p38丝裂素活化蛋白激酶/核转录因子-κB转导通路在脓毒症所致内皮细胞凝血功能障碍中的作用

The role of p38 mitogen-activated protein kinase/nuclear factor-κB transduction pathway on coagulation disorders due to endothelial injury induced by sepsis

目的 探讨p38丝裂素活化蛋白激酶/核转录因子-κB（p38MAPK/NF-κB）是否参与了脓毒症所致的内皮细胞凝血功能障碍.方法 选取22例脓毒症患者血浆刺激人脐静脉内皮细胞（HUVEC）,以8名健康人血浆刺激HUVEC 60 min作为阴性对照,肿瘤坏死因子-α（TNF-α）作为阳性对照.用酶联免疫吸附法（ELISA）、蛋白质免疫印迹法（Western blotting）、免疫荧光法检测p38MAPK和NF-κB的磷酸化.结果 脓毒症患者血浆TNF-α水平（ng/L）明显高于健康对照者（155.68±89.74比5.00±0.47,P〈0.01）.与20%的健康人血浆比较,用20%的脓毒症患者血浆刺激HUVEC后,组织因子（TF,μg/L）于180 min时达高峰（5.87±0.14比1.25±0.11,P〈0.01）,血管性血友病因子（vWF,μg/L）于120 min达到高峰（9.59±0.07比3.59±0.06,P〈0.01）并随后下降.用20%的脓毒症患者血浆刺激HUVEC后p38MAPK和NF-κB发生活化,p38MAPK的活化早于NF-κB（2 min比5 min）;加入p38MAPK特异性抑制剂SB239063后,NF-κB的磷酸化和核移位受阻.结论 p38MAPK/NF-κB转导通路在脓毒症引起的凝血功能障碍中具有一定的作用.

Objective To determine the activation status of p38 mitogen-activated protein kinase （p38MAPK）/nuclear factor-κB （NF-κB） in coagulation disorders due to endothelial injury induced by sepsis. Methods Human umbilical vein endothelial cells （HUVECs） were exposed to plasma obtained from 22 patients suffering from sepsis. Plasma was also obtained from 8 healthy individuals to serve as negativecontrol, and tumor necrosis factor-α （TNF-α） was used as positive control. Phosphorylation and activity of p38MAPK and NF-κB were determined with enzyme-linked immunosorbent assay （ELISA）, Western blotting, and immunofluorescence assay. Results The level of TNF-α （ng/L） in sepsis plasma was significantly higher than that in healthy plasma （155.68±89.74 vs. 5.00±0.47, P〈0.01）. Compared with healthy plasma in 20% concentration it was found when HUVECs were treated with sepsis plasma in 20% concentration, tissue factor （TF, μg/L） reached the peak at 180 minutes （5.87±0.14 vs. 1.25±0.11, P〈0.01）, von Willebrand factor （vWF, μg/L） reached the peak at 120 minutes （9.59±0.07 vs. 3.59±0.06, P〈0.01）, then they began to decline. When HUVECs were treated with sepsis plasma in 20% concentration increased phosphorylation and activity of p38MAPK and NF-κB, phosphorylation of p38MAPK occurred before phosphorylation of NF-κB （2 minutes vs. 5 minutes）. When the inhibitor of p38MAPK （SB239063） was added, NF-κB phosphorylation （activation） and NF-κB nuclear translocation were inhibited. Conclusion This study demonstrates that p38MAPK/NF-κB transduction pathway plays an important role in septic coagulopathy.