Atherosclerosis (AS) is a severe disease worldwide, which leads to alterations and lesions in the inner walls of the blood vessels. Injury to the vascular endothelium may be the initiating event in the etiology of AS.1
Endothelial cells cover the inner side of blood vessels. They constitute an active and selective barrier between blood and tissues, with a thromboresistant and hemocompatible capability under normal conditions. Endothelial dysfunctions, especially elicited by oxidized low-density lipoprotein (ox-LDL), play a critical role in the pathogenesis of AS.2,3 For the treatment of AS, statins are widely used. Although considered effective, their side effects are also reported.4 New drugs with better efficacy and less toxicity await further exploration and development. Herbal drugs from Traditional Chinese Medicines might serve as good candidates.
In recent years, the clinical importance of herbal drugs has received considerable attention. Flavonoids existing in herbal drugs and diets are known to possess a wide range of biologic functions. As reviewed by Middleton, the plant flavonoids exhibited strong antioxidant activity and were applied in inflammation, heart disease, and cancer on mammalian cells.5 Many flavonoids also showed inhibitory activity toward cyclic nucleotide phosphodiesterases.6 The consumption of fruits, vegetables, and beverages containing flavonoids may lower the risk of cardiovascular diseases, including AS and hypertension.7,8
Since the 1950s, many medicinal preparations of Seabuckthorn, also called Hippophae rhamnoides L, from wild and cultivated materials have been widely used in cardiovascular disorders, radiation damage, and inflammation treatment.9-11 Flavonoids, such as quercetin, isorhamnetin, kaempherol, etc, have been identified in seabuckthorn.12 The antioxidant and free radical scavenging properties, inhibition of LDL oxidation capabilities, antitumor activity as well as the activity against ox-LDL induced cell apoptosis of quercetin have already been reported recently.13-15
However, the intracellular factors and enzymes with which the flavonoids from seabuckthorn (FSBT), quercetin and isorhamnetin interfered to prevent the cyto-toxicity of ox-LDL are still unclear. This promoted us to investigate whether FSBT protect EA.hy926 from ox-LDL injuries via modulated the expression of endothelial constitutive NO synthase (eNOS) and lectin-like low-density lipoprotein receptor-1 (LOX-1), a recently identified receptor for ox-LDL in endothelial cells.16-19 And it also promoted us to uncover whether the antioxidant enzyme superoxide dismutase (SOD), superoxide, and NO contribute to the effects of FSBT.
FSBT was supplied by Zhejiang Conba Pharmaceutical Co Ltd, China; Low-density lipoprotein was supplied by Institute of Biochemistry, Peking Union Medical college; Dulbecco modified Eagle medium (DMEM), DPPH (2, 2-diphenyl-1-picrylhydrazyl), dimethylsulfoxide (DMSO), quercetin, epicatechin, and cytochrome C were obtained from Sigma (St Louis, MO); Isorhamnetin was provided by National Institute for the Control of Pharmaceutical and Biological Products (NICPBP, Beijing, PRC); lactate dehydrogenase (LDH) and SOD assay kits were purchased from Nanjing Jiancheng Bioengineering Institute; the primers, Taq polymerase, dNTP, Rnasin, MMLV were provided by Shanghai Sangon Biological Engineering Co Ltd; Rabbit anti-NOS3 antibody, second antibody, ABC kit were provided by Santa Cruz Biotechnology Inc.
Endothelial cell line EA.hy926 is the kind gift of Dr Edgell, and is well characterized for its endothelial biology.20,21 Cells were grown in DMEM supplemented with 10% fetal bovine serum. The cultures were maintained in a humidified atmosphere containing 5% CO2 at 37°C. After reaching 80% to 90% confluence in plates, the medium was replaced by serum-free DMEM and used for experiments.
Components Identification (HPLC/ESI/MS Assay)
FSBT was supplied by Zhejiang Conba Pharmaceutical Co Ltd, China. It was separated and identified by High pressure liquid chromatography. The assay was performed on a Shimadzu LC-10AT liquid chromatograph system equipped with 2 LC-10ATvp pump and SPD-10Avp UV-VIS detector. Data were processed with N2000 Shimadzu chromatocorder. The sample was separated by a C18 (Particle size 5 μm) column (250 × 4.6 mm, ID) using methanol: 0.4% H3PO4 = 60:40 as mobile phase at flow rate of 0.5 mL/min and was detected at 270 nm with injection volume of 10 μL. All HPLC analysis were performed at 20 ± 1°C. The mass spectrometer was equipped with an electro-spray ionization source. The analyzing conditions were as following: ion polarity positive, capillary 3.5 kV, skimmer 50 V, dry inert gas N2, gas temperature 300°C, scan begin m/z 100.00, and scan end m/z 800.00.
Oxidation of Low-density Lipoprotein
Ox-LDL was prepared according to the method of Sternberg.22 A 10-mg sample of LDL was dialyzed against Tris/NaCl buffer (50 mmol/L Tris in 0.15 mol/L NaCl, pH 8.0) to remove the ethylenediaminetetraacetic acid (EDTA). Tris/NaCl buffer was added to the dialyzed LDL to adjust the protein concentration to 30 mg/mL. One milliliter of 10 μmol/L CuSO4 was added to 1 mL of dialyzed LDL. Oxidation at 37°C for a period of 18 hours was followed by the addition of 300 μmol/L EDTA to stop the reaction. The ox-LDL was then dialyzed at 4°C with 4 L Tris/NaCl buffer, filtered with a 0.22 μm filter, and stored in nitrogen at 4°C.
Oxidation was monitored by the estimation of thiobarbituric acid reactive substances using malondialdehyde assay kit according to the manufacturer's instructions.
DPPH Radical Scavenging Activity of FSBT, Quercetin, and Isorhamnetin
The DPPH assay was used to measure the antiradical activity of FSBT and 2 major flavonoids contained in it according to the method in.23 Briefly, DPPH solution was freshly prepared by dissolving 4.5 mg of DPPH powder in 100 mL of methanol, and the sample solution was prepared by dissolving 2.5 mg of FSBT in 1 mL of DMSO. Then, 15 μL of the sample solution was added to 985 μl of DPPH solution at room temperature. The absorbance was measured at 517 nm after 2 minutes of the reaction. Quercetin and isorhamnetin, with the end concentration of 15 μg/mL, were also evaluated and DMSO was used as negative control. The results were expressed as percentage of DPPH elimination.
Cell Viability and LDH Release Assay
The viability of cells was determined by trypan blue dye exclusion test. Trypan blue is an acid dye that stains nonviable cells.24 EA.hy926 cells were cultured in 24-well plates at a density of 1 × 105 cells/mL and allowed to grow to the desired confluence. The cells were pretreated for 20 minutes with 9.38 to 37.5 μg/mL FSBT, 15 μg/mL quercetin, 15 μg/mL isorhamnetin, or corresponding amount of solvent and were then exposed to 100 μg/mL ox-LDL for another 24 hours. In other group, the cells were treated with FSBT, quercetin, and isorhamnetin without ox-LDL addition. After incubation, the cells were digested by Puck EDTA and stained by 0.4% trypan blue dye solution. The percentage of LDH released into the media was measured as following: after incubation the cells were lysed in the ice-cold lysis buffer, and then the lysates and the supernatants were analyzed to determine LDH activity using an analysis kit according to the manufacturer's instructions. Data were presented as the percentage of cells excluding trypan blue dye and the percentage of LDH released to the supernatants.
SOD and NO Release Assay
EA.hy926 cells were cultured after the procedure as described in the Cell Viability and LDH Release Assay section. After incubation, the supernatant was collected from plates and the SOD content was determined using SOD assay kit according to the manufacturer's instructions. The NO released into the media was measured by the Griess assay, briefly, 100 μL of supernatant was mixed with 100 μL Griess reagent (1% sulfanilamide and 0.1% N-naphthylethyl-ethylenediamine dihydrochloride in 5% phosphoric acid were mixed at a ratio of 1:1), the absorbance was measured at 550 nm, and NO concentration was determined using a curve calibrated on sodium nitrite standards.
Superoxide generation was detected by following the SOD sensitive cytochrome C reduction method as previously described.25 Cells were grown in 24-well plates in a density of 105 per well. After incubation cells were mixed with 26.5 μmol/L cytochrome C with or without SOD (20 μg/mL) in DMEM without serum at 37°C. Cells were stimulated by ox-LDL just before the addition of cytochrome C, and the reaction was terminated in 2 hours. In other groups, cells were pretreated with different concentrations of FSBT, quercetin, or isorhamnetin for 20 minutes before the addition of ox-LDL. The supernatants were collected and the absorbance at 550 nm was recorded in UV-visible spectrophotometer. Superoxide production was estimated on the absorption peak at 550 nm indicating the concentration of reduced cytochrome C. Results are expressed in nmol of superoxide generated by per 105 cells.
Reverse Transcription Polymerase Chain Reaction for eNOS mRNA and LOX-1mRNA Expression
After incubation, the cells were washed twice with phosphate buffered saline (PBS) and the total mRNA was extracted by Trizol. Thereafter, it was reverse-transcripted as following program: 25°C 10 minutes, 42°C 1 hour, 72°C 15 minutes, and the cDNA product was stored at −80°C.
For the PCR, 1.5 μL of the cDNA product of each sample was amplified with Taq DNA polymerase, using a primer pair26 specific to human eNOS (forward primer, 5′-GCT GCG CCA GGC TCT CAC CTT C-3′, reverse primer, 5′-GGC TGC AGC CCT TTG CTC TCA A-3′, 36 cycles, 554 bp), LOX-1 (forward primer, 5′-TTG CTG CAC ACA ATC TAG CA-3′, reverse primer, 5′-CGA GCA TCA AGA TGG AGA CA-3′, 30 cycles, 394 bp) and β-actin (forward primer, 5′-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3′, reverse primer, 5′-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3′, 610 bp). The annealing temperatures for eNOS and LOX-1 were 62.8°C and 57.3°C, respectively. Finally, the PCR amplified samples were visualized on 1.5% agarose gels using Quantity One to analyze.
Western Blot Detection of eNOS
After incubation, the cells were lysed in the ice-cold lysis buffer, and the protein concentration of the lysates was determined by Lowry method. Cell lysates from each experiment group (30 μg per lane) were electrophoretically separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. After incubation in blocking solution (4% nonfat milk), membranes were incubated with 1:500 dilution primary antibody to human eNOS (Santa) for overnight at 4°C. Membranes were washed and incubated with 1:5000 dilution of the second antibody for 1 hour. Relative intensities were analyzed by Quantity One software.
Immunocytochemical Detection of eNOS
Endothelial cells were planted on glass slides in a 24-well plates at 5 × 104 cells per well and were allowed to grow to confluence. Cells were then treated with 37.50 μg/mL of FSBT, 15 μg/mL quercetin, or 15 μg/mL isorhamnetin for 20 minutes before incubation with ox-LDL for 24 hours. In other groups, the cells were treated with FSBT or corresponding amount of solvent without ox-LDL addition. At the end of incubation, cells grown on the slides were washed twice gently with PBS at 4°C for 5 minutes and fixed with 4% paraformaldehyde for 10 minutes. Washed again in PBS, slides were immersed in cold 30% (vol/vol) H2O2 for 5 minutes followed by normal goat serum for 20 minutes at room temperature. Next, slides were covered with eNOS antibody (1:500 dilution in PBS) and left for overnight at 4°C. Then the slides were incubated with the secondary antibody and the avidin-biotin-antibody after the kit instructions. Afterwards, slides were incubated in fresh 3, 3-diamino-benzidine solution until suitably stained (about 10 min), and were counterstained with hematoxylin, and then photographs were taken. Negative control without first antibody incubation was systematically included.
All data are represented in the statistics of 3 independent experiments in the form of mean ± SD. The significance of the difference was analyzed by Student-Newman-Keuls test. A value of P < 0.05 was considered statistically significant.
We scanned the absorbance at different wavelengths between 200 to 400 nm and chosen 270 nm to analyze the composition of FSBT. At this wavelength most of the flavonoids have absorbance. HPLC chromatogram of FSBT is shown in Figure 1A. The peak 1, with retention time of 5.7 minutes, is the absorbance of vehicle DMSO and there are 2 other major peaks corresponding to components in FSBT (2 and 3) with retention time of 13.6 and 22.4 minutes, respectively. The retention times of these 2 peaks are consistent with those of the standard sample quercetin and isorhamnetin, which are 13.5 and 22.4 minutes, respectively. Mass spectrometer was used to confirm the components of FSBT. The 2 components with peaks 2 and 3 were shown to have the m/z values of 301 and 315 corresponding to [M-H]− of quercetin and isorhamnetin, respectively (data not shown). We thus draw the conclusion that the 2 major components of FSBT are quercetin (peak 2) and isorhamnetin (peak 3), which consisted 57.7% and 25.4% of FSBT, respectively. Their structures are shown in Figures 1B and C. We also excluded the existence of epicatechin, which were reported to be endothelium preserving in vitro,27 by the measurement on standard samples. The retention time of epicatechin was 4.7 minutes under the assay conditions above.
DPPH Radical Scavenging Activity of FSBT, Quercetin, and Isorhamnetin
DPPH has an active free radical when dissolved in methanol. The radical is stable with a maximum absorbance at 517 nm and can undergo reduction by antioxidant. Because of the ease and convenience of this reaction, it is now of wide use in the free radical-scavenging activity assessment.23 FSBT (37.50 μg/mL) is an excellent DPPH radical-scavenger, which scavenges 46.5% of DPPH in 2 minutes under the experimental conditions, approximately half of the antiradical capability of 15 μg/mL quercetin (91.8%) and higher than 15 μg/mL isorhamnetin (38.0%).
Cell Viability and LDH Release Assay
Lipid oxidation of LDL catalyzed by copper ions is 36.9 times more than that of native LDL, the malondialdehyde is 0.70 ± 0.12 nmol/mg protein for native LDL and is 25.94 ± 1.90 nmol/mg protein for oxidized LDL. Trypan blue dye exclusion test and the percentage of LDH released into the media were used in the present study to evaluate whether FSBT, quercetin, and isorhamnetin protected endothelial cell line EA.hy926 from ox-LDL induced cell injuries. As shown in Table 1, ox-LDL treatment for 24 hours significantly decreased the cell viability and promoted LDH release (P < 0.01). Pretreatment of FSBT significantly increased the cell viability and suppressed the LDH release in a concentration dependent manner. Both 15 μg/mL quercetin and 15 μg/mL isorhamnetin exhibited the protective effects similar to FSBT. Although quercetin shows a higher efficacy than isorhamnetin in inhibition of LDH release. Each of FSBT, quercetin, and isorhamnetin alone has no effect on cell viability and LDH release (data other than FSBT with concentration of 37.50 μg/mL are not shown).
NO and SOD Release Assay
NO and SOD are the most important substances endogenously generated to maintain the functional integrity of endothelium monolayer to prevent vascular leakage and formation of the AS.28 NO and SOD contents were also measured to determine the effects of FSBT on ox-LDL injured cells. As shown in Table 2, incubation of endothelial cells with ox-LDL resulted in the remarkable reduction of NO and SOD content, whereas FSBT pretreatment attenuated the ox-LDL induced decreases of NO and SOD. Both 15 μg/mL quercetin and 15 μg/mL isorhamnetin exhibited effects similar to FSBT. Although quercetin has shown a higher efficacy in SOD generation than isorhamnetin, FSBT and isorhamnetin alone has no effect on NO and SOD release (only data of FSBT with concentration of 37.50 μg/mL have been shown), whereas quercetin alone slightly increased the NO release as reported before29 (data not shown).
Endothelial cell line EA.hy926 generated small quantities of superoxide in the absence of ox-LDL (Fig. 2). The basal superoxide production may represent a nonspecific effect related to the interaction between the cells or between cells and other particles in the incubation medium. Incubation of endothelial cell line EA.hy926 with ox-LDL significantly (P < 0.01) increased the generation of superoxide (3.04 times more than that of control), whereas pretreatment of FSBT concentration-dependently inhibited this effect of ox-LDL, whereas FSBT alone has no effect on superoxide generation. Both 15 μg/mL quercetin and 15 μg/mL isorhamnetin showed the similar superoxide-suppressive effects. However, each of FSBT, quercetin, and isorhamnetin alone has no effect on superoxide production (data other than FSBT with concentration of 37.50 μg/mL are not shown).
Modulation of eNOS and LOX-1 Expression by FSBT, Quercetin, and Isorhamnetin
It is confirmed in present study that ox-LDL reduces eNOS expression and increases LOX-1 expression.16-19 The mRNA expression of LOX-1 and eNOS (Fig. 3A) was measured by reverse transcriptase-polymerase chain reaction, eNOS protein expression (Fig. 3B) was measured by Western blot analysis in cell lysates. Treatment of endothelial cell line EA.hy926 with 100 μg/mL ox-LDL for 24 hours increased LOX-1 mRNA significantly to about 2.5 times more than that of control and decreased eNOS mRNA to about 58.9% that of control (Fig. 3A, lane 2) accompanied with a significant decrease of eNOS protein expression (Fig. 3B, lane 2). Pretreatment of FSBT prevented ox-LDL mediated decrease in eNOS and increase in LOX-1 expression in a concentration dependent manner (Figs. 3A, B, lanes 3-5). FSBT alone showed no effect on eNOS mRNA and LOX-1 mRNA expression while slightly increased eNOS protein expression (only data at the concentration of 37.50 μg/mL are shown. Figs. 3A, B, lane 6). Similar effects were shown with 15 μg/mL of quercetin and isorhamnetin (Figs. 3A, B, lanes 7, 8), with the former shown a higher efficacy on eNOS protein expression than the latter (Fig. 3B, lanes 7, 8).
Immunocytochemical Detection of eNOS
Figure 4 illustrates eNOS immunoreactivity in ox-LDL injured EA.hy926 pretreated with FSBT, quercetin, and isorhamnetin, or treated with FSBT alone. Consistent with the data from Western analysis, immunocytochemistry demonstrated notable eNOS staining intensity in control group, which was treated with the vehicle only (Fig. 4B). Moreover, the staining was localized within the cytoplasm of the cells. The eNOS staining intensity was remarkably decreased after treatment with ox-LDL for 24 hours (Fig. 4C). Pretreatment of FSBT (37.50 μg/mL) on EA.hy926 markedly reduced the inhibition effect of ox-LDL (Fig. 4D). Although FSBT (37.5 μg/mL) alone has no effect on eNOS protein expression, as shown in Figure 4E, the cells exhibited almost the same intensity of staining as control group consistent with the results from Western blot. Both 15 μg/mL quercetin and isorhamnetin showed inhibitory effects on ox-LDL induced eNOS down-regulation, with quercetin exhibited a stronger effect (Figs. 4F, G). The negative control with non-immune serum was entirely negative for staining, assuring the specificity of this reaction (Fig. 4A).
The present study demonstrated that FSBT, quercetin, and isorhamnetin protected EA.hy926 from ox-LDL induced cell death and secretion disorders. Study on EA.hy926 also showed that FSBT attenuated ox-LDL mediated LOX-1 up-regulation and increased ox-LDL induced eNOS down-regulation, indicating this receptor and synthase are involved in the protective mechanisms. The mechanisms also involve the improvement of cellular antioxidant defense and the decrease of superoxide production. Quercetin and isorhamnetin showed the similar effects as FSBT, implicated that they may be partially responsible for the protective effects of FSBT.
The previous in vitro studies revealed that flavonoids may have considerable antioxidant activity in various chemical oxidation systems.30-32 In the present study, 2 major components were identified, they are quercetin and isorhamnetin. DPPH assay showed powerful antioxidant capacity of FSBT, quercetin, and isorhamnetin in the cell-free systems, implicating this effect of FSBT may at least partially due to the later 2 major flavonoids.
The antioxidant enzyme SOD plays an important role in maintaining physiologic levels of superoxide. Cellular levels of superoxide are normally low due to the action of SOD, which catalyzes rapidly the conversion of superoxide to H2O2 and oxygen. Agents that inhibit superoxide production or enhance cellular antioxidant defenses can protect cells from the harmful influence of oxygen radicals.33 In this study, we demonstrated that ox-LDL (100 μg/mL) increased the generation of superoxide, decreased the secretion of SOD. FSBT, with strong free radical scavenging capability, increased the cellular SOD, suppressed superoxide overproduction, and consistently inhibited the ox-LDL mediated decrease of cell viability. Thus, we hypothesize that the protective effects of FSBT may partially relate to the antioxidant activity and the cellular antioxidant defense improvement capability of it.
More importantly, a significant (P < 0.01) inhibition of ox-LDL induced LOX-1mRNA up-regulation and eNOS (mRNA and protein) down-regulation were observed after pretreatment with FSBT, indicating that this receptor and synthase are also involved in the protective mechanisms. The similar effects were observed with the pretreatment of quercetin and isorhamnetin, implied that they may partially contribute to the protective effect of FSBT. Endothelial dysfunction or activation elicited by ox-LDL is assumed to be the key step in the initiation of AS.34,35 LOX-1, a lectinlike receptor for ox-LDL recently been identified in endothelial cells,16,26-28 can be activated by shear stress, endothelin, angiotensin-α and ox-LDL. Activation of this receptor initiates intracellular signaling pathways, and involves in eNOS expression,19,36-38 superoxide generation, endothelium secretory activities alteration, and leads to endothelial activation, dysfunction, and apoptosis.39-42 Among the effects of ox-LDL, the reduced expression of eNOS and after NO reduction may be the basis of the alteration in endothelial biology.43 The direct relationship between LOX-1 down-regulation and eNOS up-regulation cannot be discerned from the present study, but previous studies have shown that treatment of HCAECs with a specific antisense of LOX-1mRNA decreases apoptosis, and leads to expression of adhesion molecules and deposition of inflammatory cells.18 All these features of cell injury correlate with diminished eNOS expression.
We recognized that large concentrations were required to show the unique effects of FSBT in this study. These concentrations were necessary to demonstrate and amplify the effects of different compounds in vitro experiments. Some investigators who examined the cyto-protective effects of the extracts from seabuck-thorn have used high concentrations.44,45 These high concentrations gave the above effects of FSBT on EA.hy926, whereas they did not affect the cell viability (more than 97% viable).
Because cardiovascular disease is the major cause of mortality in patients with obesity and insulin resistance,46 the development of effective treatments to control these adverse outcomes is necessary. Also, a better understanding of the mechanisms, with which the agents play their role and show their effects, is needed. The present work provides insight into the effects and partial mechanisms of vascular protective properties of FSBT on EA.hy926.
The present study in endothelial cell line EA.hy926 provides evidence that FSBT inhibits ox-LDL induced cell injuries. These inhibitory effects are possessed by inhibition of superoxide over production, enhancement of cellular antioxidant defenses, attenuation of ox-LDL induced LOX-1 up-regulation, and increase of ox-LDL reduced eNOS expression. The similar effects were observed for quercetin and isorhamnetin, the 2 major flavonoids contained in FSBT, thereby may provide part of the pharmacologic basis for the clinical application of Traditional Chinese Medicines for treatment of AS.
The authors thank Dr C.J.S. Edgell (University of North Carolina at Chapel Hill, NC) for kindly providing the cell lines EA.hy926 cell. They also thank Zhejiang Conba Pharmaceutical Co Ltd for supplying FSBT.
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