HUVECs与重组HMGB1（0 - 75 ng/ml）孵育24小时，收集培养基上清液，用LiquiChip系统检测IL-8和MCP-1。在用15 ng/ml HMGB1或15 ng/ml HMGB1加10 ng/ml LPS刺激后0、1、3、6、12和24小时，检测HUVECs中IL-8和MCP-1的水平。为测试MAPK抑制剂的作用，在HMGB1和LPS刺激前1小时，用抑制剂SB203580（20 μmol/L）、PD98059（20 μmol/L）和JNK抑制剂II（50 nmol/L）预处理HUVCs。
与对照水平相比，用3、15和75 ng/ml浓度的HMGB1蛋白刺激的HUVECs中IL-8和MCP-1水平显著升高（P<0.01）。自HMGB1刺激后3 - 6小时起，IL-8和MCP-1水平开始逐渐升高，并在12和24小时稳定升高，均显著高于对照组（P<0.01）。单独用LPS（10 ng/ml）或HMGB1（15 ng/ml）刺激HUVECs导致IL-8和MCP-1水平显著升高（P<0.01），LPS和HMGB1共刺激后进一步升高，提示HMGB1与LPS之间存在协同作用（P<0.01）。MAPK信号激酶抑制剂预处理可显著抑制这种协同作用，尤其是p38 MAP激酶抑制剂SB203580，MAP激酶抑制剂混合物几乎完全阻断了用HMGB1和LPS处理的HUVECs中这些趋化因子的表达。
HMGB1蛋白可激活HUVECs以剂量和时间依赖性方式产生趋化因子IL-8和MCP-1。HMGB1还与LPS协同作用诱导IL-8和MCP-1释放，这可能在脓毒症的发生发展中起重要作用。MAPK信号转导在HMGB1和LPS诱导的IL-8和MCP-1释放中起重要作用。
