© 2003 Schattauer GmbH, Stuttgart
Platelets Wound Healing and Blood andCells Inflammation/Infection
Antiinflammatory activity of astragaloside IV is mediated by inhibition of NF-qB activation and adhesion molecule expression Wei-Jian Zhang1, 2, Peter Hufnagl1, Bernd R. Binder1, Johann Wojta1 1Department
2Current
of Vascular Biology and Thrombosis Research, University of Vienna, Austria address: Linus Pauling Institute, Oregon State University, Corvallis, Oregon, USA
Summary The regulated expression of adhesion molecules on the surface of endothelial cells is a key process in the pathogenesis of inflammation.The saponin astragaloside IV (AS-IV), a 3-O-i-Dxylopyranosyl-6-O-i-D-glucopyranosylcycloastragenol purified from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. has been shown to have anti-inflammatory effects in vivo. In this study we have investigated the effect of AS-IV on cytokineand LPS-stimulated expression of adhesion molecules in and leukocyte adhesion to endothelial cells. We have demonstrated that AS-IV significantly reduced the adhesion promoting activity of LPS-stimulated HUVECs for polymorph-nuclear leukocytes (PMNs) and the monocytic cell line THP-1. Furthermore, by using specific cell ELISAs we could show that AS-IV decreased the LPS-induced expression of E-selectin and
VCAM-1 on the surface of HUVECs in a dose and time dependent manner, whereas the expression of ICAM-1 was not affected by AS-IV. AS-IV also inhibits TNFh-induced VCAM-1 expression.The saponin octyl-D-glucopyranoside had no effect on the LPS-induced expression of E-selectin and VCAM-1 excluding an unspecific detergent-like effect of AS-IV. Moreover, AS-IV significantly inhibited LPS- and TNFh-induced specific mRNA levels for E-selectin and VCAM-1. Finally, we could show that AS-IV completely abolished LPS- and TNFhinduced nuclear translocation of NF-qB and NF-qB DNA binding activity in endothelial cells.We conclude that the ability of AS-IV to inhibit the NF-qB pathway might be one underlying mechanism contributing to its anti-inflammatory potential in vivo.
Keywords Endothelial cells, adhesion molecules, lipopolysaccharide, cytokines, inflammation
Thromb Haemost 2003; 90: 904–14
Introduction The adhesion of circulating leukocytes to the endothelium directed by specific cell adhesion molecules is a critical cellular response to inflammation and tissue damage (1). Endothelial cells express an array of adhesion molecules that control processes such as leukocyte rolling along the endothelium, leukocyte attachment to the vascular wall and transmigration of
leukocytes into areas of inflammation (1). These leukocyteendothelial interactions require the regulated expression of various adhesion molecules by endothelial cells such as ICAM-1, VCAM-1 and E-selectin (2-4). Several studies have demonstrated that expression of these endothelial cell adhesion molecules is increased in inflamed tissue (5-8). Early in the inflammatory process endogenous proinflammatory mediators such as IL-1 or TNFh or exogenous proinflammatory agents such as bacterial
Correspondence to: Johann Wojta Department of Internal Medicine II Waehringer Guertel 18-20 A-1090 Vienna, Austria Tel.: +43-1-40400-2247, Fax: +43-1-40400-4216 E-mail:
[email protected]
Received March 7, 2003 Accepted after resubmission July 3, 2003 Financial support: During this study WJZ was supported by a North-South Dialogue scholarship EH-project 894 from The Austrian Academic Exchange Service (ÖAD).This work was supported by grants from The Austrian Fund for the Promotion of Scientific Research to JW (P9479) and BRB (P8011 and P8854). DOI: 10.1160/TH03-03-0136
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endotoxin – lipopolysaccharide (LPS) – lead to activation of endothelial cells. Activation of endothelial cells by cytokines or LPS results in an increased expression of cell adhesion molecules such as E-selectin, VCAM-1 and ICAM-1 on their surface (for reviews see ref. 9, 10). Thus as a result of endothelial cell activation, leukocytes adhere to the endothelium and transmigrate into the target tissue at local sites of inflammation. This sequence of events is crucial for the development of the inflammatory response and therefore constitutes a prime target for therapeutical intervention in a variety of inflammatory disorders. We could show recently that the saponin Notoginsenoside R1 (NG-R1) purified from the Chinese medical herb Panax notoginseng, which is used to treat cardiovascular disease in traditional Chinese medicine can counteract endotoxin induced endothelial cell activation - at least in part - by interference with the NF-qB pathway. NG-R1 inhibited the endotoxin-induced expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in vitro and significantly reduced lethality in an endotoxin-mouse model in vivo (11, 12). In addition we could show that a saponin structurally related to NG-R1, namely astragaloside IV (AS-IV), a 3-O-i-D-xylopyranosyl-6O-i-D-glucopyranosylcycloastragenol purified from the Chinese medical herb Astragalus membranaceus (Fisch) Bge., affects the fibrinolytic system of endothelial cells by up-regulating tissue type-PA and down-regulating PAI-1 (13). In these studies, however, we did not determine the effect of saponins on other major markers of endothelial cell activation and inflammation, namely adhesion molecules such as E-selectin, VCAM-1 and ICAM-1. AS-IV has been shown to have antiinflammatory effects in vivo (14-19) and it was therefore the aim of the present study to investigate whether AS-IV could affect the cytokine- and endotoxin stimulated expression of adhesion molecules in and leukocyte adhesion to endothelial cells and to determine its possible mode of action.
Materials and methods Materials Chemically pure Astragaloside IV was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and was dissolved and diluted in incubation medium to yield final concentrations of 1 to 100 µg/ml. Stock solution of AS-IV was made in DMSO. In no case was the final concentration of DMSO added to cells greater than 0.1%. LPS (from Escherichia coli, serotype 026:B6, >10,000 endotoxin units per mg lipopolysaccharide), prepared by phenolic extraction procedure, was obtained from Sigma (MO, USA). Human recombinant TNFh was purchased from Boehringer, Mannheim (Germany). SDS (Bio-Rad, USA), morpholinopropane sulfonic acid (Serva, Germany), PIPES
(Sigma), Seakem LE Agarose (FMC Bioproducts, ME, USA), [h-32P] dATP (ICN Pharmaceuticals, CA, USA) were obtained from the sources indicated. Other materials used in the methods described below have been specified in detail in the pertinent references. Cell culture Culture of HUVECs HUVECs were isolated from fresh umbilical cords by mild collagenase treatment and characterized and cultured as described (20). Cells were grown to confluence at 37°C in a humidified 95% air-5% CO2 atmosphere in Medium 199 (M199; Sigma) supplemented with 20% heat-inactivated supplemented calf serum (SCS; HyClone, UT, USA), 100 µg/ml streptomycin, 100 IU/ml penicillin, 250 ng/ml fungizone, 1 mM glutamine (JHR Biosciences, KS, USA), 2 IU/ml heparin (Liquemin Roche; Hoffmann-La Roche, Switzerland), 50 µg/ml endothelial cell growth supplement (ECGS; Technoclone, Austria). Cells were confirmed to be endothelial by their cobblestone morphology, positive immunofluorescence with anti-von Willebrand Factor VIII antibodies and by uptake of acetylated low density lipoprotein. Primary cultures were harvested at confluence with 0.05% trypsin-0.02% EDTA (JRH Biosciences) and plated at a split ratio of 1:3 in 75 cm2 flasks. Subconfluent cells were allowed to grow to confluence under the same conditions, harvested during exponential cell-growth phase with trypsin/EDTA and frozen in 1-ml aliquots of medium 199 containing 10% DMSO in liquid nitrogen. For experiments, vials were thawed at 37°C and cells were grown in 24- or 96-well plates, or 75 and 225 cm2 flasks (Costar) in M199 containing SCS, ECGS, and heparin at concentrations as described above until confluence was reached. All cells used in this study were between passage 2 and 3. Culture of THP-1 cells The human monocytic cell line THP-1 cells was purchased from the American Type Culture Collection (MD, USA) and grown in RPMI-1640 media (Sigma) containing 10% SCS, 100 µg/ml streptomycin, 100 IU/ml penicillin, 250 ng/ml fungizone, 1 mM glutamine, 5 $ 10 –5M 2-mercaptoethanol and routinely subcultured at a 1:5 ratio three times per week. Isolation of human polymorph-nuclear leukocytes (PMNs) Human PMNs were isolated from heparinized (100 U/mL) peripheral venous blood of healthy donors as described (21). Briefly, red cells were sedimented with 6% dextran (MW 500,000; Pharmacia, Sweden) in Hank’s balanced salt solution (HBSS; Sigma) for 60 min at room temperature, leukocyte-rich plasma was collected and PMNs were then isolated by centrifugation, washed and resuspended to 1x106cells/ml in RPMI-1640 with 10 mmol/L HEPES.
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Endotoxin assays All solutions that came into contact with cells prior to endotoxin stimulation were assayed at
