山莨菪碱对血管内皮细胞组织因子和纤溶酶原激活物抑制剂1表达的影响及其机制研究

目的 通过研究山莨菪碱对内毒素脂多糖(LPS)致血管内皮细胞组织因子(TF)和纤溶酶原激活物抑制剂1(PAI—1)表达的影响，探讨山莨菪碱治疗感染性休克的机制。方法 用酶消化法培养人脐静脉内皮细胞(HUVEC)；ELISA方法检测HUVEC条件培养液PAI—1蛋白量：用一步凝固法检测HUVEC TF活性；Northern blot检测HUVEC TF和PAI—1的mRNA表达；为了评估上述作用是否通过NF—κB途径而转导，用电泳迁移变动检测法(EMSA)检测HUVEC核提取物NF—κB DNA结合活性。结果 LPS能使HUVEC PAI—1蛋白和TF活性及其mRNA表达显著增强，加入山莨菪碱后，LPS的这种作用明显减弱，并与山莨菪碱呈量效关系。山莨菪碱能完全阻止LPS致内皮细胞核提取物NF—κB DNA结合活性。结论 山莨菪碱治疗感染性休克的机制之一可能是通过拮抗LPS致HUVEC TF和PAI—1的表达。而且这种拮抗作用可能通过NF—κB途径来转导。

Study on Effect of Anisodamine on Expressions of Tissue Factor and Plasminogen Activator-1 Inhibitor in Vascular Endothelial Cells and Its Mechanisms

OBJECTIVE: To explore the mechanism of anisodamine in treating infectious shock through studying effect of anisodamine on endotoxin lipopolysaccharide (LPS) induced expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in vascular endothelial cells (EC). METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured by trypsin digestion method. PAI-1 was measured in the conditioned medium of HUVEC by a specific enzyme-linked immunosorbent assay (ELISA), whereas TF activity was measured in the lysates of these cells by using a single step clotting assay. Specific mRNA expressions were determined by Northern blotting. In order to evaluate a possible contribution of the nuclear factor-kappa B (NF-kappa B) pathway on the transductive effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-kappa B binding oligonucleotides. RESULTS: LPS could significantly strengthen the expression of HUVEC PAI-1 protein and TF activity and its mRNA, this effect of LPS could be markedly weakened after adding Anisodamine dose-dependently. Anisodamine could also completely block the LPS induced NF-kappa B DNA binding activity in nuclear extracts from HUVEC. CONCLUSION: The possible mechanism of anisodamine in treating infectious shock may be through antagonizing LPS induced HUVEC TF and PAI-1 expression, and the antagonism might be, at least partially, transduced by path of NF-kappa B.