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目的研究重组人高迁移率族蛋白1(HMGB1)诱导内皮细胞释放趋化因子白介素-8(IL-8)和单核细胞趋化蛋白-1(MCP-1)的规律及其与脂多糖(LPS)的协同作用;探讨丝裂原活化的蛋白激酶(MAPK)信号通路在上述作用中的地位。方法用LiquiChip液相蛋白芯片系统检测不同浓度重组HMGB1(0~75ng/ml),或者HMGB1(15ng/ml)刺激后不同时间点人脐静脉内皮细胞(HUVEC)分泌IL-8、MCP-1的水平变化以及HMGB1(15ng/ml)与LPS(10ng/ml)共同刺激后IL-8、MCP-1的水平变化;探讨MAPK信号通路在HMGB1诱导内皮细胞释放趋化因子中的作用时首先加入抑制剂SB203580(20mol/L)、PD98059(20mol/L)和JNKinhibitorII(50nmol/L)预处理细胞1h,再加入HMGB1和LPS刺激。结果在HMGB1蛋白刺激后3~6h,IL-8和MCP-1水平开始增加,12~24h持续增高(P<0.01);随着HMGB1浓度的增加,IL-8和MCP-1水平也明显升高,与基础值相比差异有统计学意义(P<0.01)。如果用LPS(10ng/ml)和HMGB1(15ng/ml)共同刺激HUVEC,IL-8和MCP-1的生成量较单独刺激时大大增加(P<0.01),二者存在协同效应(P<0.01)。SB203580、PD98059及JNKinhibitorII对HMGB1和LPS协同诱导趋化因子的释放均有不同程度的抑制作用,其中以p38MAPK抑制剂SB203580的抑制作用最为明显;同时用SB203580、PD98059和JNKinhibitorII预处理细胞则完全抑制趋化因子的释放。结论HMGB1蛋白以时间和剂量依赖方式诱导HUVEC表达趋化因子IL-8和MCP-1的上调;并协同LPS刺激HUVEC释放趋化因子IL-8和MCP-1从而加重炎症反应。MAPK信号通路在HMGB1与LPS协同诱导内皮细胞释放趋化因子的过程中发挥了重要作用。
Objective To study the regulation of the release of chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) from endothelial cells by recombinant human HMGB1 (HMGB1) LPS); explore the status of mitogen-activated protein kinase (MAPK) signaling pathway in these roles. Methods LiquiChip liquid phase protein chip system was used to detect the secretion of IL-8 and MCP-1 by human umbilical vein endothelial cells (HUVECs) at different time points after stimulation with different concentrations of HMGB1 (0-75ng / ml) or HMGB1 (15ng / ml) Level and the levels of IL-8 and MCP-1 after co-stimulated by HMGB1 (15ng / ml) and LPS (10ng / ml); To investigate the role of MAPK signaling pathway in HMGB1-induced chemotaxis release by endothelial cells The cells were pretreated with SB203580 (20mol / L), PD98059 (20mol / L) and JNKinhibitorII (50nmol / L) for 1h, then stimulated by HMGB1 and LPS. Results The levels of IL-8 and MCP-1 began to increase 3 to 6 hours after HMGB1 stimulation, and continued to increase from 12 to 24 hours (P <0.01). The levels of IL-8 and MCP-1 also increased significantly with the increase of HMGB1 concentration High, compared with the base value difference was statistically significant (P <0.01). When HUVECs were stimulated with LPS (10ng / ml) and HMGB1 (15ng / ml), the production of IL-8 and MCP-1 were significantly increased (P <0.01) ). SB203580, PD98059 and JNK inhibitor H1 inhibited the release of chemokines induced by HMGB1 and LPS to some extent, of which inhibition by p38 MAPK inhibitor SB203580 was the most obvious. Pretreatment with SB203580, PD98059 and JNK inhibitor H1 completely inhibited the chemotaxis The release of the factor. Conclusion The HMGB1 protein induces the up-regulation of HUVECs expression of chemokines IL-8 and MCP-1 in a time-and dose-dependent manner. Synergistically, LPS stimulates the release of chemokines IL-8 and MCP-1 from HUVECs to exacerbate the inflammatory response. MAPK signaling pathway plays an important role in the process of synergistic induction of chemokines by endothelial cells by HMGB1 and LPS.