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The ginseng root has a long history of medical use in China for more than 2000 years as a tonic to combat stress agents. Ginsenosides are the most active components isolated from ginseng,［1］ which can protect against myocardial ischemia-reperfusion damage and protect endothelium against electrolysis-induced free radical injury.［2］ Ginsenoside Rg1 (Rg1) is a steroidal saponin of high abundance in ginseng and can relax aorta and pulmonary vessels via the action of nitric oxide release.［2］ Rg1 can also stimulate the formation of nitric oxide in cultured bovin aortic endothelial cells. Cardiovascular protection by ginsenosides may be partly mediated by the release of nitric oxide (NO), a potent antioxidant.［2］ However, the molecular mechanism of endothelial protection by Rg1 is not well known up to now. Rg1-induced alterations in gene expression in endothelial cells may help to understand its action and classify its molecular mechanism of endothelial protection. In this study, we systematically investigated Rg1-induced alterations in gene expression in cultured human umbilical vein cells by using DNA microarray technology.
DNA microarrays provide a powerful means to query the relative transcript abundance of many genes in parallel.［3］ With the development of DNA microarray technology, oligonucleotide-based DNA microarrays are useful for the analysis of gene expression.［4］ Our lab has developed an oligonucleotide microarray platform, which has been proven to be a useful tool to study the gene expression profile.［5］ Based on this, a cardiovascular oligonucleotide microarray which contains approximately 400 cardiovascular diseases related genes was constructed and employed to investigate Rg1-induced alterations in gene expression in TNF-αstimulated endothelial cells. The vascular endothelium is an important regulatory in maintaining cardiovascular homeostasis and the endothelial dysfunction is present in several cardiovascular diseases. Endothelial dysfunction is an early marker of atherosclerosis and is often defined as the impaired release of nitric oxide (NO) by the endothelium.［6］ In this study, TNF-α stimulation that decreased NO production in HUVEC was used to set up an in vitro model of endothelium dysfunction. The effect of Rg1 on NO production in endothelium dysfunction model was detected. And then Rg1-induced alterations in gene expression in TNF-α stimulated HUVEC was investigated by home-made cardiovascular oligonucleotide microarrays.
METHODS
Drugs and reagents
Ginsenoside Rg1 was a standard product from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing. NO assay kit was purchased from Nanjing Jiancheng Institute of Biological Engineering, China. TRIzol reagent, Superscript ⅡTM Reverse Transcription kit and Medium199 were purchased from Gibco BRL, USA. Cy3-dUTP and Cy5-dUTP were purchased from Amersham Pharmacia Biotech, USA. TNF-α and collagenase type Ⅰ were purchased from Sigma Co, USA. Other analytically pure reagents were domestically produced.
Cell culture
Human umbilical vein endothelial cells (HUVECs) were prepared according to a modified version of the protocol of Jaffe et al.［7］
Treatment of TNF-α and gingsenoside Rg1 and assay of NO
After HUVECs reaching confluence, cells were harvested by trypsin digestion, and then transferred to a 96 well microplate and cultured till 80% confluent. The cell monolayers were washed twice with phosphate buffered saline (PBS) and then 0.1 ml serum-free M199 medium containing the indicated concentration of TNF-α were added to each well. After cultured for 6 hours, the medium was collected and the amount of NO released by HUVECs was determined using a NO assay kit according to the manufacturer's protocol. And then a dose of 2.5 ng/ml TNF-α was chosen to perform an in vitro endothelial dysfunction model. HUVECs were seeded into another 96 well microplate; 0.1 ml serum-free M199 medium containing the indicated concentration of Rg1 were added to each well. After 18-hour, TNF-α (2.5 ng/ml) were added to the medium and co-cultured for 6 hours, and then the medium was collected. The NO production in the medium was detected by the same NO assay kit.
Construction of oligonucleotide microarray
Selection of detected genes
To detect the expression alterations of some cardiovascular disease related genes, a low density oligonucleotide microarray was constructed. About 400 cardiovascular disease related genes were identified from Cardio (Cardiovascular Disease Related Database). Homo sapiens mRNA sequences of these genes were picked from GenBank.
Design of oligonucleotide probes
Forty-mer oligonucleotide probes were designed by a self-made software-Mprobe.［8］ The probe with the highest predicted specificity was selected to represent its source sequence. To normalize varying efficiencies of labeling and detection, several housekeeping genes (genes encoding ribosomal protein S32, aldolase B, GAPDH, ALB, highly basic protein, and β-actin), positive control (the firefly luciferase gene) and negative control (rice gene and rpoB) were added.
Sy nthesis of probes and printing
Olignucleotide probes were synthesized using PE8909 DNA Synthesizer (Applied BiosystemsPE, USA). The 5' terminals of probes were modified with amino-linker (MMT-Amidite) at the last step of synthesis. Oligonucleotide probes were solubilized at 0.5 μg/μl in 3×SSC and were spotted on aldehyde coated glass microscope slides by using PixSys 7500 (Cartesian Technologies, USA), and then the slides were stored at room temperature. The slides were washed in 0.2% SDS for 3 minutes, in pure water for 2 minutes, and dried in the air before use.
Preparation of total RNA
HUVECs of endothelial dysfunction group were exposed to 2.5 ng/ml TNF-αfor 6 hours without Rg1 pretreatment, while HUVECs of Rg1 group were pretreated with 20 μg/ml Rg1 for 18 hours and then exposed to 2.5 ng/ml TNF-α for another 6 hours. Total RNA was extracted from HUVECs of endothelial dysfunction group and HUVECs of Rg1 group respectively by the TRIzol reagent according to the manufacturer's instructions. The amount and the quality of RNA were checked using electrophoresis on a 1% formamide agarose gel.
Preparation of fluorescent labeled cDNA
The fluoresent labelled probes were prepared essentially as preveously described.［9］ Fifty to 100 μg total RNA was used in the labelling reaction; 0.3 μg in vitro transcripted firefly luciferase mRNA was spiked into the total RNA as positive control to assist in quantification and estimation of experimental variation introduced during labelling and analysis.［10］
Reverse labelling was employed to eliminate the Cy-dye labelling difference, and three replicates were performed to decrease random error.
Hybridization and washing
The two cDNAs pools from endothelial dysfunction group and Rg1 group were mixed and the methods of hybridisation and washing were performed as preveously described.［5］
Scanning and data analysis
Hybridized slides were scanned using a GenePix 4000B simultaneous dual wave length scanner (Axon Instruments Inc, USA). The data obtained were analysed using GenePix pro 4.0 software (Axon Instruments Inc, USA). Intensity values were normalized using positive controls and housekeeping genes. After the quality of each point was evaluated, the data sets of selected genes (median of ratios ≥1.5 or ≤0.66) were extracted into a Microsoft Excel.
Statistical analysis
Data were expressed as mean±standard error. The statistical analysis was evaluated by Student's t test. A P value <0.05 was considered significant.
RESULTS
The effect of ginsenoside Rg1 on the nitric oxide production in TNF-α stimulated HUVECs
TNF-α treatment decreased NO production in HUVECs significantly ( Fig. 1 ), indicating that the endothelial dysfunction may occur. Then a dose of 2.5 ng/ml was chosen to perform an in vitro endothelial dysfunction model in order to investigate the effects of Rg1. Rg1 (1.25-20 μg/ml) increased the NO production in TNF-α stimulated HUVECs in a dose dependent manner ( Fig. 2 ). A dose of 20 μg/ml Rg1 was chosen to perform oligonucleotide microarray analysis.
Ginsenoside Rg1-induced alterations in gene expression in TNF-α stimulated endothelial cells
Fig. 3 is an example of false-color images, where hybridization with cDNAs derived from cells of two groups is indicated by Cy-5 fluorochrome (red) and Cy-3 fluorochrome (green) signals, respectively. Frame rounds with indicated colors were drawn to highlight quality control points ( Fig. 3 ), which showed the microarray results with satisfied specificity and sensitivity. Fig. 4 shows one of scatter plots of partial genes spotted on our microarray. The two lines in the graph represent 1.5- and 0.5-fold changes in expression. Those points outside the two lines represent genes with altered expression levels. Those genes present outside at lest three times were picked up and classified in Table . Table displays the regulated genes by Rg1. It shows that Rg1 affected the mRNA levels of genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in TNF-α stimulated HUVECs.
DISCUSSION
Endothelial cells (EC), which are the essential constituents of vessel, are known to modulate vascular reactivity by releasing vasoactive substances, such as nitric oxide (NO). Constitutively expressed eNOS produces low concentrations of NO, which is necessary for a good endothelial function and integrity.［11,12］ So we chose NO production in EC as an indicator of endothelial dysfunction in this study. TNF-α, a pro-inflammatory cytokine,decreases eNOS mRNA and eNOS protein in EC.［13］ In this study, TNF-α treatment resulted in decrease of NO production in EC, indicating that endothelial dysfunction may occur. Rg1 can attenuate the effect of TNF-α on NO production in EC, and microarray analysis showed an increased mRNA level of eNOS in Rg1 treated EC. These findings imply that the endothelial protection mechanism by Rg1 may involve the increased expression of eNOS and enhanced NO production in EC.
In addition to discovering upregulation of eNOS mRNA by Rg1, microarray analysis showed that Rg1 affected mRNA level of about 30 genes, which were involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction. Microarray analysis provides us with lots of information about important genes and valuable insights into the action of Rg1.
Vascular endothelium is a major target of actions of TNF-α and the activation by TNF-α enhances expression of cell surface adhesion molecules, which in turn facilitates the attachment of blood leukocytes to endothelial surfaces. Leukocyte adhesion to the endothelium via adhesion molecules is one of the earliest events in atherosclerosis. This study demonstrates that Rg1 can down-regulate the expression of adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin. This result indicates that Rg1 can decrease leukocytes adherence to the endothelium and may protect EC from TNF-α activation.
Rg1 can also regulate the expression level of some important genes that are involved in coagulation, such as the downregulation of PAI-1. Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type plasminogen activators (t-PAs) and urokinase-type plasminogen activators (u-PAs). PAI-1 regulates intravascular fibrinolysis and tissue proteolysis, and thereby controls thrombus dissolution. EC produces low amounts of PAI-1 under normal resting conditions. PAI-1 expression is up-regulated by inflammatory mediators -TNF-α.［14］ Rg1 can upregulate the expression of PAI-1 mRNA, which indicates that Rg1 may have the potential to attenuate the prothrombotic effect of TNF-α.
Another interesting set of genes is a set of growth factors such as VEGF, bFGF, EGF, TGF-β1 and IGF. It is reported that vascular endothelial growth factor (VEGF) induces vascular smooth muscle cells (VSMC) migration but has no significant activity on proliferation.［15］ Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) can mediate the migration and proliferation of VSMC.［16］ TGF-β1 can promote VSMC proliferation.［17］ Migration and proliferation of vascular smooth muscle cells (VSMC) contribute to angiogenesis and the lesions of atherosclerosis. Rg1 can downregulate the expression of VEGF, bFGF, TGF-β1 and EGF, which implies that Rg1 can inhibit VSMC migration and proliferation and can have beneficial effect on post-angioplasty resteonosis.
Endothelin type-B receptor (ETB) is another gene in which we are interested very much. ETB receptors are non-specific receptors for ET-1, 2 and 3. The activation of endothelial ETB receptors stimulates the release of NO and prostacyclin, prevents apoptosis, and inhibits ECE-1 expression in endothelial cells.［18］ Our results showed Rg1 upregulated the mRNA level of ETB, thus suggesting that stimulation of ETB receptors may be another mechenism of endothelial protection by Rg1.
In summery, priming of quiescent EC with inflammatory cytokine-TNF-α can switch on the EC transcription machinery, the genes and protein products of which can activate EC. Microarray analysis demonstrates that Rg1 can attenuate the effects of TNF-α by altering the expression level of adherence molecules, growth factors and coagulation-related molecules. Rg1 may have a potential to protect EC from TNF-α activation. Ginsenoside Rg1 can enhance NO production and the expression level of eNOS in TNF-α stimulated EC. Ginsenoside Rg1 altered the expression level of genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in TNF-α activated ECs. Microarray analysis provides us with valuable insights into the atheroprotection mechanism by ginsenoside Rg1 from a large scale.
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