Anisodamine counteracts lipopolysaccharide-induced tissue factor and plasminogen activator inhibitor-1 expression in human endothelial cells:: Contribution of the NF-κB pathway
Anisodamine counteracts lipopolysaccharide-induced tissue factor and plasminogen activator inhibitor-1 expression in human endothelial cells:: Contribution of the NF-κB pathway
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DOI：
10.1159/000051025
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发表时间：
2001-01-01
影响因子：
1.7
通讯作者：
Wojta, J
中科院分区：
文献类型：
--
作者：
Ruan, QR;Zhang, WJ;Wojta, J
在这项研究中，我们的目的是探讨山梨碱治疗细菌性休克的疗效是否--至少部分地--是通过直接干扰脂多糖(LPS)诱导的内皮细胞激活而实现的。因此，我们研究了山梨碱对LFS诱导的纤溶酶原激活物抑制物-1(PAI-1)和组织因子(TF)表达的影响，这是内皮激活的两个主要标志。采用酶联免疫吸附试验(ELISA)检测人脐静脉内皮细胞(HUVEC)条件培养液中PAI-1的表达，用一步凝血法测定细胞裂解液中的TF活性。用人纤溶酶原激活物-1或转铁蛋白的特异性探针进行Northern印迹分析，结果证实了这些方法的结果。为了评估核因子-kappaB通路在观察到的效应中的可能作用，用HUVEC核提取液和核因子-kappaB结合的寡核苷酸进行了凝胶迁移率改变分析(EMSA)。1ug/mlLPS处理HUVEC后，PAI-1和TF活性较未加内毒素处理的HUVEC明显升高。山梨碱可剂量依赖性地抑制脂多糖诱导的PAI-1和Tf的上调。单独山梨碱对PAI-1和TF在这些细胞中的组成性表达没有影响。Northern印迹结果也证实了这些作用在特异性PAI-1和TFm RNA表达水平上的作用。此外，EMSA显示，山梨碱可完全阻断内毒素与山梨碱共同作用的HUVEC核提取液中的核因子-kappaB DNA结合活性。因此，我们提供的证据表明，山莨菪碱通过抑制脂多糖诱导的内皮细胞PAI-1和TF的表达来抵消这些细胞的激活。它对核因子-kappaB途径的干扰可能--至少在部分逆转录中--参与了这一效应。异丹明拮抗内毒素对内皮细胞的作用可能是其治疗细菌性休克的机制之一。版权所有(C)2001 S.Karger AG，巴塞尔。
In this study we aimed to investigate whether the therapeutic efficacy of anisodamine in the treatment of bacteraemic shock could - at least in part - be brought about by its direct interference with the lipopolysaccharide (LPS)-induced activation of endothelial cells. Thus, we investigated the effect of anisodamine on LFS-induced expression of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF), two major markers of endothelial activation. PAI-1 was measured in the conditioned media of human umbilical vein endothelial cells (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. Results obtained in these assays were confirmed on the level of specific mRNA expression by Northern blotting using specific probes for human PAI-1 or TF. In order to evaluate a possible contribution of the NF-kappaB pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-kappaB-binding oligonucleotides. When HUVEC were treated with 1 mug/ml LPS a significant increase in PAI-1 and TF activity was observed compared with cells incubated without LPS. Anisodamine dose-dependently inhibited this LPS-induced upregulation of PAI-1 and TF. Anisodamine alone had no effect on the constitutive expression of PAI-1 and TF in these cells. These effects were also confirmed on the level of specific PAI-1 and TF mRNA expression by Northern blotting. Furthermore, we could show by EMSA that anisodamine completely abolished LPS-induced NF-kappaB DNA binding activity in nuclear extracts from HUVEC treated with LPS together with anisodamine. Thus, we provide evidence that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced PAI-1 and TF expression in these cells. Its interference with the NF-kappaB pathway might - at least in pa rt - contribute to th is effect. The ability of an isodamine to counteract LPS effects on endothelial cells might be one underlying mechanism explaining its efficacy in the treatment of bacteraemic shock. Copyright (C) 2001 S. Karger AG, Basel.