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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:465-474.)
© 1997 American Heart Association, Inc.

Articles

Notoginsenoside R1 Counteracts Endotoxin-Induced Activation of Endothelial Cells In Vitro and Endotoxin-Induced Lethality in Mice In Vivo

Wei-Jian Zhang; Johann Wojta; ; Bernd R. Binder

From the Department of Vascular Biology and Thrombosis Research, University of Vienna, Austria (W-J.Z., J.W., B.R.B.), and the Department of Physiology, Beijing University of Traditional Chinese Medicine, PRC (W-J.Z.).

	Abstract

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Abstract In this study we investigated a possible counteracting activity of notoginsenoside R1 (NG-R1) on lipopolysaccharide (LPS)-induced effects in vitro and in vivo. The upregulation of plasminogen activator inhibitor-1 (PAI-1) antigen due to LPS (1 µg/mL for 12 hours) in human umbilical vein endothelial cells (HUVECs) was prevented when the cells were incubated simultaneously with 100 µg/mL NG-R1 (PAI-1 antigen: LPS-treated cells, 969±54 ng/10⁵ cells; control cells, 370±15 ng/10⁵ cells; LPS+NG-R1–treated cells, 469±29 ng/10⁵ cells; n=6). The 2.5- and 3.4-fold (2.2- and 3.2-kb) increases in PAI-1 mRNA levels induced by LPS (1 µg/mL for 6 hours) were reduced to 1.4- and 2.6-fold increases in the presence of both LPS and 100 µg/mL NG-R1. LPS-induced tissue factor (TF) activity in HUVECs was also counteracted when the cells were coincubated with both LPS and 100 µg/mL NG-R1 for 6 hours (TF activity: LPS-treated cells, 88.6±6.5 mU/10⁶ cells; control cells, 0.7±0.01 mU/10⁶ cells; LPS+NG-R1–treated cells, 56.0±1.9 mU/10⁶ cells). The 26-fold increase in TF mRNA levels induced by LPS (1 µg/mL for 2 hours) was reduced to a 13-fold increase in the presence of both LPS and 100 µg/mL NG-R1. PAI activity levels in the plasma of mice 4 hours after injection of LPS (10 ng/g body wt) increased 2.3-fold compared with a control group. In contrast, PAI activity from LPS+NG-R1 (1 µg/g body wt NG-R1)–treated animals was at control level (PAI-1 activity: LPS-treated group, 11.3±3.1 U/mL; control group, 4.9±0.3 U/mL; LPS+NG-R1–treated group, 4.3±1.0 U/mL; n=5 to 8). The production of TNF- induced by 1 µg/mL LPS by cultured human whole-blood cells was inhibited by 46% when the cells were incubated together with 100 µg/mL NG-R1. NG-R1 protected mice from the lethal effects of LPS. The 78% lethality induced by LPS/galactosamine was reduced to 23% when NG-R1 was administered simultaneously (P<.01 by ² test). To extend this study to inflammatory cells, the effect of NG-R1 on LPS stimulation of the monocytic cell line THP-1 was investigated. NG-R1 inhibited the LPS-induced degradation of IB- and superinduced LPS-induced IB- mRNA, indicating that the effect of NG-R1 is not restricted to endothelial cells and is at least in part mediated by interference with the NF-B/IB- pathway.

Key Words: notoginsenoside R1 • PAI-1 • tissue factor • endotoxin • TNF-

	Introduction

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Lipopolysaccharide-producing Gram-negative bacteria are the most frequent cause of septic shock, which affects 200 000 patients annually, resulting in more than 100 000 deaths in the United States.¹ Septic shock is a serious clinical condition with a mortality of 50% to 80%.^{2 3 4 5 6 7} LPS released into the circulation is responsible for endothelial cell activation, resulting in complications of septic shock such as hypotension and DIC.⁸ These pathological consequences are brought about by initiation by LPS of a cascade of events, including the release of cytokines and the activation of the coagulation, fibrinolytic, and complement systems.⁹

Recent observations have contributed significantly to our understanding of the molecular and cellular events occurring in the initial stages of this syndrome. It has been shown in vitro that LPS binds to LPS-binding protein present in serum and that this complex binds to a receptor, CD14, present primarily on the surface of monocytes, which then become activated and release TNF-, the primary mediator of endotoxin shock.^{10 11 12 13} Such release of TNF- would subsequently lead to activation of endothelial cells, resulting in an increased expression of TF, PAI-1, and adhesion molecules as well as in the increased release of cytokines such as IL-1, IL-6, and IL-8 by these cells.^{14 15 16} Endothelial cells, however, which do not express membrane-bound CD14, are also responsive to LPS directly.¹⁶ Recent evidence suggests that such activation occurs via LPS binding to soluble CD14 present in serum, with the resulting complex then binding to a cellular receptor.^{17 18} Therefore, like TNF-, LPS can also directly increase the expression of PAI-1 and TF by endothelial cells, thereby leading to an antifibrinolytic and procoagulatory phenotype.^{14 15}

Reports show that the Chinese herb Panax notoginseng, which has been used by traditional Chinese medical doctors for thousands of years as a drug to treat cardiovascular diseases and relieve blood stasis and pain, can reduce inflammatory reactions in patients and animal models and can ameliorate the symptoms of experimental DIC.^{19 20 21 22 23 24} We recently showed that the dammarene-type saponin 20(`S)-protopanaxatrol NG-R1 purified from Panax notoginseng can modulate the fibrinolytic capacity of endothelial cells in vitro by increasing the expression of TPA and decreasing PAI-1 activity.^{25 26} It was therefore the aim of this study to investigate whether the LPS-induced thrombogenic changes in the fibrinolytic and coagulation systems of endothelial cells that are brought about by increased expression of PAI-1 and TF and are thought to contribute significantly to the pathogenesis of DIC after septicemia are counteracted by NG-R1.^{14 15} We showed not only that NG-R1 inhibits PAI-1 and TF induction by LPS in endothelial cells and TNF- generation in a human whole-blood assay but also that NG-R1 was efficient in ameliorating the LPS-induced PAI-1 response in mice in vivo and in significantly reducing LPS-related lethality in a mouse LPS shock model. In addition, we showed that NG-R1 effects are also operative in the monocytic cell line THP-1 and are at least partially caused by inhibition of IB- degradation and increased synthesis of IB- protein.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
NG-R1 judged to be chemically pure by high-performance liquid chromatography was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products. A stock solution was prepared by dissolving 100 mg/mL NG-R1 in 96% ethanol. This solution was further diluted in incubation medium to yield final concentrations of 0.01 to 100 µg/mL. As a control, 96% ethanol alone diluted 1:1000 in incubation medium was shown not to have any effects in the experiments described below. LPS (from Escherichia coli, serotype 026:B6, >10 000 endotoxin units/mg LPS) prepared by the phenolic extraction procedure was obtained from Sigma Chemical Co. A stock solution of 1 mg/mL in distilled water was stored at -70°C. SDS (Bio-Rad), morpholinopropane sulfonic acid (Serva), guanidine thiocyanate (Fluka), PIPES (Sigma), Seakem LE agarose (FMC Bioproducts), and dCTP (Aloha-³²P) (ICN Radiochemicals) 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 Endothelial Cells
Endothelial cells were isolated from fresh human umbilical cord veins with collagenase (Sigma) by a technique similar to that described by Jaffe et al.²⁷ Cells from four to six cords were pooled and plated in 75-cm² tissue culture flasks (Costar) coated with 1% calf skin gelatin (Sigma). Cells were grown to confluence at 37°C in a humidified 95% air/5% CO₂ atmosphere in medium 199 (Sigma) supplemented with 20% heat-inactivated SCS (HyClone; lot No. 21512059, endotoxin test <0.125 endotoxin units/mL), 100 µg/mL streptomycin, 100 IU/mL penicillin, 250 ng/mL fungizone, 1 mmol/L glutamine (all JHR Biosciences), 2 IU/mL heparin (Liquemin Roche, Hoffmann–La Roche), and 50 µg/mL ECGS (Technoclone). Cells were confirmed to be endothelial by their cobblestone morphology, positive immunofluorescence with anti–von Willebrand factor VIII antibodies,²⁸ and uptake of acetylated low-density lipoprotein.²⁹ 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 the 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 12-well plates (3.8 cm², Costar) in medium 199 containing SCS, ECGS, and heparin at concentrations as described above until confluence was reached. Average cell densities at confluence were 3x10⁴ cells/well. Cells used in this study were between passages 2 and 3. The cells were always fed with fresh medium the day before the experiment.

For experiments, confluent cultures were rinsed twice with HBSS (Sigma). Thereafter, 1 mL/well medium 199 containing 1.25% SCS (for TPA and PAI-1 experiments) or 20% SCS (for TF activity determination), 50 µg/mL ECGS with or without the indicated concentrations of LPS, and/or NG-R1 was added up to 24 hours. At the indicated times, the conditioned media were collected after removal of cell debris by centrifugation and stored at -70°C until use for determination of TPA and PAI-1 antigen and PAI-1 activity (see below). Alternatively, for TF activity determination, the cells were washed three times with clotting buffer (12 mmol/L sodium acetate, 8 mmol/L sodium barbital, 130 mmol/L sodium chloride, pH 7.4) and scrape-harvested into 300 µL clotting buffer. The scraped cells were frozen and thawed three times. The cell lysate was assayed in a one-stage clotting assay for TF activity (see below). The total cell number was determined with a hemocytometer after trypsinization. For Northern blotting analysis, cells were grown in 75-cm² tissue culture flasks. At the indicated times, total cellular RNA was isolated by acid guanidinium thiocyanate–phenol-chloroform extraction as described by Chomczynski and Sacchi.³⁰ The final RNA pellet was resuspended in 10 to 20 µL 0.5% SDS, and the concentration was determined spectrophotometrically at a wavelength of 260 nm.

Culture of THP-1 Cells
THP-1 cells originally derived from a human monocyte leukemia were purchased from American Type Culture Collection and grown in suspension culture in RPMI 1640 medium (Sigma) containing 10% SCS, 100 µg/mL streptomycin, 100 IU/mL penicillin, 250 ng/mL fungizone, 1 mmol/L glutamine, and 5x10^-5 mol/L 2-mercaptoethanol and routinely subcultured at a 1:5 ratio three times per week.

Whole-Blood TNF- Release Assay
The induction of TNF- by LPS in whole blood was measured as originally described.³¹ Briefly, heparinized venous blood from three healthy male volunteers (age, 32±8 years) was diluted 1:5 in RPMI 1640 culture medium containing 100 IU/mL penicillin, 100 µg/mL streptomycin, 250 ng/mL amphotericin B, and 1 mmol/L glutamine. Diluted blood (500 µL/well) was cultured in 24-well plates (Costar) for 24 hours at 37°C in a humidified 95% air/5% CO₂ atmosphere in the absence or presence of different concentrations of LPS without or with 100 µg/mL NG-R1. After incubation, the culture supernatant was collected after removal of cell debris by centrifugation and stored at -70°C until further analysis.

Animal Experiments
Male BALB/c mice (18 to 30 g body weight) were obtained from the Forschungsinstitut für Versuchstierzucht und Haltung (Himberg, Austria). All experiments were performed under ether (Merck) anesthesia. Mice were injected via lateral tail vein with LPS (10 ng/g) and/or NG-R1 (1 µg/g) or only saline in a volume of 5 µL/g. The control group received an injection of saline. At the times specified, blood was obtained and anticoagulated by 1:10 dilution in 3.8% (wt/vol) sodium citrate. Platelet-free plasma was prepared by centrifugation at 4°C for 30 minutes at 2500g and stored at -70°C until assayed.

The galactosamine-sensitized mouse model as described by Galanos et al³² was used to investigate whether NG-R1 has an effect on LPS-induced lethality. Male C3H/He mice (20 to 25 g body weight) at 10 weeks of age were purchased from the Forschungsinstitut für Versuchstierzucht und Haltung. All animals were kept under specific pathogen–free conditions up to the time of the experiment. Mice were injected intraperitoneally with 8 mg/mouse D-galactosamine (Sigma)+1.5 mg/mouse LPS or D-galactosamine/LPS+1.5 mg/mouse NG-R1. Half of the total dosage of NG-R1 was injected 1 hour before and the other half simultaneously with galactosamine/LPS. The mice were monitored for signs of endotoxemia and lethality at least four times daily for 2 days and periodically thereafter.

Endotoxin Assay
All solutions that came into contact with cells before endotoxin stimulation were assayed at <0.05 ng/mL endotoxin with the Coatest endotoxin kit (Kabi Diagnostica) performed according to the supplier's instructions. Briefly, before testing, the samples or standards were incubated at 37°C for 3 to 5 minutes. Then 100 µL of samples or standards was incubated with 100 µL limulus amoebocyte lysate at 37°C for exactly 10 minutes. Subsequently, 200 µL of the substrate solution (S-2423, Ac-Ile-Glu-Arg-pNA HCl), prewarmed at 37°C, was added to the mixture and incubated at 37°C for exactly 3 minutes. The reaction was stopped by the addition of 200 µL 20% acetic acid. The absorbance (A) of the samples and the standards against water or the blank was measured at 405 nm in a photometer, and the endotoxin concentrations of the test samples were calculated from the standards according to the formula endotoxin (ng/mL)=0.05/A75-A25x(Ax+A75-3A25/2), where A25 is the absorbance for the 0.025-ng/mL standard, A75 is the absorbance for the 0.075-ng/mL standard, and Ax is the absorbance for the test sample. Alternatively, the endotoxin concentration was determined with a standard curve: The absorbance for the standards was plotted against the concentrations of endotoxin. The endotoxin concentrations in the test samples were read from the standard curve.

Assays for PAI-1 Antigen and PAI-1 Activity
PAI-1 antigen was determined by a specific, commercially available ELISA (Technoclone) according to the manufacturer's instructions. The test range for this assay was 1.0 to 30 ng/mL. The PAI-1 ELISA measures free, complexed, and latent PAI-1. The PAI-1 activity assay was performed by addition of exogenous TPA to plasma or conditioned media samples and quantification of the amount of PAI-1 by measurement of the decrease in TPA activity with an enzymatic assay using plasminogen and a synthetic plasmin substrate (Technoclone). PAI-1 activity was defined in IU of TPA inhibited.

Measurement of TF Activity
Cell lysate (100 µL) was incubated with 100 µL of 20 mmol/L CaCl₂ at 37°C for 5 minutes in prewarmed plastic tubes in a Coagulometer (H. Amelung GmbH). Clotting was initiated by the addition of 100 µL of prewarmed citrated normal human plasma or coagulation factor X–deficient plasma (Sigma). TF activity was quantified by means of a standard curve (log-log plot) constructed with rabbit brain thromboplastin (Sigma), and 100 mU activity was defined as a clotting time of 20 seconds in a standard assay with normal human plasma. The coagulant activity observed reflects TF activity, because no procoagulant activity was detected from endothelial cells when factor X–deficient plasma was used instead of normal plasma.

Assay for TNF-
The concentration of TNF- in conditioned media was determined by a specific enzyme immunoassay (Innotest hTNF- kit; Innogenetics) according to the manufacturer's instructions. The sensitivity of the assay is 4 pg TNF-/mL. Briefly, 50 µL of incubation reagent was added to each well of the 96-well microtiter plate coated with rabbit anti–human TNF- polyclonal antibody. Subsequently, 50 µL of diluted samples or standards (recombinant TNF-) was added to each appropriate test well and incubated for 2 hours at 37°C. After four washes, 100 µL/well of a biotinylated neutralizing mouse monoclonal anti–TNF- antibody was added, and the plate was incubated at 37°C for 60 minutes followed by four washes. Then 100 µL of peroxidase-conjugated streptavidin was added to each well and incubated for 30 minutes at 37°C. After four wash steps, 100 µL/well of TMB chromogen solution (tetramethylbenzidine dissolved in DMSO) was added, and the plate was incubated for 30 minutes at 20°C to 25°C. Thereafter, the stop solution (sulfuric acid, 2N) was added, and the plate was measured in an automated reader (Anthos reader 2001) at 450 nm. Purified recombinant TNF- was used as standard.

Quantification of PAI-1, TF, and IB- mRNA Levels by Northern Blot Analysis
For Northern blot analysis, RNA samples were electrophoresed in a 1.2% agarose gel followed by capillary transfer of the fractionated RNA to a Duralon-UV membrane (Stratagene). RNA blots were placed in Seal-a-Meal bags and prehybridized in (in mmol/L) PIPES 50, NaCl 100, sodium phosphate 50, and EDTA 1 containing 5.0% SDS for 3 hours at 57°C. The prehybridization buffer was then discarded and replaced with fresh prehybridization buffer containing 10⁶ cpm/mL of the ³²P-labeled cDNA probes for either human PAI-1 (1.4 kb EcoRI/Bgl II fragment of a human PAI-1 cDNA of the 3.2-kb transcript), human TF (0.64-kb EcoRI/EcoRI fragment of human TF cDNA), pig IB-,³³ or rat GAPDH (1.2-kb Pst I fragment of a rat GAPDH cDNA). The cDNA fragments were radiolabeled by random priming (Random Prime DNA Labeling Kit; Boehringer Mannheim). Hybridization was carried out in a water bath overnight at 57°C. After hybridization, blots were removed from the bag and rinsed for 10 minutes in 100 mL 5% SDS/0.2xSSC at room temperature. Thereafter, blots were washed for 20 minutes in 400 mL 5% SDS/1xSSC at the hybridization temperature. After hybridization, the RNA blots were air-dried and exposed to XAR-5 x-ray films (Eastman Kodak) at -70°C. To quantify differences in the specific mRNA expression, the developed films were scanned with a densitometer (Hirschmann Elscript 400). The scanning data for each specific mRNA message were compared with the intensity of the GAPDH message.

Western Blot Analysis
Preparation of Cell Lysates
THP-1 cells were incubated in suspension culture in RPMI 1640 medium containing 10% SCS, 100 µg/mL streptomycin, 100 IU/mL penicillin, 250 ng/mL fungizone, 1 mmol/L glutamine, and 2x10^-5 mol/L 2-mercaptoethanol with or without the 1 µg/mL LPS and/or 100 µg/mL NG-R1 for the indicated times. The cells were preincubated with medium alone or with 100 µg/mL NG-R1 for 1 hour. Thereafter, medium or 1 µg/mL LPS was added to the pretreated cells. After stimulation, cells were rinsed twice with ice-cold PBS containing 1 mmol/L sodium orthovanadate (Na₃VO₄) and 5 mmol/L EDTA and then lysed for 20 minutes on ice in a buffer containing 1% Triton X-100, 20 mmol/L Tris-HCl, pH 8.0, 137 mmol/L NaCl, 10% glycerol, 1 mmol/L Na₃VO₄, 2 mmol/L EDTA, 1 mmol/L PMSF, 20 mmol/L leupeptin, and 0.15 U/mL aprotinin. These conditions have been shown to block in vitro phosphorylation and dephosphorylation after cell lysis.³⁴ After centrifugation at 10 000g at 4°C for 15 minutes, the supernatant containing solubilized protein was recovered, frozen in small aliquots, and stored at -70°C until use.

Electrophoresis and Immunoblotting
Protein samples were prepared for electrophoresis by mixing a concentrated sample buffer to obtain a final concentration of 62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 100 mmol/L DTT, 10% glycerol, and 0.01% bromphenol blue, followed by boiling for 2 minutes. Samples were then separated on 10% SDS-PAGE with the buffer system as described.³⁵ After SDS-PAGE, the separating gel was soaked in transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, pH 8.4, containing 20% methanol) for 10 minutes, then the proteins were transferred to nitrocellulose membrane for 2 hours at 120 mA by the semidry blotting system³⁶ with the same transfer buffer as above. Subsequently, the nitrocellulose membrane was blocked with TBS (20 mmol/L Tris-HCl, pH 7.5, 0.5 mol/L NaCl) containing 1.5% SMP for 1 hour at room temperature. The membrane was washed three times with TBS before overnight incubation with a rabbit polyclonal anti–IB- antibody (0.1 mg/mL) (Santa Cruz Biotechnology Inc) that was diluted 1:500 in TBS containing 1.5% SMP. Thereafter, the membrane was washed three times with TTBS, followed by two washes with TBS before incubation with a horseradish peroxidase–conjugated donkey anti-rabbit antibody (Amersham Life Science) (1:1000 dilution in TBS+1.5% SMP) for 2 hours at room temperature. The membrane was rinsed three times with TTBS, followed by two washes with TBS before color development with a chemiluminescent substrate (Enhanced Chemiluminescence Detection System, Amersham). The intensity of the bands on the Hyperfilm MP (Amersham) was quantified by densitometry as described above for the Northern blots.

Statistical Analysis
The results are reported as mean±SD. A Student's unpaired t test and ² test were used to determine significance levels.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of NG-R1 on LPS-Induced Upregulation of PAI-1 Antigen and Activity and PAI-1 mRNA in Cultured HUVECs In Vitro
As shown in the inset of Fig 1A, a gradual increase in PAI-1 antigen levels was observed in cells after exposure of HUVECs to LPS. The effect was also dose dependent with respect to LPS concentrations (0.01 to 1 µg/mL), and the PAI-1 antigen level increased to 2.7-fold control levels after 12 hours of treatment with 1 µg/mL LPS (Fig 1A; LPS-treated cells, 946±42 ng·10⁵ cells^-1·12 h^-1; control cells, 347±34 ng·10⁵ cells^-1·12 h^-1; n=6). The upregulation of PAI-1 antigen measured after exposure of the cells in response to different concentrations of LPS for 12 hours was antagonized by simultaneous treatment with 100 µg/mL NG-R1, resulting in an 88.3%, 81.6%, and 83% inhibition at 0.01, 0.1, and 1 µg/mL LPS, respectively (Fig 1A). The extent of the antagonism was dose dependent with respect to the NG-R1 concentrations after exposure of the cells to 1 µg/mL LPS, resulting in a 32.4%, 56.4%, 71%, and 83% inhibition at 0.1, 1, 10, and 100 µg/mL of NG-R1, respectively (Fig 1B). The PAI-1 activity of the cells changed parallel to the PAI-1 antigen changes (control cells, 5.48±0.78 IU·10⁵ cells^-1·12 h^-1; LPS-treated cells, 8.22±0.18 IU·10⁵ cells^-1·12 h^-1; LPS+NG-R1–treated cells, 4.77±0.26 IU·10⁵ cells^-1·12 h^-1, n=6).

View larger version (29K): [in this window] [in a new window]   Figure 1. Time and dose dependence of PAI-1 antigen production after exposure of cultured HUVECs to LPS and/or NG-R1. Confluent HUVECs were incubated with 1 µg/mL LPS alone (inset, solid circles) or medium alone (inset, open circles) or with 0.01 to 1 µg/mL LPS alone (open bars) or along with 100 µg/mL NG-R1 (hatched bars) for 12 hours (A) or with 1 µg/mL LPS along with 0.1 to 100 µg/mL NG-R1 for 12 hours (B). At indicated times, conditioned media were harvested and analyzed for PAI-1 antigen as described under "Methods." Percent inhibition was calculated and plotted against concentrations of NG-R1 (B, inset). Data shown are mean±SD of six independent wells. *P<.05, ** P<.01, ***P<.001 vs cell treated with LPS alone.

The 2.5- and 3.4-fold increases in PAI-1 mRNA levels (2.2 and 3.2 kb) induced by LPS were reduced to 1.37- and 2.6-fold increases in the presence of both LPS (1 µg/mL) and NG-R1 (100 µg/mL) (Fig 2).

View larger version (33K): [in this window] [in a new window]   Figure 2. Northern blots and bar graph showing effect of NG-R1 (NR1) on LPS-induced upregulation of PAI-1 mRNA levels in cultured HUVECs. Confluent HUVECs were incubated for 6 hours in absence (cont.) or presence of 1 µg/mL LPS, 100 µg/mL NG-R1, or LPS+NG-R1. Northern blot analysis of RNA extracts from untreated and treated HUVECs was performed with 32P-labeled cDNA probes for PAI-1 and GAPDH mRNA. Intensity of bands present on autoradiogram was assessed by densitometry. Results are expressed as percentages of control values after normalization to GAPDH mRNA and represent mean of two independent experiments.

Effects of NG-R1 on LPS-Induced TF Expression in Cultured HUVECs
As shown in the inset of Fig 3A, only a little TF activity was detected in untreated HUVECs (0.78±0.15 mU/10⁶ cells, n=9). The TF activity in cultured HUVECs quickly increased after exposure to LPS (1 µg/mL) and reached a maximum level (88.6±6.5 mU/10⁶ cells, n=6) at 6 hours, then diminished after 8 hours and returned to basal levels by 24 hours. However, when LPS was added simultaneously with 100 µg/mL NG-R1, TF expression in HUVECs after exposure of the cells to different concentrations of LPS was significantly inhibited, by 79.4%, 54.3%, 39.4%, and 37.1% at 0.1, 0.25, 0.5, and 1 µg/mL LPS, respectively (Fig 3A). The extent of the inhibition was also dependent on the concentration of NG-R1 used. At 0.1, 1, 10, and 100 µg/mL NG-R1, an inhibition of 0.68%, 21%, 33.6%, and 37.1%, respectively, was seen (1 µg/mL LPS-treated cells, 88.6±6.5 mU/10⁶ cells; LPS+100 µg/mL NG-R1–treated cells, 56.0±1.9 mU/10⁶ cells) (Fig 3B).

View larger version (29K): [in this window] [in a new window]   Figure 3. Time and dose dependence of TF activity after exposure of cultured HUVECs to LPS and/or NG-R1. Confluent HUVECs were incubated with 1 µg/mL LPS alone (inset, solid circles) or medium alone (inset, open circles) or with 0.1 to 1 µg/mL LPS alone (open bars) or along with 100 µg/mL NG-R1 (hatched bars) for 6 hours (A) or 1 µg/mL LPS along with 0.1 to 100 µg/mL NG-R1 for 6 hours (B). At indicated times, cell lysates were collected and assayed for TF activity as described under "Methods." Percent inhibition was calculated and plotted against concentrations of NG-R1 (B, inset). Data shown are mean±SD of six independent wells. ***P<.001 vs cell treated with LPS alone.

TF mRNA has one major TF transcript (2.4 kb), representing the mature TF mRNA, and two minor transcripts (3.5 and 3.1 kb), which have been reported to result from alternative splicing.³⁷ The minor transcripts have been observed in several cultured cell types but not in tissue. As shown in Fig 4, TF mRNA was barely detectable in untreated and NG-R1–treated control cells. A significant increase in TF mRNA, however, was observed after exposure of HUVECs to LPS, reaching a 26-fold (2.4+3.1+3.5 kb) induction over control levels at 2 hours. The augmented TF mRNA levels induced by LPS were inhibited by coincubation with NG-R1. TF mRNA was reduced to 50% over control levels. Also, the basal expression of TF-specific mRNA was reduced to 40% in cells treated with NG-R1 alone compared with untreated control cells.

View larger version (33K): [in this window] [in a new window]   Figure 4. Northern blots and bar graph showing effects of NG-R1 (NR1) on LPS-induced TF mRNA levels in cultured HUVECs. Confluent HUVECs were incubated for 2 hours in absence (cont.) or presence of 1 µg/mL LPS, 100 µg/mL NG-R1, or LPS+NG-R1. Northern blot analysis of RNA extracts from untreated and treated HUVECs was performed with 32P-labeled cDNA probes for TF and GAPDH mRNA. Intensity of bands present on autoradiogram was assessed by densitometry. Results are expressed as percentages of control values after normalization to GAPDH mRNA and represent mean of two independent experiments.

Effects of NG-R1 on LPS-Induced Antifibrinolytic Activity in Mice In Vivo
As shown in Fig 5, injection of LPS into mice resulted in a rapid increase in plasma PAI activity. At an LPS dosage of 10 ng/g body wt, a significant increase was already seen 2 hours after injection (control group, 1.5±0.9 U/mL; LPS-treated group, 10.7±1.7 U/mL; n=5 or 6, P<.001), and peak values of PAI activity were reached 4 hours after injection. After 12 hours, PAI activity levels in plasma of LPS-treated mice returned to values seen in control animals. In contrast to the marked 2.3-fold increase in the plasma PAI activity levels seen 4 hours after injection of LPS alone, the administration of the same dose of LPS into animals simultaneously receiving intravenous NG-R1 (1 µg/g body wt) resulted in complete inhibition of the increase in PAI activity plasma levels seen in LPS-treated mice (LPS-treated group, 11.3±3.1 U/mL; LPS+NG-R1–treated group, 4.3±1.0 U/mL, P<.01 compared with LPS-treated group; control group, 4.9±0.3 U/mL; n=5 to 8), whereas after 2 hours no significant inhibition effect of NG-R1 was seen.

View larger version (17K): [in this window] [in a new window]   Figure 5. Time course of PAI activity in mouse plasma after injection of 10 µg/kg body wt LPS IV (solid triangles), 1 mg/kg body wt NG-R1 (solid circles), LPS+NG-R1 (open triangles), or saline (open circles). Data are mean±SD (n=5 to 8) of PAI activity in plasma from mice bled at various times after injection.

NG-R1 Inhibits LPS-Induced Production of TNF- From Leukocytes in Cultured HWBCs Ex Vivo
From the data presented above, we could not evaluate whether the amelioration of LPS-induced effects by NG-R1 was due to inhibition of endothelial cell activation alone or whether NG-R1 would be also effective in preventing LPS-induced activation of monocytes. Therefore, using a human whole-blood assay, we investigated whether NG-R1 would affect LPS-induced TNF- generation by human leukocytes. As shown in Fig 6, LPS at a concentration of 1 ng/mL stimulated TNF- release from leukocytes in cultured HWBCs up to 300 pg/mL TNF-. The production of TNF- induced by LPS in the supernatant of HWBCs was inhibited by 46% when the cells were incubated together with 100 µg/mL NG-R1 (TNF- concentration in the supernatant of HWBCs treated with 1 ng/mL LPS, 297±192 pg/mL; 1 ng/mL LPS+100 µg/mL NG-R1, 162±137 pg/mL; n=6, P<.01). When LPS was used at a concentration of 10 ng/mL, the TNF- production by HWBCs was inhibited by 22% (TNF- concentration in the supernatant of HWBCs treated with 10 ng/mL LPS, 3399±617 pg/mL; 10 ng/mL LPS+100 µg/mL NG-R1, 2840±847 pg/mL; n=9, P<.01). When LPS was added at a concentration of 100 ng/mL, no significant inhibition in TNF- production was achieved with NG-R1 at a concentration of 100 µg/mL (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 6. Effects of NG-R1 on LPS-stimulated TNF- release from leukocytes in cultured HWBCs. Heparinized venous blood from three healthy male volunteers (age, 32±8 years) was diluted 1:5 in RPMI 1640 culture medium. Diluted blood (500 µL/well) was cultured for 24 hours at 37°C in 24-well plates in the presence of 1 ng/mL LPS alone (open bars) or together with 100 µg/mL NG-R1 (solid bars). After incubation, cultured supernatant was collected and concentration of TNF- in supernatant was measured as described under "Methods." No TNF- was detected in supernatant of HWBCs treated with either medium or NG-R1 alone. Data are mean±SD from six independent wells. **P<.01 vs cells treated with LPS alone.

NG-R1 Inhibits LPS-Induced Lethality in Mice
Having shown the effect of NG-R1 on LPS-induced activation of endothelial cells and leukocytes, we aimed to confirm these effects seen in vitro in an in vivo model. The results in Fig 7 demonstrate that NG-R1 protects mice from the lethal effects of LPS. The 78% lethality (14 of 18 mice died) induced by LPS/galactosamine was inhibited to 23% (3 of 13 mice died) when NG-R1 was administered together with LPS+galactosamine (P<.01 by ² test). The animals initially showed signs of endotoxemia in both groups, including immobility, lethargy, piloerection, mild febrile shivering, and diarrhea. These signs in NG-R1+LPS+galactosamine–treated mice were milder than in the mice treated with LPS+galactosamine alone. Thereafter, most of the animals that had been treated with NG-R1+LPS+galactosamine gradually recovered, and on the third day they appeared normal. There were no visible toxic symptoms in mice treated with NG-R1 alone. After the third day, surviving mice in all groups appeared normal, and none of the mice died within the observation period (3 weeks).

View larger version (14K): [in this window] [in a new window]   Figure 7. Prevention of LPS- and D-galactosamine–induced lethal toxicity by NG-R1 in male C3H/He mice. Mice were injected with 1.5 mg/mouse LPS+8 mg/mouse D-galactosamine IP (open circles) or LPS+galactosamine+1.5 mg/mouse NG-R1 IP (solid circles). Mice were monitored for signs of endotoxemia and lethality at least four times daily for 2 days and periodically thereafter. **P<.01 vs LPS+galactosamine–treated mice.

NG-R1 Inhibits LPS-Induced Degradation of IB- Protein in THP-1 Cells
The nuclear translocation of NF-B by LPS is accompanied by the rapid degradation of cytoplasmic IB-. We examined the protein levels of IB- in the cytoplasm of the monocytic cell line THP-1. The results of Western blot analysis of THP-1 cell cytoplasmic extracts an IB-–specific antibody used are shown in Fig 8. A 37-kD protein representing IB- was detected in the cytoplasmic extracts from unstimulated cells. Treatment of the cells with LPS resulted in 65% degradation of cytoplasmic IB- within 30 minutes. When the cells were preincubated with NG-R1 for 1 hour followed by addition of 1 µg/mL LPS for 30 minutes, however, the 65% degradation of IB- protein induced by LPS was abolished (Fig 8).

View larger version (39K): [in this window] [in a new window]   Figure 8. Western blot and bar graph showing effects of NG-R1 on LPS-induced degradation of IB- protein in cultured THP-1 cells. THP-1 cells were preincubated with medium alone or 100 µg/mL NG-R1 for 1 hour followed by addition of 1 µg/mL LPS or medium alone. Thereafter, cells were incubated for 30 minutes. After incubation, cells were lysed and 30 µg protein was loaded to each of the three lanes, and extracts were subjected to SDS-PAGE and blotted onto nitrocellulose paper. Immunoreactive proteins were detected by enhanced chemiluminescence after incubation with a polyclonal anti–IB- antibody in combination with a second antibody linked to horseradish peroxidase. Intensity of bands was assessed by densitometry. Results are given as percent of control.

NG-R1 Superinduces LPS-Stimulated IB- mRNA in THP-1 Cells
As shown in Fig 9, a significant increase in IB- mRNA was observed after exposure of THP-1 cells to LPS, reaching a 2- and 3-fold induction over control levels at 2 and 4 hours of incubation, respectively. When the cells were preincubated with NG-R1 for 1 hour followed by addition of LPS for another 2 or 4 hours, however, IB- mRNA levels were superinduced to 2.7- and 4.1-fold above control levels, respectively. NG-R1 alone also induced a 1.7-fold increase of IB- mRNA levels over control after 4 hours of incubation.

View larger version (16K): [in this window] [in a new window]   Figure 9. Northern blots and bar graph showing effects of NG-R1 on LPS-induced IB- mRNA levels in cultured THP-1 cells. THP-1 cells were incubated for 2 (A) or 4 (B) hours in the absence (lane 1) or presence of 100 µg/mL NG-R1 (lane 2) or 1 µg/mL LPS (lane 3) or preincubated with 100 µg/mL NG-R1 for 1 hour followed by addition of 1 µg/mL LPS for 2 or 4 hours (lane 4). Northern blot analysis of RNA extracts from untreated and treated THP-1 cells was performed with 32P-labeled cDNA probes for IB- and GAPDH mRNA. Intensity of bands on autoradiogram was assessed by densitometry. Results are given as percent of control after normalization to GAPDH mRNA and represent mean of two independent experiments.

NG-R1 Does Not Interfere With LPS Binding to HUVECs and THP-1 Cells
When LPS was measured in the conditioned medium of HUVECs or THP-1 cells incubated with LPS or with LPS+NG-R1 for 1, 2, 5, 10, 30, and 60 minutes, no significant difference in the LPS content at the end of the respective incubation period was found between cells treated with LPS alone or cells treated with LPS+NG-R1 (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Experimental and clinical studies have shown that LPSs present in the outer membrane of Gram-negative bacteria, called endotoxins, play a pivotal role in triggering the development of the clinical and laboratory manifestations of Gram-negative septicemia.^{38 39} The host response to Gram-negative septicemia is complex and may culminate in irreversible shock and death of the host. This shock state is frequently associated clinically with organ dysfunction, especially of lungs and kidneys, and the development of DIC, including widespread vascular injury, focal thrombosis of small vessels, and ischemic necrosis.

Death in septic shock is a result of multiple organ failure brought about in part by thrombotic occlusion of blood vessels of the microvasculature due to activation of the coagulation system and a decrease in fibrinolytic capacity.⁴⁰ There is ample evidence that these changes are brought about by LPS-induced activation of endothelial cells, which respond to LPS treatment in vitro with an increased expression of PAI-1 and TF, thereby changing from an anticoagulatory, profibrinolytic resting state to a procoagulatory, antifibrinolytic activated state.^{8 9} In accordance with these data, we could show that LPS treatment of cultured HUVECs resulted in a significant increase in PAI-1 and TF expression by such cells. However, when HUVECs were treated with LPS+NG-R1, the 3-fold upregulation of PAI-1 antigen and the >100-fold increase of TF activity induced by LPS alone were considerably attenuated, with about 83% and 37% suppression at 100 µg/mL NG-R1, respectively. This counteracting activity of NG-R1 on LPS-induced effects on PAI-1 and TF expression was also reflected at the levels of specific mRNA expression, as determined by Northern blotting. The 2-fold increase in PAI-1 mRNA levels and the 26-fold increase in TF mRNA levels induced by LPS were reduced to 1.37-fold and 13-fold above control levels, respectively, when cells were incubated with LPS+100 µg/mL NG-R1. This reduction in LPS-stimulated mRNA levels could be due to either a reduction in the transcription rate, eg, by activation of a repressor, or to an increase in mRNA degradation, resulting in a shorter half-life of these mRNAs.

Markedly increased levels of PAI-1 in plasma were observed in experimental animals after LPS infusion and in patients with septicemia.^{15 41 42 43} It has also been demonstrated that high serum PAI-1 levels even may serve as a prognostic marker for survival in septic shock.⁴⁴ In this study, we demonstrate that treatment of mice with LPS resulted in increased plasma levels of PAI activity in these animals, with peak values reached 2 to 6 hours after LPS injection. The 2.3-fold increase of PAI activity levels in the plasma of mice 4 hours after injection of LPS was completely abrogated in animals treated with LPS and NG-R1 simultaneously. Thus, the ameliorating effect of NG-R1 on LPS-induced increased PAI-1 levels was not restricted to the in vitro situation but rather was also operative in mice in vivo. It is noteworthy that 2 hours after LPS treatment, NG-R1 did not significantly reduce PAI plasma levels. This might be due to different pharmacokinetics of LPS and NG-R1; eg, one could speculate that NG-R1 has to be "processed" in the organism before being capable of counteracting LPS-induced effects.

In an in vivo model, as described above, LPS would activate not only endothelial cells but also monocytes. Current hypotheses for the pathogenesis of septic shock hold that microbial products such as LPS induce massive production and release of TNF- by monocytes, which in turn induces IL-1 production and release by macrophages and endothelial cells. Both TNF- and IL-1 have profound effects on vascular endothelial cells, leading to cell adhesion, vascular leakage, and shock.⁴⁵ TNF-, a product of stimulated monocytes and macrophages synthesized and released as a consequence of microbial stimulation and tissue injury, was initially identified in the circulation of animals after the injection of endotoxin.⁴⁶ The levels of circulating TNF- also increased rapidly in human subjects injected with LPS.^{47 48} Thus, TNF- seems to play a key role in septic shock. Therefore, we analyzed the effect of NG-R1 on TNF- production by leukocytes. Using cultured HWBCs ex vivo, we provide evidence that NG-R1 inhibits the LPS-induced production of TNF- by these cells. The production of TNF- by HWBCs treated with 1 ng/mL LPS was inhibited by 46% when the cells were incubated simultaneously with 100 µg/mL NG-R1. In this respect, it is noteworthy that the plasma levels of LPS in septic humans rarely exceed 1 ng/mL.⁴⁹ Thus, our data suggested that at such concentrations of LPS, NG-R1 has an inhibitory effect on TNF- production that may also be crucial in decreasing the in vivo toxicity of LPS. Therefore, we aimed to confirm the significance of these in vitro findings in vivo. Using a mouse model, we showed that mice challenged with LPS+galactosamine suffered from a mortality rate of 78% within 48 hours, whereas the mortality rate was significantly reduced to 23% when NG-R1 was administered simultaneously with LPS+galactosamine to mice.

Finally, we provide evidence that NG-R1, although it does not affect LPS binding to endothelial cells and the monocytic cell line THP-1, interferes with the NF-B pathway activated by LPS in inflammatory cells. Proteolytic degradation of IB- is a requirement for the activation and translocation of the aforementioned transcription factor.^{50 51} Thus, activation of NF-B is accompanied by a simultaneous degradation of IB- in LPS- or cytokine-activated cells.⁵¹ Here, we demonstrate that NG-R1 counteracts the LPS-induced decrease in IB- in the monocytic cell line THP-1 and, in addition, causes a superinduction of the LPS-induced increase in IB-–specific mRNA in these cells. Therefore, it is likely that NG-R1 attenuates LPS effects in inflammatory cells by raising the cytosolic levels of IB-, thereby preventing nuclear translocation of NF-B. The early increase in IB- protein seen 90 minutes after the addition of NG-R1 suggests a mechanism independent of new protein synthesis, because the increase in IB-–specific mRNA is seen only 3 to 5 hours after NG-R1 is added to the cells. The latter mechanism might, however, contribute to the sustained capacity of NG-R1 in, eg, counteracting LPS-induced mortality in the mouse model. In fact, it was shown recently that in endothelial cells, overexpression of IB- reduces the expression of activation markers such as vascular cell adhesion molecule-1, IL-1, -6, and -8, and TF.⁵²

In conclusion, we provide evidence that the saponin NG-R1 purified from the traditional Chinese herbal drug Panax notoginseng can counteract LPS-induced activation of endothelial cells and mononuclear cells in vitro, as demonstrated by its ability to counteract LPS-induced PAI-1 and TF expression in endothelial cells and TNF- production by human blood cells, as well as by reducing LPS-induced mortality in vivo in a mouse model. These effects might be at least partially due to interference of NG-R1 with the NF-B/IB- pathway. NG-R1 might therefore offer a therapeutic approach in the treatment of septic shock. In fact, extracts from the traditional Chinese herb Panax notoginseng, from which NG-R1 was purified, are used in traditional Chinese medicine to reduce inflammatory reactions and have been shown to ameliorate the symptoms of experimental DIC.^{19 20 21 22 23 24} The exclusive use of total extracts of Panax notoginseng in those studies does not allow us to compare plasma concentrations of NG-R1 with concentrations of NG-R1 used in our experiments. However, it is of considerable interest in this respect that although the clinical and pathological picture of septic shock has been known for many years, only a few substances that counteract LPS-induced effects have as yet been described. Neutralization of either TNF- or IL-1ß prevents lethality in animal models of sepsis.^{46 53 54} Only limited success of the use of antibodies against endotoxin has been reported recently.^{55 56} An effective inhibitor of LPS-induced endothelial cell activation and TNF- production by leukocytes such as NG-R1, which has been shown to reduce LPS-induced mortality in mice, could therefore be useful in reducing mortality due to septic shock in humans.

	Selected Abbreviations and Acronyms

 

DIC = disseminated intravascular coagulation ECGS = endothelial cell growth supplement HUVEC = human umbilical vein endothelial cell HWBC = human whole blood cell IB- = specific inhibitor of NF-B IL = interleukin LPS = lipopolysaccharide NG-R1 = notoginsenoside R1 PAI-1 = plasminogen activator inhibitor-1 SCS = supplemented calf serum SMP = skim milk powder TBS = Tris-buffered saline TF = tissue factor TNF- = tumor necrosis factor- TPA = tissue plasminogen activator TTBS = TBS+0.05% Tween-20

	Acknowledgments

 
During this study, Dr Zhang was supported by a North-South Dialogue scholarship (EH-project 894) from the Austrian Academic Exchange Service. This work was supported by grants from the Austrian Fund for the Promotion of Scientific Research to Drs Wojta (P9479) and Binder (P8011 and P8854). The authors acknowledge the artwork of T. Nardelli.

	Footnotes

 
Reprint requests to Johann Wojta, PhD, Department of Vascular Biology and Thrombosis Research, Schwarzspanierstr 17, A-1090 Vienna, Austria.

Received June 21, 1996; accepted December 5, 1996.

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