山莨菪碱下行调节培养的内皮细胞表达纤溶酶原激活物抑制剂1

Anisodamine Downregulates PAI-1 Expression of Cultured Endothelial Cells

为了探讨山莨菪碱对正常及内毒素脂多糖刺激过的内皮细胞纤溶酶原激活物抑制剂 1表达的作用及机制。本文采用酶消化法培养人脐静脉内皮细胞 ;用酶联免疫吸附试验 (ELISA)方法检测人脐静脉内皮细胞条件培养基纤溶酶原激活物抑制剂 1和组织型纤溶酶原激活物蛋白量 ;用Northern印迹方法检测人脐静脉内皮细胞纤溶酶原激活物抑制剂 1的mRNA表达 ;用电泳迁移检测法对人脐静脉内皮细胞的核因子κB核内转移情况进行研究。结果发现 ,脂多糖能使人脐静脉内皮细胞纤溶酶原激活物抑制剂 1蛋白及mRNA表达显著增强 ,但加入山莨菪碱后 ,脂多糖的这种作用明显减弱。而且 ,山莨菪碱还能抑制人脐静脉内皮细胞纤溶酶原激活物抑制剂 1基础水平的表达。经脂多糖刺激的人脐静脉内皮细胞核提取物与核因子κB探针结合明显增强 ,而山莨菪碱则能阻止脂多糖致核因子κB的核内转移现象。提示山莨菪碱不仅下行调节脂多糖所致内皮细胞纤溶酶原激活物抑制剂 1的蛋白分泌和mRNA表达 ,而且下行调节其纤溶酶原激活物抑制剂 1基础水平表达 。

Aim To investigate the effects and mechanisms of anisodamine on plasminogen activator inhibitor type 1 (PAI 1) expression in normal endothelial cells (EC) and lipopolicaccharide (LPS) treated EC. Methods Human umbilical vein endothelial cells (hUVEC) were cultured by trypsin digestion method. PAI 1 and tissue plasminogen activator (tPA) proteins in hUVEC conditioned medium were measured by sandwich enzyme linked immunosorbent assay (ELISA), and their mRNA expressions were determined by Northern blot analysis. Using electrophoretic mobility shift assay (EMSA), we assessed hUVEC nuclear factor kappa B (NF κB) nuclear translocation. Results LPS treatment of cultured hUVEC resulted in a significant increase in PAI 1 protein as well as mRNA expression by these cells. However, when hUVEC were incubated with LPS plus anisodamine, the upregulation of PAI 1 by LPS was abated. Moreover anisodamine was able to decrease the basal level of PAI 1 protein and mRNA as compared to control. Nuclear extracts prepared from LPS stimulated hUVEC demonstrated increased binding to the NF κB oligonucleotide as compared to unstimulated cells, and anisodamine could block those binding in the presence of LPS. Conclusion Anisodamine downregulated both basal and LPS induced PAI 1 protein and mRNA expression in EC, and the mechanism of the modulation might be via NF κB pathway.