Article pubs.acs.org/JAFC
Mulberry Water Extracts Inhibit Atherosclerosis through Suppression of the Integrin‑β3/Focal Adhesion Kinase Complex and Downregulation of Nuclear Factor κB Signaling in Vivo and in Vitro Kuei-Chuan Chan,†,‡ Hsieh-Hsun Ho,§ Ming-Cheng Lin,†,‡ Chi-Hua Yen,‡,∥ Chien-Ning Huang,†,‡ Hui-Pei Huang,*,⊥,# and Chau-Jong Wang*,§,⊥ †
Department of Internal Medicine, ∥Department of Family and Community Medicine, and ⊥Department of Medical Research, Chung-Shan Medical University Hospital, Number 110, Section 1, Jianguo North Road, Taichung 402, Taiwan ‡ School of Medicine, Institute of Medicine, §Institute of Biochemistry and Biotechnology, and #Department of Biochemistry, School of Medicine, Chung-Shan Medical University, Number 110, Section 1, Jianguo North Road, Taichung 402, Taiwan ABSTRACT: Previous studies have shown that mulberry water extracts (MWEs), which contain polyphenolic compounds, have an antiatherosclerotic effect in vivo and in vitro through stimulating apoptosis of vascular smooth muscle cells (VSMCs). Histological analysis was performed on atherosclerotic lesions from high-cholesterol diet (HCD)-fed rabbits after treatment with 0.5−1% MWEs for 10 weeks. Immunohistochemistry showed that the expressions of SMA, Ras, and matrix metalloproteinase-2 in the VSMCs were dose-dependently inhibited after MWE treatment. The antimigratory effects of MWEs on A7r5 VSMCs were assessed by western blot analysis of migration-related proteins, visualization of F-actin cytoskeleton, and reverse transcription polymerase chain reaction. The results showed that MWEs inhibited VSMC migration through reducing interactions of the integrin-β3/focal adhesion kinase complex, alterations of the cytoskeleton, and downregulation of glycogen synthase kinase 3β/ nuclear factor κB signaling. Taken together, MWEs inhibited HCD-induced rabbit atherogenesis through blocking VSMC migration via reducing interactions of integrin-β3 and focal adhesion kinase and downregulating migration-related proteins. KEYWORDS: atherosclerosis, vascular smooth muscle cells, migration, mulberry water extracts, integrin-β3, FAK, NF-κB
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INTRODUCTION Migration of vascular smooth muscle cells (VSMCs) from the media into the intima and their subsequent proliferation are important processes in neointima formation in atherosclerosis and restenosis after percutaneous coronary interventions.1,2 VSMC migration has been reported to depend upon phosphoinositide-3-kinase (PI3K) signaling and an increased activity of matrix metalloproteinase (MMP)-2, with Akt kinase being activated by the PI3K signaling pathway.3−5 Cytoskeletal proteins, including focal adhesion kinase (FAK) and PI3K, are recruited to focal adhesion complexes during cell migration, and the inhibition of integrin-β3 has been shown to result in a significant decrease in migration.2 PI3K-induced activation of Akt has been reported to mediate the phosphorylation of glycogen synthase kinase 3β (GSK3β), resulting in the inhibition of its activity.6,7 GSK3β has been reported to mediate the disassembly of focal adhesions and plays a positive role in activating Rac, thereby promoting cell migration.8−10 FAK and the FAK/Src complex play a role in integrin signaling, which induces rearrangement of the actin cytoskeleton. Actin filaments play an important role in structure, motility, division, and contraction of both muscle and nonmuscle cells.11 Increased FAK phosphorylation has been observed in migrating endothelial cells, and inhibition of FAK activity has been shown to block their migration into wounded monolayers of cells.12,13 By dephosphorylation of FAK, activated Ras can promote tumor cell migration.14 Raf is an important downstream effector of Ras. Ras-related small GTPases of the Rho family, including Rho, Rac, and Cdc42, are known to be involved in the © 2014 American Chemical Society
regulation of the actin cytoskeleton. Rho and Rho kinase can also mediate VSMC migration, and RhoA has been reported to be important in the regulation of the actin cytoskeleton.15−19 Increased expression of RhoB has been reported to inhibit migration of VSMCs.1 Nuclear factor κB (NF-κB) signaling is activated by RhoA, Rac1, and Cdc42 and inhibited by RhoB, and NF-κB plays a key role in the processes of inflammation, atherosclerosis, and angiogenesis via regulating MMP and vascular endothelial growth factor (VEGF).20,21 IκB is a specific inhibitor of NF-κB.22 Overexpression of the MMP inhibitor, tissue inhibitor of metalloproteinase (TIMP)-2, has been shown to reduce invasion and angiogenesis.23,24 Inhibition of VEGF has been reported to prevent angiogenesis.25 Although previous studies have shown that mulberry water extracts (MWEs) have antiatherosclerotic effects in vivo and in vitro through stimulation of the apoptosis of VSMCs26 and inhibition of low-density lipoprotein oxidation,27 the effects of MWEs on VSMC migration are still not fully understood. The aim of this study was to investigate whether MWEs inhibit VSMC migration and the underlying mechanism.
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MATERIALS AND METHODS
Preparation of MWEs. Dried Morus alba L. (mulberry) fruit (100 g) was freeze-dried from 500 g of fresh fruit. The 100 g of dried fruit was Received: Revised: Accepted: Published: 9463
June 21, 2014 September 3, 2014 September 8, 2014 September 8, 2014 dx.doi.org/10.1021/jf502942r | J. Agric. Food Chem. 2014, 62, 9463−9471
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stirred with water (1000 mL) and then centrifuged (6000 rpm) for 10 min to remove the sediment. The supernatant was then lyophilized (at −80 °C for 12 h) to obtain the MWEs and stored at −20 °C. High-Performance Liquid Chromatography (HPLC). Using a Hewlett-Packard Vectra 436/33N system and a diode array detector, the components of the MWEs were determined by HPLC analysis28 employing a 5 μm RP-18 column (4.6 × 150 mm inner diameter). After filtering through a 0.22 μm filter disk, the MWEs (25 mg/mL) were injected into the column. Two systems of mobile phase (A, 2% acetic acid/water; B, 0.5% acetic acid in water/acetonitrile) were used. The peak purity was confirmed with chromatography at 280 nm and ultraviolet (UV) spectra. The total phenolic compound content was measured according to the Folin−Ciocalteu method. The samples (20 μL/1.6 mL of water) were added in 100 μL of Folin−Ciocalteu reagent (Sigma, St. Louis, MO) and 300 μL of sodium carbonate (20%), then mixed, and incubated at 40 °C for 40 min. Absorption at 725 nm was measured. Animals and Diet. A total of 30 male New Zealand white rabbits weighing 2000−2500 g (Animal Center of Chung Shan Medical University) were used in this study. They were individually housed in metal cages under the standard laboratory conditions (22 ± 2 °C, 55 ± 5% relative humidity, and 12 h light/12 h dark cycle) with free access to food and water. According to a suitable pharmacological dose of MWE for humans in a daily diet, five groups (n = 6) of animals were described as follows: group I, standard chow; group II, standard chow with 1% MWEs in the daily diet; group III, high-cholesterol diet (HCD) (containing 95.7% standard Purina chow (Purina Mills, Inc., Gray Summit, MO), 3% lard oil, and 1.3% cholesterol) for 10 weeks; and groups IV and V, HCD with 0.5 and 1.0% MWEs, respectively, in the daily diet. During the experimental period, all of the animals were handled according to the guidelines of the Institutional Animal Care and Use Committee of Chung Shan Medical University. After 10 weeks, the rabbits were sacrificed by exsanguination after deep anesthesia with pentobarbital (30 mg/kg intravenous) via the marginal ear vein. Serum was stored at −80 °C prior to serum lipid analysis and measurement of serum values. The aortic arch and thoracic aortas were carefully removed to protect the endothelial lining, collected, and freed of adhering soft tissue. Evaluation of Atherosclerotic Lesions. Sudan IV is a fat-soluble dye often used for staining triglycerides, lipids, and lipoproteins present in cells and tissues. Aortic arches were rapidly dissected and stained with Sudan IV and then kept at −80 °C or in 10% neutral-buffer formalin. The sections of the aortic arch were stained with hematoxylin and eosin or α-smooth muscle actin antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX) by immunohistochemistry. Cell Cultures. Thea rat A7r5 thoracic aorta smooth muscle cells, obtained from American Type Culture Collection (ATCC, CRL-1444, Manassas, VA), were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine, 1% penicillin−streptomycin, and 1.5 g/L sodium bicarbonate (all from Gibco/BRL, Gaithersburg, MD) at 37 °C in a humidified atmosphere of 5% CO2. The medium was changed every 2 or 3 days, and the cells were subcultured when confluence was achieved. Wound Healing. The 1 × 106/mL A7r5 cells were cultured in 6-well plates for 48 h. A line was drawn on the underside of the well with a yellow P200 pipet tip. These lines served as fiducial marks for the wound areas to be analyzed. The non-adhering cells were washed out with phosphate-buffered saline (PBS), and the remaining cells were treated with MWEs (0.05−1.0 mg/mL). Under a 40× lens, images of the linear wounds were taken in nine fields per well at 0, 12, 24, 36, 48, 60, 72, 84, and 96 h. The migrated cells were counted per well, and the counts were averaged.29 MMP Gelatin Zymography. The levels of MMP-2 and MMP-9 released in the cultured medium were assayed by gelatin zymography as described previously.30 MWE-treated A7r5 cells were plated onto 6-well culture plates (5 × 105/well) and incubated with 1 mL of 0.5% FBS in Dulbecco’s modified Eagle’s medium for 24 h. The culture medium was prepared with a 5× loading buffer containing 0.01% sodium dodecyl sulfate (SDS) without β-mercaptoenthanol and subjected to electrophoresis with 8% SDS polyacrylamide gels containing 0.1% gelatin.
Following electrophoresis, the gels were washed twice with 2.5% Triton X-100 on a gyrating shaker for 30 min at room temperature to remove SDS. The gels were then incubated in 50 mL of reaction buffer (40 mM Tris-HCl, 10 mM CaCl2, and 0.01% NaN3) at 37 °C overnight on a rotary shaker, stained with Coomassie Brilliant Blue R-250, and destained with methanol/acetic acid/water (50:75:87.5, v/v/v). Gelatinolytic activities were detected as horizontal white bands on a blue background. Western Blot Analysis. Analysis of TIMP-2, VEGF, FAK, integrinβ3, c-Raf, Src, Ras, PI3K, Akt, Cdc42, RhoA, RhoB, Rac1, p-FAK, IκB, and NF-κB (all from Santa Cruz Biotechnology, Dallas, TX), PI3K (Becton-Dickinson, San Jose, CA), and p-Akt and β-actin (Sigma, St. Louis, MO) was performed using western blot analysis.31. After the indicated treatment with MWEs (0−2.0 mg/mL) for 48 h, the cells were lysed and equal amounts of cell lysates (50 μg of protein) were separated by electrophoresis on 8−12% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were incubated with Tris-buffered saline containing 1% (w/v) nonfat milk and 0.1% (v/v) Tween-20 for 1 h to block non-specific binding, washed with Tween-20 for 30 min, incubated with the appropriate primary antibody for 2 h, incubated with horseradish-peroxidaseconjugated second antibody (Sigma, St. Louis, MO) for 1 h, developed using enhanced chemiluminescence (ECL, Millipore, Bedford, MA), and analyzed by densitometry using AlphaImager Series 2200 software. The results were representative of at least three independent experiments. Immunoprecipitation Assay. Approximately 0.5 mg of the lysate protein was immunoprecipitated32 using monoclonal anti-integrin-β3 (Santa Cruz Biotechnology, Dallas, TX) antibodies. Immune complexes were harvested with protein-A-conjugated sepharose beads. After centrifugation at 2500 rpm, the eluates were analyzed by immunoblotting against FAK, Src, Ras, and PI3K antibodies. Electrophoretic Mobility Shift Assay of NF-κB. An electrophoretic mobility shift assay33 was performed using a Lightshift kit from Promega. A total of 10 μg of nuclear protein was mixed with the binding reaction buffer containing 10 mM Tris, 50 mM KCl, 1 mM dithiothreitol (DTT), 5 mM MgCl2, 2 μg poly(dI·dC), and 2 pmol of oligonucleotide probe and incubated for 20 min at room temperature. Specific binding was confirmed using a 200-fold excess of unlabeled probe as a specific competitor. Protein−DNA complexes were separated by 6% nondenaturing acrylamide gel electrophoresis and then transferred to positively charged nylon membranes and cross-linked in a Stratagene cross-linker. Gel shifts were visualized with streptavidin−horseradish peroxidase, followed by chemiluminescent detection. Visualization of the F-Actin Cytoskeleton. A7r5 cells (5 × 104 cells/well) were treated with 0.1−1.0 mg/mL MWEs for 48 h, then fixed in 4.6% formaldehyde in PBS for 10 min, and permeabilized in 0.2% Triton X-100/PBS for 5 min. The cells were stained with phalloidin− fluorescein isothiocyanate (FITC, A12379, Invitrogen, Grand Island, NY) to visualize polymerized F-actin microfilaments under a Nikon upright fluorescence microscope (100× objective)34 (red color). Then, they were extensively washed with PBS, briefly counterstained with 1 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei, and photographed (blue color). Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis. Total RNA was isolated from cells using a RNA Isolation Kit (Ultraspec, Biotecx, Houston, TX) according to the instructions of the manufacturer and quantified spectrophotometrically. For reverse transcription, total RNA (4 μg) was used as templates in the raction buffer containing oligo dT primer and 4 units/μL RTase (MMLV) in an Applied Biosystems GeneAmp PCR system 2700 at 42 °C for 60 min and then at 99 °C for 5 min to inactivate the RT. PCR master mix containing 1 mM deoxyguanosine triphosphate, 1 mM deoxyadenosine triphosphate, 1 mM deoxycytidine triphosphate, 1 mM deoxythymidine triphosphate, Dyna PCR buffer II, 2 units/μL AmpliTaq DNA polymerase, and the following primers was added at a 0.5 μM concentration: murine MMP, upstream primer (5′-ACACCCAGTACTCATTCCCTG-3′) and downstream primer (5′-GTCCTGACCAAGGATATAGCC-3′); VEGF, upstream primer (5′-TGCACCCACGACAGAAGGGGA-3′) and downstream primer (5′9464
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TCACCGCCTTGGCTTGTCACA-3′); GADPH, upstream primer (5′-ACCACAGTCCATGCCATCAC-3′) and downstream primer (5′TCCACCACCCTGTTGCTGTA-3′). The complementary DNAs (cDNAs) in the samples were amplified for 30 cycles as follows: 1 min at 94 °C as an initial step, followed by 30 thermal cycles of 60 s at an annealing temperature and 2 min at 72 °C, and finally 20 min at 72 °C. The RT-PCR products were analyzed by agarose gel electrophoresis, and the intensity of the bands corresponding to MMP-2, VEGF, and GADPH were measured using Fujifilm Image Gauge software (version 3.1, Fujifilm Co., Ltd., Tokyo, Japan).35 Statistical Analysis. Data were shown as the means (standard deviation) of three independent experiments. Statistical comparisons were evaluated by one-way analysis of variance (ANOVA). A p value of less than 0.05 was considered to be statistically significant.
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RESULTS Polyphenolic Compounds of MWEs. The main polyphenolic constituents of MWEs have been identified as gallic acid (0.31%), protocatechuic acid (2.92%), catechin (0.54%), epigallocatechin gallate (2.68%), caffeic acid (1.10%), epicatechin (1.21%), p-coumaric acid (0.35%), rutin (3.22%), ferulic acid (0.27%), gossypin (0.26%), hesperetin (0.34%), resveratrol (0.35%), quercetin (0.50%), naringenin (0.52%), and hydroxyflavin (0.58%) (Table 1). The other constituents include protein (1.77%), fat (4.40%), polysaccharide (24.83%), and unknown components without standards (data not shown).
Figure 1. Histological analysis of a representative atherosclerotic lesion from HCD-fed rabbits treated for 10 weeks with 0.5 and 1% MWEs. The same group of slices were from the same area of the artery and stained with different antibodies. Primary antibodies were detected with α-actin, Ras, and MMP-2. Semi-quantitative computer-assisted image analysis of the atherosclerotic lesion was performed on six randomly selected 400× magnified images of slides from individual animals.
Table 1 HPLC assay polyphenolic compound
gallic acid protocatechuic acid catechin epigallocatechin gallate caffeic acid epicatechin p-coumaric acid rutin ferulic acid gossypin hesperetin resveratrol quercetin naringenin hydroxyflavin total
MWEs (%) 0.31 ± 0.05 2.92 ± 0.45 0.54 ± 0.03 2.68 ± 0.20 1.10 ± 0.24 1.21 ± 0.17 0.35 ± 0.04 3.22 ± 1.01 0.27 ± 0.01 0.26 ± 0.02 0.34 ± 0.10 0.35 ± 0.23 0.50 ± 0.07 0.52 ± 0.17 0.58 ± 0.12 15.13 ± 0.93
B of Figure 2 show that MWEs could effectively dosedependently inhibit migration of the VSMCs. MWEs Inhibited MMP Activity and VEGF Secretion of the A7r5 Cells. A7r5 cells were treated with or without MWEs for 0−48 h. The 0.5% FBS starvation medium of the A7r5 cells was changed after treatment with MWEs, and gelatin zymography was then performed to analyze the activities of MMP-9 and MMP-2. Figure 3A shows that more MMP-2 was secreted by the A7r5 cells than MMP-9. After treatment with MWEs, the activities of MMP-2 and MMP-9 decreased timedependently. In time-dependent assays, A7r5 cells were treated with 1.0 mg/mL MWEs at the indicated times. Cell lysates (50 μg) were prepared and subjected to western blot analysis. Proteins were detected by TIMP-2 and VEGF antibodies, and albumin and β-actin were used for equal loading. The activity of TIMP-2 was time-dependently increased after MWE treatment (Figure 3B), and the activity of VEGF was time-dependently decreased (Figure 3C). MWEs Inhibited the Activities of FAK and Akt in A7r5 Cells. Cultured cells were treated with MWEs (0.5−2 mg/mL) for 48 h. A7r5 cells were treated with MWEs (1.0 mg/mL) for 0− 48 h, and cell lysates (50 μg) were prepared and subjected to western blot analysis. Proteins were detected by phospho-FAK, integrin-β3, PI3K, Akt, and GSK3β antibodies, and β-actin was used for equal loading. As shown in panels A and B of Figure 4, the activities of FAK-p, PI3K, Akt-p, GSK3β-p, and c-Raf were dose- and time-dependently decreased by MWE treatment. Total cell lysates were then immunoprecipitated with anti-integrin-β3 antibodies, and the precipitated complexes were examined by immunoblotting with anti-FAK, Src, Ras, and PI3K antibodies. MWE treatment dose-dependently decreased the activities of Src, Ras, and PI3K (Figure 4C). MWEs Inhibited the Activity of NF-κB in A7r5 Cells. A7r5 cells were treated with MWEs (0−1.5 mg/mL) for 48 h, and cultured cells were treated with MWEs (1.0 mg/mL) for 0− 48 h. Cell lysates (50 μg) were prepared and subjected to western
retention time (RT) (min) 7.85 15.17 23.49 23.95 26.08 27.27 31.57 33.44 34.00 41.72 42.85 45.17 51.83 56.28 57.44
MWEs Inhibited Proliferation of VSMCs and the Expression of SMA, Ras, and MMP-2 in Neointima. Histological analysis was performed on a representative atherosclerotic lesion from a HCD-fed rabbit after treatment with 0.5−1% MWEs for 10 weeks. As shown in Figure 1, hematoxylin and eosin staining showed that the MWEs dosedependently inhibited HCD-fed-induced intimal hyperplasia of vessel walls. After immunohistochemical staining, the expressions of SMA, Ras, and MMP-2 in the VSMCs were also found to be dose-dependently inhibited after MWE treatment. MWEs Effectively Inhibited VSMC (A7r5 Cell) Migration. Monolayers of growth-arrested A7r5 cells treated with or without different concentrations of MWEs (0.1−1.0 mg/mL) were scraped, and the number of cells in the denuded zone (i.e., wound) were analyzed at the indicated times (0−96 h) under light microscopy and Boyden chamber assay. Both panels A and 9465
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Figure 2. Effect of MWEs on the migration of A7r5 cells. Monolayers of growth-arrested A7r5 cells treated with or without different concentrations of MWEs (0.1−1.0 mg/mL) were scraped, and the number of cells in the denuded zone (i.e., wound) was analyzed at the indicated times (0−96 h) under (A) light microscopy and (B) Boyden chamber assay. Quantitative assessment of the mean number of cells at the indicated times in the denuded zone represents the average of three independent experiments ± standard deviation.
blot analysis. As shown in panels A and B of Figure 5, MWE treatment dose- and time-dependently decreased the activity of NF-κB and increased the activity of IκB. The cell lysates were then immunoprecipitated with anti-NF-κB and immunoblotted with anti-IκB. β-actin was used for equal loading. MWE treatment dose-dependently increased the combination of IκB and NF-κB (Figure 5C). Thereafter, the A7r5 cells were treated with or without MWEs for 12 h, and then nuclear extracts were prepared. C23 was used as a nuclear control. NF-κB DNA binding activity was analyzed by an electrophoretic mobility shift assay. MWE treatment dose-dependently inhibited the activity of NF-κB in nuclei (Figure 5D). Finally, total RNA was extracted. Expressions of MMP-2 and VEGF mRNA were analyzed by RTPCR with GAPDH as the loading control. Figure 5E shows that the mRNA expressions of MMP-2 and VEGF were dosedependently decreased by MWE treatment. MWEs Inhibited the Expressions of Small G Proteins and Cytoskeletal F-Actin in A7r5 cells. A7r5 cells were treated with MWEs (0−2 mg/mL) for 48 h. The A7r5 cells were then treated with MWEs (1.0 mg/mL) for 0−48 h. Cell lysates (50 μg) were prepared and subjected to western blot analysis. Proteins were detected by specific antibodies (Cdc42, Ras, RhoA, and Rac1), and β-actin was used for equal loading. As shown in panels A and B of Figure 6, MWE treatment dose- and timedependently decreased the expressions of Ras, RhoA, cdc42, and Rac-1 and increased the activity of RhoB. Finally, cultured cells were treated with different concentrations of MWEs or without MWEs for 48 h. The cells were fixed and labeled for F-actin with phalloidin−TRITC (red) and for nucleic acid with DAPI (blue).
Figure 3. Effects of MWEs on MMP activity and VEGF secretion of A7r5 cells. A7r5 cells were treated with or without MWEs for 0−48 h. The 0.5% FBS starvation medium of the A7r5 cells was changed after treatment with MWEs, and (A) gelatin zymography was then performed to analyze the activities of MMP-9 and MMP-2 as described in the Materials and Methods. In time-dependent assays, A7r5 cells were treated with 1.0 mg/mL MWEs at the indicated times. Cell lysates (50 μg) were prepared and subjected to western blot analysis. Proteins were detected by (B) TIMP-2 and (C) VEGF antibodies. Albumin and βactin were used for equal loading. The results from three separate experiments were similar.
Immunofluorescence microcopy of phalloidin (F-actin)- and DAPI (nuclei)-stained A7r5 cells revealed a marked increase in phalloidin staining in the control group (medium with FBS) compared to the starvation group (medium without FBS) (Figure 6C). After MWE treatment in the control group, the phalloidin staining decreased significantly again and was similar to that of the starvation group. Taken together, these results demonstrate that MWEs are a positive regulator of actin organization.
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DISCUSSION Nelumbo nucifera leaf extracts, which contain a large amount of phenolic acids and flavonoids, including gallic acid, rutin, 9466
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Figure 4. Effect of MWEs on the activities of FAK and Akt in A7r5 cells. (A) Cultured cells were treated with MWEs (0.5−2 mg/mL) for 48 h. (B) A7r5 cells were treated with MWEs (1.0 mg/mL) for 0−48 h. Cell lysates (50 μg) were prepared and subjected to western blot analysis. (C) Proteins were detected by phospho-FAK, integrin-β3, PI3K, Akt, and GSK3β antibodies. β-actin was used for equal loading. Total cell lysates were immunoprecipitated with anti-integrin-β3 antibodies, and the precipitated complexes were examined by immunoblotting with anti-FAK, Src, Ras, and PI3K antibodies. The results from representative experiments were normalized to immunoprecipitated integrin-β3 expression. The results from three separate experiments were similar. 9467
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Figure 5. Effect of MWEs on the activity of NF-κB in A7r5 cells. (A) A7r5 cells were treated with MWEs (0−1.5 mg/mL) for 48 h. (B) Cultured cells were treated with MWEs (1.0 mg/mL) for 0−48 h. Cell lysates (50 μg) were prepared and subjected to western blot analysis. (C) Cell lysates were also immunoprecipitated with anti-NF-κB and then immunoblotted with anti-IκB. β-actin was used for equal loading. (D) A7r5 cells were treated with or without MWEs for 12 h, and then nuclear extracts were prepared. C23 was used as the nuclear control. NF-κB DNA binding activity was analyzed by an electrophoretic mobility shift assay as described in the Materials and Methods. “S” represented starvation, and “P” represented nuclear extracts incubated with unlabeled oligonucleotides (free probe), to confirm the specificity of binding. (E) Total RNA was extracted. Expressions of MMP-2 and VEGF mRNA were analyzed by RT-PCR. GAPDH served as the loading control. The results from three separate experiments were similar.
modulation of smooth muscle cell proliferation and migration.36 A previous study showed that red wine polyphenolic compounds
quercetin, catechin, epicatechin, and epigallocatechin gallate, have been reported to inhibit neointimal hyperplasia through 9468
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Figure 6. Effect of MWEs on the expressions of small G proteins and cytoskeletal F-actin in A7r5 cells. (A) A7r5 cells were treated with MWEs (0−2 mg/mL) for 48 h. (B) A7r5 cells were treated with MWEs (1.0 mg/mL) for 0−48 h. Cell lysates (50 μg) were prepared and subjected to western blot analysis. Proteins were detected by specific antibodies (Cdc42, Ras, RhoA, and Rac1). β-actin was used for equal loading. (C) Cultured cells were treated with different concentrations of MWEs or without MWEs for 48 h. The cells were fixed and labeled for F-actin with phalloidin−TRITC (red) and for nucleic acid with DAPI (blue).
and green tea polyphenols are able to inhibit the proliferation and migration of VSMCs and the expressions of VEGF and MMP2.37 Gallic acid has been reported to have a hypolipidemic effect
on high-fat-diet-fed mice, and the antioxidative properties of protocatechuic acid has been reported to contribute to its suppression of platelet-derived growth factor (PDGF)-induced 9469
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migration and proliferation in VSMCs.38 Catechins have been reported to be able to suppress VSMC proliferation and migration via the inhibition of βPDGF receptors.39 Quercetin, catechin, epicatechin, and epigallocatechin gallate have also been reported to inhibit the activity of xanthine oxidase, thereby reducing oxidative stress on vascular walls.40 Quercetin has also been reported to inhibit the proliferation and migration of VSMCs by suppressing the phosphorylation of mitogenactivated protein kinase.41 In addition, quercetin 3-O-β-Dglucuronide has been shown to inhibit PDGF-induced migration of rat aortic smooth muscle cells by suppression of PDGFinduced c-Jun N-terminal kinase (JNK) and Akt activations.42 MWEs are also rich in phenolic acids and flavonoids, including gallic acid, caffeic acid, protocatechuic acid, catechin, epigallocatechin gallate, epicatechin, rutin, quercetin, and naringenin. As we know, protein, fat, and polysaccharide were not reported to have an antiatherosclerotic effect before. Therefore, the inhibitory effect of MWEs on VSMC migration may be attributed to the polyphenol compounds in MWEs. We found that 0.5−1.0% MWEs effectively reduced HCD-fedinduced intimal hyperplasia in rabbits (Figure 1), which is consistent with the results of our previous study.26 This further suggests that 2.5−5.0 g/day of MWEs may be enough to prevent atherosclerosis in humans. In the present in vivo and in vitro studies, we found that MWEs effectively inhibited migration of A7r5 VSMCs (Figure 2), which is important to prevent neointima formation in atherosclerosis. It is known that MMP and VEGF are vital regulators of VSMC migration and angiogenesis, and our results showed that MWEs could timedependently decrease the activities of MMP-2, MMP-9, and VEGF (Figure 3). On the other hand, the activity of TIMP-2 (a MMP inhibitor) was time-dependently increased after MWE treatment. These results indicate that MWEs can effectively inhibit the activities of MMP-2, MMP-9, and VEGF and thereby inhibit VSMC migration and angiogenesis. Previous reports have shown that PI3K can induce the activation of Akt, thus mediate the phosphorylation of GSK3β, and inhibit its activity, thereby preventing migration. The FAK/Src complex plays a role in integrin signaling and actin cytoskeleton rearrangement. In addition, Ras-related small GTPases of the Rho family are involved in the regulation of the actin cytoskeleton, and Raf is an important downstream effector of Ras. Our results showed that the activities of FAK-p, PI3K, Akt-p, GSK3β-p, Src Ras, c-Raf, and integrin-β3 were all decreased after MWE treatment (Figure 4), which suggests that MWEs can inhibit VSMC migration via inhibition of the FAK, PI3K/Akt, and Ras pathways. Previous studies have reported that NF-κB may regulate angiogenesis via MMP induction and that IκB is an inhibitor of NF-κB. Our results showed that MWEs dose- and time-dependently decreased the activity of NF-κB and increased the activity of IκB (Figure 5). In addition, the combination of IκB and NF-κB was increased after MWE treatment. MWEs also dose-dependently inhibited the activity of NF-κB in nuclei. These results indicate that MWEs inhibit VSMC migration via the NF-κB pathway. Previous reports have also shown that NF-κB is activated by RhoA, Rac1, and cdc42 and inhibited by RhoB. Rho and Rho kinase can also mediate VSMC migration, and RhoA has been reported to regulate the actin cytoskeleton. Our results showed that the activities of Ras and Ras-related small GTPases, including RhoA, cdc42, and Rac, were all significantly decreased after MWE treatment (Figure 6); however, the activity of RhoB was increased after MWE treatment. These results indicate that MWEs can inhibit VSMC migration via Ras and Ras-related
small GPTase pathways. We also found that MWEs were a positive regulator of actin organization. In conclusion, MWEs inhibit VSMC migration via reducing the interactions between the integrin-β3 and FAK complex and downregulating GSK3β and NF-κB signaling.
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AUTHOR INFORMATION
Corresponding Authors
*Telephone: 886-4-24730022, ext. 11671. Fax: 886-4-23248110. E-mail:
[email protected]. *Telephone: 886-4-24730022, ext. 11670. Fax: 886-4-23248167. E-mail:
[email protected]. Funding
This study was supported by a grant from the Ministry of Science and Technology (NSC99-2320-B-040-013-MY3), Taiwan. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED FAK, focal adhesion kinase; GSK3β, glycogen synthase kinase 3β; HCD, high-cholesterol diet; MMP, matrix metalloproteinase; MWE, mulberry water extract; NF-κB, nuclear factor κB; RT-PCR, reverse transcription polymerase chain reaction; TIMP, tissue inhibitor of metalloproteinase; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cell
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