The adhesion of circulating leukocytes, especially monocytes, to vascular endothelium is a critical early event in the development of atherosclersis.1,2 This process depends on the interaction between cell adhesion molecules expressed on the surface of endothelial cells, such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and their cognate ligands on leukocytes.3,4 These cell adhesion proteins are normally at low level on the endothelial cell surface but are greatly induced by various proinflammatory cytokines such as IL-1 and TNFα.5,6
It has been shown that the signal transduction pathways for expression of intercellular cell adhesion molecules induced by TNFα include the activation and translocation of the redox-sensitive transcription factor NF-κB.7,8 NF-κB is present in the cytoplasm of unstimulated cells in an inactive form, complexed to the inhibitory protein IκB. Cellular activation by cytokines such as TNFα induces phosphorylation and degradation of IκB, with subsequent liberation and translocation of NF-κB to the nuclei,9 where it regulates many genes including adhesion molecules.7,10
Salvia miltiorrhoza Bunge (SM), a Chinese medicinal herb, has long been used for the treatment of atherosclerosis. It was also reported that it is effective in improving blood fluidity,11 inhibiting platelet aggregation,12 and protecting against peroxidative damage.13 Recent study indicated that it significantly decreased adhesion of leukocytes to TNFα-induced endothelial cells.14 However, its effect on adhesion molecule expression and the mechanism are not well elucidated.
In this paper, we tried to examine the ability of ESM to modulate the expression of adhesion molecules by TNFα-induced endothelial cells. We also attempted to find out whether the modulation is NF-κB dependent. We reported here that ESM is effective in inhibiting up-regulation of adhesion molecules in TNFα-induced endothelial cells; it also significantly inhibits the activation of transcription factor NF-κB. These results suggested that inhibition of ESM on NF-κB activation might be involved in its modulation on adhesion molecules.
MATERIALS AND METHODS
Endothelial growth medium was purchased from Clonetics (San Diego, CA). Trypsin/EDTA, penicillin, streptomycin, gelatin, L-Buthionine-(S,R)-sulfoximine (BSO), and o-phenyleneseven diamine dihydrochloride (OPD) were obtained from Sigma (St Louis, MO). Recombinant human tumor necrosis factor α (TNFα) was obtained from Pepro Tech EC Ltd (UK). Antibodies for ICAM-1, VCAM-1, NF-κB, p65, β-actin, and ECL reagent kit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals used were of the highest grade available commercially.
Preparation of Salvia miltiorrhoza Bunge Extract
Salvia miltiorrhoza Bunge was obtained from the Salvia miltiorrhoza Bunge GAP (Good Agricultural Practices) planting base of Tianjin Tasly Group Co, Ltd in Shangluo, Shaanxi province. For the extract preparation, the fresh air-dried Salvia miltiorrhoza Bunge was ground, passed through a 30-mesh screen, and extracted with water by refluxing for 2 hours and filtered. The filtrate was concentrated under reduced pressure. The concentration of danshensu and tanshinone II in the Salvia miltiorrhoza Bunge extract were more than 1% and 0.2%, respectively, as determined by HPLC described in quality standardization for composite Danshen Pills on Chinese Pharmacopoeia 2000. The extract was stored at −20°C before use. Aliquot portions of these extract residues were weighed and dissolved in medium for use on each day of our experiment. All experiments were performed with the same batch of Salvia miltiorrhoza Bunge extract.
Human umbilical vein endothelial cells (HUVEC) were isolated from freshly obtained human umbilical cords by collagenase type II treatment as previously described.15 The cells were cultured in gelatin-coated culture flasks containing endothelial growth medium supplemented with 15% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, at 37°C in a humidified environment containing 5% CO2. The endothelial cells were identified by their typical “cobblestone” morphology, and staining of Von Willebrand factor by immunocytochemistry. The purity of HUVEC in cultures was higher than 95%, and passages from 1 to 3 were used in this study.
Cell Viability Assay Using MTT
The 3-(4,5-dimethylthinazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure cell viability. Briefly, endothelial cells were counted and seeded onto gelatin-coated 96-well culture plates at a density of 5 × 104 cells/well. After incubation with various concentrations of ESM for 24 hours or TNFα for 6 hours, each well was washed twice with PBS to remove the medium; then 100 μL of MTT (0.5 mg/mL) was added to each well, and incubation continued at 37°C for an additional 4 hours. After that, 150 μL dimethyl sulfoxide (DMSO) was added to each well, and the absorbance (570 nm) was read on a microplate reader (BioRad 3550, Bio-Rad Laboratories). The absorbance was used as a measurement of cell viability, normalized to cells incubated in control medium, which were considered 100% viable.
Adhesion assays were performed by rose Bengal staining assay16 with slight modification. Briefly, endothelial cells were distributed into gelatin-coated 96-well culture plates and were allowed to reach confluence. Endothelial cell monolayers were divided into 5 groups: (1) exposed to media alone as a negative control; (2) exposed to media alone, then added HL-60 cells as an unstimulated control; (3) incubated with ESM (400 μg/mL) for indicated times, then added HL-60 cells; (4) incubated with TNFα (100 ng/mL) for 6 hours, then added HL-60 cells; (5) incubated with various concentrations of ESM for indicated times followed by TNFα (100 ng/mL) incubation for 6 hours, then added HL-60 cell. On the conclusion of varying incubations, for groups 2 to 5, HL-60 cells (3 × 106/mL) were added to endothelial cell monolayer in a volume of 100 μL each well. After coincubation at 37°C for 40 minutes, the wells were gently rinsed with PBS to remove nonadherent cells. HL-60 adhesion to endothelial cells was evaluated by rose Bengal staining, and absorbance value was measured with a microplate reader at 570 nm.
HL-60 cell adhesion (expressed as ΔA570 nm in Fig. 2B, see below) was calculated as follows:
Percentage inhibition of ESM on HL-60 adhesion was calculated as follows:
ELISA was performed on endothelial cell monolayers in gelatin-coated 96-well culture plates as described.17 Briefly, the endothelial cell monolayers were fixed for 30 minutes at 4°C with 4% paraformaldehyde in PBS and blocked with 2% BSA in PBS at 4°C overnight. The plates were incubated with specific primary antibodies of mouse anti-ICAM-1 or mouse anti-VCAM-1 for 2 hours at 37°C and then incubated for 1 hour with horseradish peroxidase-conjugated secondary antibody of goat anti-mouse IgG. After every step, the plates were washed 3 times with 0.05% Tween-20 in PBS. The expression of ICAM-1 and VCAM-1 was quantified by the addition of the peroxidase substrate o-phenyleneseven diamine dihydrochloride (OPD). The absorbance of each well was measured at 492 nm in a microplate reader. For negative groups, primary antibodies were replaced with PBS; the value (absorbance of sample subtracts that of negative group) was used as a measurement of adhesion molecule expression, normalized to cells incubated in control medium, which were considered 100%.
Western Blotting Assay for ICAM-1 and VCAM-1
For Western blotting, the endothelial cell monolayers were washed with PBS, centrifuged at 3000 rpm for 1 minute at 4°C, then lysed for 30 minutes at 0°C with lysis buffer (0.5 M NaCl, 50 mM Tris, 1 mM EDTA, 0.05% SDS, 0.5% TritonX-100, and 1 mM PMSF) and centrifuged at 15,000 rpm for 30 minutes at 4°C. The supernatants were applied to 8% SDS-PAGE and transferred to nitrocellulose membranes, which were then treated with 5% skim milk/TBS-Tween 20 (0.05%) at 4°C overnight. The membranes were incubated for 2 hours at room temperature with specific primary antibodies of mouse anti-ICAM-1 or mouse anti-VCAM-1. After washing, the membranes were incubated for 1 hour at room temperature with horseradish peroxidase-conjugated secondary antibody of goat anti-mouse IgG. Antigen detection was performed via ECL reagent and exposure to Kodak film. The band intensities were quantified using scanning densitometry. Actin expression was used as an internal standard for each sample. We have confirmed the specificity of these antibodies by omitting primary antibodies in preliminary experiments.
Western Blotting Assay for NF-κB p65 in the Nuclei
Endothelial cell monolayers were incubated with various concentrations of ESM for 18 hours followed by incubation with TNFα (100 ng/mL) for 30 minutes, and then the cells were washed with PBS. After centrifugation at 3000 rpm/min for 1 minute at 4°C, they were resuspended in 80 μL of buffer A (20 mmol/L Hepes, 25% glycerol, 1.5 mmol/L MgCl2·6H2O, 0.02 mol/L KCl, 0.2 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF), left on ice for 15 minutes at 4°C, followed by centrifugation at 6000 rpm for 2 minutes at 4°C. The nuclear pellets were resuspended in 70 μL of buffer B (20 mmol/L Hepes, 25% glycerol, 1.5 mmol/L MgCl2·6H2O, 1.2 mol/L KCl, 0.2 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF), shaken for 30 minutes at 4°C, then centrifuged at 15,000 rpm for 30 minutes at 4°C. The supernatants were applied to 10% SDS-PAGE and transferred to nitrocellulose membranes, which were then treated for 2 hours with specific rabbit antibody to NF-κB p65 and horseradish peroxidase-conjugated goat anti-rabbit second antibody for 1 hour. The protein belts were detected with ECL regent.
Endothelial cells were grown on gelatin-coated plates and treated as above experiment. Following fixing with 2% paraformaldehyde at 4°C for 30 minutes, the cells were treated with 0.2% Triton X-100. After treatment with blocking buffer (10% goat serum) for 30 minutes, the cells were incubated with specific primary antibody of rabbit anti-NF-κB p65 protein for 3 hours at room temperature, followed by incubation with peroxidase-conjugated secondary antibody of goat anti-rabbit IgG for 1 hour. After staining, the plates were dehydrated and mounted and observed under a microscope. The immunohistochemical specificity of antibody was confirmed by omitting the primary antibody in this protocol without final staining observed.
Statistical analysis was carried out using ANOVA and Dunnett test. For all tests, values of P < 0.05 were considered statistically significant.
Effect of ESM and TNFα on Cytotoxicity
Cell viability was assessed by incubating endothelial cells with various concentrations of ESM or TNFα for indicated times (Fig. 1). Treatment with 50, 100, 150, 200 ng/mL TNFα for 6 hours did not result in cytotoxicity (Fig. 1A). When incubated with 50, 100, 200, 400μg/ml ESM for 24 hours, cell viability did not show marked changes compared with media alone (Fig. 1B), significant cytotoxicity was observed at 800 μg/mL ESM for 24 hours; therefore, 400 μg/mL ESM was used as the maximum dose throughout subsequent experiments.
Effect of ESM on TNFα Induced Endothelial Cell Adhesion to HL-60 Cells
HL-60 cell is human monocyte-like cell; it was used to investigate HUVEC adhesion to leukocytes in previous studies.18,19 We examined the effect of TNFα on the adhesion of endothelial cells to HL-60 cells. In the unstimulated control group, HL-60 cell adhesion to endothelial cell was minimal, but it was dose-dependently increased by incubation with TNFα for 6 hours (Fig. 2A). Treatment with 100 ng/mL of TNFα was found to give almost maximal adhesion of HL-60 cells to endothelial cells, and this condition was therefore used in subsequent experiments, unless otherwise stated.
Next we determined the effect of ESM on TNFα-induced endothelial cell adhesion to HL-60 cells. In preliminary experiments, endothelial cells were incubated with ESM at the indicated times before addition of TNFα, simultaneously with TNFα or at the indicated times after addition of TNFα, we found ESM inhibited HL-60 cell adhesion to endothelial cells only when it was added before induction with TNFα. Figure 2B shows that pretreatment with ESM time- and dose-dependently reduced HL-60 cell adhesion to TNFα-induced endothelial cells. For example, pretreatment with higher concentrations of ESM (200 and 400 μg/mL) for 12 hours had caused reduction of 15.9% and 24.2%, respectively, whereas for 18 hours of pretreatment with ESM (200 and 400 μg/mL), the percentage inhibitions were 35.0% and 47.8%. Treatment with ESM alone had no effect on HL-60 cell adhesion. To obtain a visual picture, the adhesion morphology was taken under the microscope. As shown in Figure 3, HL-60 cells demonstrated very low basal adhesion to unstimulated or ESM alone-treated endothelial cell monolayers (Fig. 3A,B). In response to TNFα, however, a significant increase of HL-60 cell adherence to the endothelial monolayers was observed (Fig. 3C). Cytokine-induced cell adhesion was markedly reduced by pretreatment with ESM (400 μg/mL) (Fig. 3D).
Effect of ESM on Expression of Adhesion Molecules ICAM-1 and VCAM-1 in TNFα-Induced Endothelial Cells
With regard to the role of adhesion molecules in the process of leukocyte adhesion to endothelial cells, we evaluated the effect of ESM on the expression of ICAM-1, VCAM-1 in TNFα-induced endothelial cells by cell ELISA (Fig. 4). The results show that treatment of endothelial cells with TNFα resulted in increase in adhesion molecules ICAM-1 and VCAM-1 compared with cells treated with media only. ESM time-dependently inhibited enhanced adhesion molecules expression, with a maximal inhibitory effect detected at 18 hours of pretreatment (Fig. 4A). This effect was also concentration dependent (Fig. 4B). The results show that regulation of ESM on adhesion molecules expression may be responsible for its inhibition of HL-60 cell adhesion.
To conform these findings, Western blotting analysis was performed. As illustrated in Figure 5, substantially intensified belts of ICAM-1 and VCAM-1 were found for TNFα-stimulated groups, whereas the light belts of these adhesion molecules were seen for groups pretreated with ESM. Data of these experiments demonstrated that ESM dose-dependently attenuated expression of ICAM-1 and VCAM-1 in TNFα-stimulated endothelial cells, which is consistent with the results in Figure 4B.
Effect of ESM on Translocation of NF-κB p65 in TNFα-Induced Endothelial Cells
Nuclear translocation of NF-κB heterodimers from cytoplasm is prerequisite for activation of NF-κB-regulated genes. To determine whether NF-κB activation and nuclear translocation were involved in the regulation of ESM on adhesion molecule expression, we studied the effects of ESM on the translocation of NF-κB p65 by immunocytochemistry. As demonstrated in Figure 6B, TNFα-induced endothelial cells showed marked NF-κB p65 staining in the nuclei, whereas ESM-pretreated cells showed weaker staining in the nuclei (Fig. 6C). These data suggested that TNFα treatment facilitated the translocation of the NF-κB p65 protein from cytoplasm to nuclei, and ESM clearly prevented the TNFα-induced translocation of the NF-κB p65 protein.
In addition to the immunocytochemistry assay, we further studied NF-κB p65 protein levels in the nuclei of TNFα-induced endothelial cells by Western blotting. Consistent with in situ findings in Figure 6, a higher level of NF-κB p65 protein was found in the nuclei of TNFα induced endothelial cells compared with the control group, whereas ESM (200, 400 μg/mL) obviously reduced nuclear NF-κB p65 protein levels (Fig. 7).
BSO Reverses Inhibition of VCAM-1 Expression by ESM in TNFα-Induced Endothelial Cells
Because glutathione (GSH), which is present in human cells and plays a pivotal role in cellular antioxidant defenses, has been implicated in the regulation of NF-κB activation,7 we investigated the role of GSH in the inhibition of adhesion molecules by ESM. As shown in Figure 8,presence of 300 μM L-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of γ-glutamyl-cysteinyl synthetase, significantly attenuated the inhibition of VCAM-1 expression by ESM but had no effect on ICAM-1 expression.
Several lines of clinical evidence strongly show that preparations of SM are very effective in prevention and treatment of atherosclersis.20 Atherosclerosis is characterized by endothelial cell injury, which in turn leads to the up-regulation of adhesion molecule expression and resultant adhesion of leukocytes to the endothelium. Our results demonstrated that ESM significantly reduced the adhesion of HL-60 to TNFα-induced endothelial cells and effectively inhibited VCAM-1 and ICAM-1 expression. We also found that ESM significantly inhibited the activation of nuclear transcription factor NF-κB p65 by blocking its translocation into nuclei.
TNFα provides cell signals resulting in the activation of the redox-sensitive transcription factor NF-κB,21 and then NF-κB translocates from cytosol to the nucleus, where it binds to DNA and regulates transcription of many target genes including ICAM-1 and VCAM-1.7-10 Our data demonstrated that there was little NF-κB protein in nuclei of unstimulated cells, but the level of NF-κB protein was substantially increased on TNFα stimulation (Figs. 6 and 7). When HUVECs were preincubated with ESM, NF-κB protein level in nuclei was decreased, indicating that ESM inhibits NF-κB nuclear translocation.
Regulation of adhesion molecule expression is under the control of the endothelial NF-κB system.7,22 Analysis of promoter elements of adhesion molecule genes has revealed that ICAM-1 and VCAM-1 contain from 1 to 3 NF-κB binding sites.23-25 This suggests that activation of NF-κB induced by TNFα would stimulate the expression of adhesion molecule genes. In our experiment, ESM significantly inhibited increases of adhesion molecules ICAM-1 and VCAM-1 expression in TNFα-induced endothelial cells; meanwhile, it effectively inhibited NF-κB activation by blocking its translocation into the nucleus at the same concentrations. These experimental results demonstrated that ESM inhibition of NF-κB activation results in, at least in part, decreases in expression of adhesion molecules ICAM-1 and VCAM-1 in TNFα-induced endothelial cells.
Our study supports the notion that NF-κB is required for activation of adhesion molecule genes in endothelial cells. However, an important question is raised about how ESM inhibited NF-κB activation in TNFα-induced endothelial cells. TNFα stimulates production of ROS, including superoxide anions, hydrogen peroxide, and hydroxyl radicals, in a variety of cell types.23-25 ROS may function as second messengers in mediating TNFα-activated signal transduction pathways that regulate the NF-κB system.26 Water extract of Salvia miltiorrhiza Bunge mainly contains Danshensu, tanshinone, and salvianolic acids.13 In many previous studies, salvianolic acids were shown to exhibit strong antioxidant ability by scavenging ROS.13,27-29 It would be reasonable to presume that ESM's inhibitory effect on NF-κB activation induced by TNFα may result from its ROS-scavenging ability. In several other cell lines, NF-κB is activated by diverse stimuli, such as TNFα, IL-1β, LPA, and inhibited by the antioxidants pyrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC).30 Our study demonstrated a similar pattern of antioxidant (ESM)-sensitive inactivation of NF-κB.
GSH, acting as an intracellular antioxidant, plays an important role in maintaining the redox potential within cells. L-Buthionine-(S,R)-sulfoximine (BSO) is a specific inhibitor of γ-glutamyl-cysteinyl synthetase, the rate-limiting enzyme in cellular GSH synthesis. In our experiment, we found that BSO reverses the inhibition of VCAM-1 by ESM (Fig. 7). It is suggested that ESM may stimulate GSH synthesis, which in turn inhibits NF-κB activation and resultant adhesion molecule expression. However, we found that BSO only partially abrogates the inhibitory effect of ESM on TNFα-induced VCAM-1 expression but had no effect on ICAM-1 expression, indicating that VCAM-1 expression is more sensitive to intracellular redox status. Our findings are consistent with previous reports. Antioxidant PDTC markedly attenuates TNFα-induced VCAM-1 expression but not ICAM-1 in HUVECs.31 Magnonol, a potent antioxidant, pretreatment of human aortic endothelial cells significantly reduces the expression of VCAM-1, but not ICAM-1, induced by TNFα.32 These data suggest that VCAM-1 and ICAM-1 expressions are not modulated by a common signaling pathway, though they may share common regulatory signals immediately after interaction of TNFα and its receptor. A selective inhibition of cytokine-induced VCAM-1 expression has been described for other substances with antioxidant properties such as verapamil,33 probucol,34 or several flavonoids.35,36 The precise mechanism responsible for this selective inhibition of VCAM-1 but not ICAM-1 is subject of further investigation.
In this study, 100-400 μg/mL of ESM could achieve significant suppression of TNFα-induced adhesion molecule expression in HUVECs. These concentrations were equivalent to 0.51-2.04 μg/mL danshensu, the main active component of ESM.37 When 10 pills of Composite Danshen, therapeutic dose for patients with angina pectoris, were administered to subjects, danshensu plasma peak concentration was shown to be 1.13 μg/mL at 2 hours after administration.38 The changes of danshensu plasma concentration follow 1-compartment pharmacokinetics with elimination rate constant K = 0.125 hours−1 (half-life 5.54 hours), and absorption rate constant Ka = 1.69 hours−1 (half-life 0.410 hours). In the above study, danshensu plasma peak concentration is in a similar magnitude to the concentrations we used, suggesting our in vitro research with HUVECs provides certain clues for action mechanisms of ESM, but the underlying pharmacological basis of ESM should be further studied.
Our results revealed that ESM, extract of Salvia miltiorrhiza Bunge, strongly inhibited TNFα-induced NF-κB activation by repressing its nuclear translocation; it also down-regulated ICAM-1 and VCAM-1 expression and resultant leukocyte adhesion to TNFα-induced endothelial cells. The inhibition of ESM on NF-κB activation may be associated with its antioxidant ability. Although the mechanism of the beneficial effect of ESM against cytokine-induced endothelial cell activation remains to be elucidated, it was suggested that ESM affects the NF-κB signaling pathway in endothelial cells. The findings of the present study may shed light on the pharmacological basis for clinical application of Salvia miltiorrhiza Bunge on the prevention and treatment of atherosclerosis.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
Salvia miltiorrhoza Bunge; tumor necrosis factor (TNF)α; endothelial cell; adhesion molecule; nuclear factor κB