Mulberry Water Extracts Inhibit Rabbit Atherosclerosis through

May 15, 2014 - Mulberry Water Extracts Inhibit Rabbit Atherosclerosis through Stimulation of Vascular Smooth Muscle Cell Apoptosis via Activating p53 ...
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Mulberry Water Extracts Inhibit Rabbit Atherosclerosis through Stimulation of Vascular Smooth Muscle Cell Apoptosis via Activating p53 and Regulating Both Intrinsic and Extrinsic Pathways Kuei-Chuan Chan,‡,§,# Hsieh-Hsun Ho,†,# Ming-Cheng Lin,‡,§ Cheng-Hsun Wu,£ Chien-Ning Huang,‡,§ Wen-Chun Chang,† and Chau-Jong Wang*,†,⊥ ‡

Department of Internal Medicine, Chung-Shan Medical University Hospital, No. 110, Sec. 1, Jianguo N. Road, Taichung 402, Taiwan † Institute of Biochemistry and Biotechnology, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, Taichung 402, Taiwan § School of Medicine, Institute of Medicine, Chung-Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, Taichung 402, Taiwan ⊥ Department of Medical Research, Chung Shan Medical University Hospital, No. 110, Sec. 1, Jianguo N. Road, Taichung 402, Taiwan £ Department of Anatomy, China Medical University, No. 91, Xueshi Road, Taichung 404, Taiwan ABSTRACT: Previous studies have shown that mulberry water extracts (MWEs), which contain polyphenolic compounds, have an antiatherosclerotic effect in rabbits. Apoptosis of vascular smooth muscle cells (VSMCs) is the key determinant of the number of VSMCs in remodeling. To improve the recovery from atherosclerosis pathology, it would be ideal to induce regression of atherosclerotic plaques and apoptosis of VSMCs. In this study, we treated high-cholesterol-diet-fed (HCD-fed) rabbits with MWEs, and we found that the MWEs effectively inhibited HCD-fed-induced intimal hyperplasia of vessel walls. We also found that MWEs initially activate JNK/p38 and p53, which in turn activate both Fas-ligand and mitochondria pathways, thereby causing mitochondria translocation of Bax and the reduction of Bcl-2 that trigger the cleavage of procaspases, finally resulting in apoptosis of VSMCs. In addition, 2.5−5.0 g/day of MWEs for humans may be enough to prevent atherosclerosis. KEYWORDS: atherosclerosis, vascular smooth muscle cells, mulberry water extracts, p53, intrinsic pathway, extrinsic pathway

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INTRODUCTION

regression of atherosclerotic plaques and restenotic lesions by inducing the apoptosis of VSMCs.1−8 In mammalian cells, apoptosis occurs through two distinct molecular pathways. The intrinsic or mitochondrial pathway is triggered by intracellular events and the release of pro-apoptotic factors such as cytochrome c from the mitochondria into the cytosol. However, the extrinsic apoptosis pathway receives signals through binding extracellular protein death ligands to pro-apoptotic death receptors. Both pathways lead to activation of caspases, leading to apoptosis. Transcription factor p53 has been shown to mediate apoptosis by activating cell surface Fas expression on human VSMCs.9 In vascular disease, the activation of p53 has been shown to increase the expression of Bax and decrease the expression of Bcl-2, thus leading to apoptosis.10,11 p53 has also been shown to induce VSMC apoptosis in vitro and to inhibit proliferation of VSMCs in vitro and in vivo.12,13 The antiproliferative activities of p53 are mediated by inducing cyclindependent kinase inhibitor p21, and p53 is essential for cell cycle control G1 arrest.14,15 By interaction with MDM2, a regulator of p53 stability, p53 can accumulate in atherosclerotic lesions to induce apoptosis.16,17 The c-Jun N-terminal kinase (JNK) and

Atherosclerosis is a systemic, chronic inflammatory disease involving the intima of large and medium arteries. It also involves many soluble mediators, including macrophage and T cell infiltration of the vascular wall, which induces migration of vascular smooth muscle cells (VSMCs) from the media to intima where these cells proliferate. During the early stages of atherosclerosis, VSMCs switch from a contractile to a synthetic state and gain the ability to proliferate in response to stimuli. Intimal hyperplasia of VSMCs is the principal cause of restenosis after a percutaneous coronary intervention (PCI). Although great improvements in restenosis rate have been achieved after drug-eluting stenting, proliferation of VSMCs with restenosis is still present. Because of compensatory enlargement of the vessel (remodeling), early atherosclerotic plaques will not compromise the vascular lumen and produce stable lesions with angina as they increase in size. Constrictive remodeling of the artery has been shown to involve restenosis after angioplasty, and this involves many biological processes including the proliferation of VSMCs. Angioplasty-induced apoptosis of VSMCs may be an important determinant of vascular remodeling and restenosis, as apoptosis of VSMCs is known to be a key determinant of the number of VSMCs in remodeling, and it has been observed in the atherosclerotic plaque. To improve atherosclerosis, it would be ideal to induce © 2014 American Chemical Society

Received: Revised: Accepted: Published: 5092

March 26, 2014 May 11, 2014 May 14, 2014 May 15, 2014 dx.doi.org/10.1021/jf501466t | J. Agric. Food Chem. 2014, 62, 5092−5101

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Animals and Diet. Thirty male New Zealand white rabbits (Da-Jung Animal Farm, Changhua, Taiwan), weighing 2000− 2500 g were used. They were individually housed in metal cages in an air-conditioned room (22 ± 2 °C, 55 ± 5%, humidity), under a 12 h light/12 h dark cycle with free access to food and water. All experimental rabbits were randomly divided into five groups, each group containing six animals. Group I rabbits were fed with standard chow. Group II rabbits were fed with standard chow with 1% MWEs in the daily diet. Group III rabbits were fed with HCD diet (containing 95.7% standard Purina chow (Purina Mills, Inc.), 3% lard oil, and 1.3% cholesterol) for 10 weeks, to provoke an atherosclerotic process. Groups IV and V were fed with HCD plus 0.5% and 1.0% MWEs, respectively, in the daily diet. The selection of the MWEs dose was based on a suitable pharmacological dose for humans in a daily diet. During the 10 week feeding period, all of the animals were handled according to the guidelines of the Institutional Animal Care and Use Committee of Chung Shan Medical University for the care and use of laboratory animals. After 10 weeks, the rabbits were sacrificed by exsanguination after deep anesthesia with pentobarbital (30 mg/kg i.v.) 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, and the specimens were collected and freed of adhering soft tissue. Evaluation of Atherosclerotic Lesions. Thoracic aortas were rapidly dissected, opened longitudinally, and stained with Sudan IV. Aortic arches were rapidly dissected and kept at −80 °C or in 10% neutral-buffer formalin. The sections of aortic arch were stained with hematoxylin and eosin or αsmooth muscle actin antibodies (Santa Cruz Biotechnology, Inc., CA). Immunohistochemistry was carried out to confirm the lesions. Experienced pathologists evaluated the presence of smooth muscle cells in the examined preparations. Cell Cultures. Cell line A7r5, a rat thoracic aorta smooth muscle cell line, was obtained from ATCC (ATCC number: CRL-1444; Manassas, VA). The A7r5 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% glutamine, 1% penicillin-streptomycin, and 1.5 g/L sodium bicarbonate (all from Gibco/BRL; Gaithersburg, MD). All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. Cell-Cycle Analysis (Flow Cytometric Analysis). Cells synchronized at the G0 phase by serum starvation for 24 h were incubated in fresh serum-containing medium to allow for cellcycle progression. At various time points after release from G0 arrest, the cells were analyzed by flow cytometry to determine cell-cycle distribution.33 The cells were seeded in 10 cm dishes at a density of 107 cells/dish and cultured in normal or high glucose Dulbecco’s modified Eagle’s medium. The cells cultured in high glucose were treated with MWEs at various concentrations (0, 2.0, 4.0, 6.0, 8.0, and 10.0 mg/mL) at 37 °C for 48 h. At the end of treatment, they were collected, fixed in 1 mL of ice-cold 70% ethanol, incubated at −20 °C for at least 24 h, and centrifuged at 380 g for 5 min at room temperature. Cell pellets were treated with l mL of cold staining solution containing 20 μg/mL propidium iodide, 20 μg/mL RNase A, and 1% Triton X-100, and they were incubated for 15 min in darkness at room temperature. Subsequently, the samples were analyzed in a FACS Calibur system (version 2.0, BD Biosciences, Franklin Lakes, NJ) using CellQuest software. The extent of apoptosis was quantified by counting the fraction of

p38 mitogen-activated protein are collectively known as stressactivated mitogen-activated protein kinases. JNK and p38 phosphorylate many known regulators of tumorigenesis, such as c-Jun, p53, Bax, Bcl-2, and Mcl-1. The activation of the p38 pathway has been shown to enhance the transcriptional activity of p53, leading to cell cycle arrest and apoptosis. JNK has also been shown to phosphorylate p53, activating transcription factor-2, c-Jun, and Elk-1 and regulating stability.18,19 Activator protein 1 (AP-1) is a transcriptional factor which is composed of proteins belonging to the c-Fos, c-Jun, ATF, and ENDOG families, and it binds DNA at a site called a TRE and activates gene transcription. The regulation of c-Jun is complex and involves both increases in c-Jun protein levels and phosphorylation of JNK. Fos family proteins cannot form homodimers to bind a TRE in the absence of Jun.20,21 Polyphenol and anthocyanin-rich mulberry has been reported to have not only antiobesity, antidiabetic, antitumor, antioxidative, and anti-inflammatory effects but also neuro-, hepato-, and cardiovascular protective characteristics.22−28 It has been reported that mulberry water extracts (MWEs) can inhibit the oxidation of low-density lipoprotein (LDL), thus preventing atherosclerosis.29 Our previous studies have shown that mulberry leaf extract (MLE) can inhibit atherosclerosis via effectively inhibiting proliferation and migration of VSMCs, and this inhibitory effect was attributed to the polyphenol component of MLE.30,31 In the early stage of atherosclerosis, stimulating apoptosis of VSMCs and thus inhibiting VSMC proliferation and migration is an important process. To examine the effect of polyphenolrich MWEs on the apoptosis of VSMCs and thus the prevention of atherosclerosis, we investigated the ability of MWEs to induce apoptosis and the underlying mechanism.

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MATERIALS AND METHODS Preparation of MWEs. MWEs were prepared from the fruit of M. alba L. (mulberry). Fresh mulberry fruit (500 g) was harvested and freeze-dried. Briefly, the dried fruit (100 g) was macerated, stirred with water (1000 mL), and centrifuged (6000 rpm, 10 min). The aqueous solution was lyophilized (−80 °C, 12 h) to obtain the MWEs and stored at −20 °C before use. High-Performance Liquid Chromatography. The components of the MWEs were determined by high-performance liquid chromatography (HPLC) analysis using a HewlettPackard Vectra 436/33N system with a diode array detector.32 The HPLC method employed a 5 μm RP-18 column (4.6 × 150 mm inner diameter). The MWEs were filtered through a 0.22 μm filter disk, and then 25 mg/mL of the MWEs was injected into the column. Chromatography was monitored at 280 nm, and UV spectra were collected to confirm peak purity. The mobile phase contained two solvents: A, 2% acetic acid/ water; B, 0.5% acetic acid in water/acetonitrile. The total phenolic compound content in each extract was spectrophotometrically determined in accordance with the Folin−Ciocalteu procedure by reading the absorbance at 725 nm against a methanol blank. Briefly, samples (20 μL, with the addition of water to 1.6 mL) were introduced into test tubes, and then 100 μL of Folin−Ciocalteu reagent and 300 μL of sodium carbonate (20%) were added. The contents of the tubes were mixed and incubated at 40 °C for 40 min. Absorption at 725 nm was measured. The powders were then resuspended in distilled water and filtered through a 0.22 μm filter for use in cell cultures. 5093

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Table 1. Polyphenolic Compounds in the MWEsa

inhibitors (20 μM of SB203580, SP600125, or PD98059; 1 μM of wortmannin; 50 μM Z-VAD-FMK; or 5 μg/mL NOK-1) for 30 min followed by treatment with MWEs (3.0 mg/mL). Transfection. A7r5 cells (3 × 105 cells/6-well dish) were pretransfected with dominant-negative (DN) JNK/p38 for 12 h. Liposome-mediated transfection was performed using LipofectAMINE Plus (Invitrogen).35 Extraction of Mitochondria Protein. Mitochondria were isolated with a mitochondria isolation kit according to the manufacturer’s instructions by using a reagent-based method (cat. no. 89874; Pierce, Rockford).36 After the indicated treatment of MWEs, we pelleted 2 × 107 cells by centrifuging the harvested cell suspension in a 2.0 mL microcentrifuge tube at ∼850g for 2 min. Then, we carefully removed and discarded the supernatant. We added 800 μL of Mitochondria Isolation Reagent A, vortexed at medium speed for 5 s, and incubated the tube on ice for exactly 2 min. We added 10 μL of Mitochondria Isolation Reagent B and vortexed at maximum speed for 5 s. Then, we incubated the tube on ice for 5 min, vortexing at maximum speed every minute. We added 800 μL of Mitochondria Isolation Reagent C, and we centrifuged the tube at 700g for 10 min at 4 °C. We transferred the supernatant to a new, 2.0 mL tube and centrifuged at 12 000g for 15 min at 4 °C. Then, we transferred the supernatant (cytosol fraction) to a new tube. The pellet contained the isolated mitochondria. Western Blot Analysis. For Western blot analysis,37 we used specific antibodies to evaluate the expressions of p-JNK, pp38, p-p53, p-Bad, Bad, Bcl-2, Mcl-1, p-Jun, Jun, Fos, activating transcription factor-2, cytochrome c, pro-caspase 3, cleavagecaspase 3, Fas-L, and C23 (all from Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), p38, JNK, and p53 (Cell Signaling), and β-actin (Sigma, St. Louis, MO, U.S.A.). After the indicated treatment of MWEs (3.0 mg/mL) for 48 h, equal amounts of cell lysates (50 μg protein) were separated by electrophoresis on 8−12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA, U.S.A.). 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 nonspecific binding, and they were then washed with Tween-20 for 30 min. Each membrane was incubated with the appropriate primary antibody for 2 h followed by horseradish peroxidaseconjugated second antibody (Sigma, St. Louis, MO, U.S.A.) for 1 h and developed by an ECL chemiluminescence kit (Millipore, Bedford, MA, U.S.A.), followed by analysis using AlphaImager Series 2200 software. The results were representative of at least three independent experiments. 4′, 6-Diamidino-2-phenylindole (DAPI) Staining. A7r5 cells (2 × 105 cells) were seeded in a 10 cm dish for 12 h and

HPLC chromatogram 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 2.92 0.54 2.68 1.10 1.21 0.35 3.22 0.27 0.26 0.34 0.35 0.50 0.52 0.58 15.13

± 0.05 ± 0.45 ± 0.03 ± 0.20 ± 0.24 ± 0.17 ± 0.04 ± 1.01 ± 0.01 ± 0.02 ± 0.10 ± 0.23 ± 0.07 ± 0.17 ± 0.12 ± 0.93

a

HPLC chromatograms of free polyphenols from MWEs (20 mg/mL). Quantitative assessment of the percentage of polyphenols in MWEs relative to the standards represents the average of three independent experiments.

cells with DNA content at the sub-G1 phase. The results were representative of at least three independent experiments. DNA Fragmentation. A7r5 cells were pretreated with or without 20 nM pifithrin-α for 30 min before the addition of MWEs (1−5 mg/mL) for 48 h. The cells were detached by trypsin−EDTA, washed with phosphate buffered saline, and then subjected to genomic DNA extraction using a DNeasy Tissue Kit (QIAGEN) according to the manufacturer’s instructions. DNA was separated by electrophoresis as previously described.34 P53, p38, JNK, ERK, PI3K, Caspase, and Fas-Ligand Inhibitor. Pifithrin-α, a p53 inhibitor, was purchased from Sigma (St. Louis, MO, U.S.A.), and diluted in dimethyl sulfoxide (DMSO). SB203580, a p38 inhibitor (Sigma, St. Louis, MO, U.S.A.), SP600125, a JNK inhibitor (CalbiochemNovabiochem, San Diego, CA), PD98059, an ERK inhibitor (Sigma, St. Louis, MO, U.S.A.), wortmannin, a PI3K inhibitor (Sigma, St. Louis, MO, U.S.A.), and Z-VAD (OMe)fluoromethyl ketone (FMK), a caspase inhibitor (Sigma, St. Louis, MO, U.S.A.) were diluted in DMSO. NOK-1 (sc-19681), a Fas-ligand (Fas-L) inhibitor (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), was diluted in phosphate-buffered saline. The cells were treated with different concentrations of the

Figure 1. Histological analysis of a representative atherosclerotic lesion from HCD-fed rabbits treated for 10 weeks with MWEs. 5094

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Figure 3. Effect of MAPK inhibitors on MWEs-induced apoptosis in A7r5 cells. Cultured cells were treated with or without 6.0 mg/mL of MWEs for 48 h and then treated in the absence or presence of various concentrations of wortmannin, PD98059, SP600125, and SB203580 for 30 min. Apoptosis was analyzed by (A) flow cytometry and (B) DAPI stain. Quantitative assessment of the percentage of A7r5 cells represents the average of three independent experiments. #p < 0.05, *p < 0.005 compared with MWEs only.

Figure 2. Effect of MWEs on the apoptosis of A7r5 cells. Cells were treated with or without MWEs (0−10 mg/mL) for 48 h, and then the apoptotic cells were stained by DAPI solution. (A) Apoptotic values were calculated as the percentage of apoptotic cells relative to the total number of cells in each random field (>100 cells). (B) Genomic DNA was analyzed by electrophoresis on 0.75% agarose gel and staining with ethidium bromide and then photographed under UV light. (C) Propidium-iodide-stained DNA histograms of the A7r5 cells were analyzed by flow cytometric analysis. Quantitative assessment of the percentage of cells in four phases represents the average of three independent experiments ± SD. *p < 0.001, **p < 0.0001 compared with the control.

variance (ANOVA). A p-value less than 0.05 was considered to be statistically significant.

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RESULTS Polyphenolic Compounds of MWEs. The main 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%), pcoumaric 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%), hydroxyflavin (0.58%), and unknown components without standards (Table 1). MWEs Inhibited Proliferation of VSMCs via Stimulating Apoptosis. Histological analysis was performed on a representative atherosclerotic lesion from HCD-fed rabbits after treatment with MWEs for 10 weeks. Figure 1 shows that the MWEs dose-dependently inhibited HCD-fed-induced

then treated with MWEs (3 mg/mL) for 48 h. The A7r5 cells were fixed in 2−4% formaldehyde in phosphate-buffered saline for 30 min and then stained with DAPI (Sigma, St. Louis, MO, U.S.A.) to visualize nuclei.38 Statistical Analysis. The results are reported as the means and standard deviations of three independent experiments, and statistical comparisons were evaluated by one-way analysis of 5095

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Figure 4. Effect of dominant-negative JNK/p38 on the related proteins of MWEs-induced apoptosis. A7r5 cells (5 × 105 cells/6-well dish) were pretransfected with dominant-negative JNK/p38 for 12 h and then were treated with MWEs (3.0 mg/mL) for 24 or 48 h. Cell lysates (50 μg) were prepared and subjected to Western blot analysis. Proteins were detected by (A) MAPK activation, (B) p53-signaling protein, (C) Bcl-2 family proteins, (D) nuclear transcriptional factors, and (E) caspase 3, FasL, and cytochrome c. β-actin was used for equal loading. #p < 0.05 compared with MWEs only, *p < 0.05 compared with MWEs only. Beta-actin and C23 were used as internal standards.

in the sub-G1 phase was analyzed by flow cytometry and DAPI staining (Figure 3). The A7r5 cells (5 × 105 cells/6-well dish) were pretransfected with dominant-negative JNK/p38 (DN-JNK/DN-p38) for 12 h and then treated with MWEs (3.0 mg/mL) for 24 h. Total cell lysates (50 μg), nuclear lysates, and cytosol lysate were prepared and subjected to Western blot analysis. Proteins were detected by specific antibodies. In Figure 3A, the MWEs-induced apoptosis was significantly increased after treatment with inhibitors of PI3K and ERK. After inhibition of JNK and p38 with DN-JNK and DN-p38, the stimulatory effect of MWEs on apoptosis was reduced, and the MWE-induced increased DNA fragmentation was also reduced (data not show). As shown in Figure 4A, after treatment with the MWEs, the expressions of phosphorylation of JNK and p38 were significantly increased. However, these increased expressions decreased after treatment with DN-JNK and DN-p38. After treatment with the MWEs, the expressions of phosphorylation of p53, Bax, and p21 were increased (Figure 4B). The increased expressions of phosphorylated p53 and p21 after treatment with MWEs were significantly reduced

intimal hyperplasia of vessel walls. In Figure 2, A7r5 cells were treated with or without MWEs (0−10 mg/mL) for the indicated times, and the apoptotic cells were stained by DAPI solution. As shown in Figure 2A, from the dose of 2 mg/mL, the MWEs effectively induced apoptosis of A7r5 VSMCs in a dose-dependent manner. DNA fragments in the A7r5 cells were then analyzed by electrophoresis on 0.75% agarose gel and staining with ethidium bromide. Propidium-iodide-stained DNA histograms of the A7r5 cells were analyzed by flow cytometry assay. DNA fragmentation of the A7r5 cells was increased as the dose of MWEs increased from 4 mg/mL (data not show). Flow cytometric analysis showed that the MWEs dose-dependently increased the percentage of apoptosis cells in the sub-G1 phase from the dose of 1 mg/mL (Figure 2B). MWEs Induced Apoptosis via JNK/p38 Signaling Pathways. The A7r5 cultured cells were treated with or without 6.0 mg/mL of MWEs for 48 h in the absence or presence of wortmannin (PI3K inhibitor), PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) for 30 min, and then the percentage of apoptotic cells 5096

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after treatment with the DN-JNK and DN-p38. The MWEsinduced increased expression of Bax was significantly reduced after inhibition of JNK but not changed after inhibition of p38. After treatment with the MWEs, the expressions of antiapoptosis proteins Bcl-2 and Mcl-1 were significantly decreased (Figure 4C). The decreased expressions of Bcl-2 and Mcl-1 after treatment with the MWEs were only partially recovered after treatment with the DN-JNK and DN-p38. After treatment with the MWEs, the expressions of Jun-p (phosphorylated Jun) and Fos in nuclei were increased (Figure 4D). Only the enhanced expression of Jun-p, but not Fos, was decreased after treatment with the DN-JNK. However, the enhanced expressions of Jun-p and Fos were decreased after treatment with the DN-p38. After treatment with the MWEs, the expressions of cytochrome c (cyto c), cleavage caspase-3 (activated caspase-3) and Fas-L in cytosol were all increased, and these increased expressions were all significantly reduced after treatment with the DN-JNK and DN-p38. MWEs Induced Intrinsic and Extrinsic Pathways via the Up-Regulation of p53. The A7r5 cells were treated with or without 6.0 mg/mL of MWEs for 48 h in the absence or presence of 20 nM pifithrin-α (p53 inhibitor) for 30 min (Figure 5). After treatment with the MWEs, the percentage of apoptotic cells in the sub-G1 phase increased significantly (Figure 5A). After treatment with the p53 inhibitor, the percentage of apoptotic cells in the sub-G1 phase decreased significantly. The increase in DNA fragmentation after MWEs treatment was reduced after treatment with the p53 inhibitor (Figure 5B). The A7r5 cells were pretreated with or without 20 nM pifithrin-α for 30 min before the addition of the MWEs (3 mg/mL) for 24 h (Figure 6). Phospho-p53, p53, and Bax expressions were determined using a specific monoclonal antibody. After treatment with the MWEs, the expressions of phosphorylated p53 and Bax were enhanced, and these increased expressions of p53 and Bax were decreased after treatment with the p53 inhibitor (Figure 6A). After MWEs treatment, the expressions of p53 in the nuclei and mitochondria were all enhanced, and these increased expressions were reduced after treatment with the p53 inhibitor (Figure 6B). The MWEs-induced increased expression of phosphorylated JNK was decreased significantly after treatment with the p53 inhibitor (Figure 6C). However, the MWEs-induced increased expression of phosphorylated p38 was not affected after p53 inhibitor treatment. We then investigated the relationship between p53 and the downstream transcription factors of JNK and p38, including Jun, Fos, AIF, and ENDOG. The MWEsinduced increased expressions of p-Jun, AIF, and ENDOG were reduced significantly after treatment with the p53 inhibitor; however, the MWEs-induced increased expression of Fos was not affected after treatment with the p53 inhibitor (Figure 6D). The MWEs-induced decreased expressions of the antiapoptotic proteins Bcl-2 and Mcl-1 were recovered after treatment with the p53 inhibitor; however, the MWEs-induced decreased expression of phosphorylated Bad was not affected by treatment with the p53 inhibitor (Figure 6E). The MWEsinduced increased expressions of cytochrome c and activated caspase 3 from the mitochondria were decreased after treatment with the p53 inhibitor (Figure 6F). The MWEsinduced increased expression of Fas-L was also decreased after p53 inhibitor treatment. Effect of Caspase Inhibitors on MWEs-Induced Apoptosis. A7r5 cultured cells were treated with or without 3.0 mg/mL of the MWEs for 48 h in the absence or presence of

Figure 5. Effect of a p53 inhibitor on MWEs-induced apoptosis in A7r5 cells. Cultured cells were treated with or without MWEs (6.0 mg/mL) for 48 h in the absence or presence of 20 nM pifithrin-α for 30 min. (A) Apoptosis was analyzed by flow cytometry. (B) Genomic DNA was analyzed by electrophoresis on 0.75% agarose gel and staining with ethidium bromide and then photographed under UV light. The results are representative of the average of three independent experiments. #p < 0.005 compared with the control, *p < 0.005 compared with MWEs only.

5 μg/mL NOK-1 (Fas-L inhibitor) or 50 μM Z-VAD-FMK (caspase inhibitor) for 30 min (Figure 7). Apoptosis was analyzed by flow cytometry, and quantitative assessment of the percentage of A7r5 cells in the sub-G1 phase was completed. The increased number of MWEs-induced apoptotic cells in the sub-G1 phase were reduced after treatment with the inhibitors of Fas-L and caspase, and especially with the caspase inhibitor.

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DISCUSSION It has been reported that polyphenols have many biochemical activities. A previous study showed that MLE can inhibit the oxidation of LDL in rabbits and humans.39 In addition, mulberry leaf polyphenols have been shown to possess an antiatherogenesis effect via inhibiting LDL oxidation and foam cell formation.40 Our previous in vitro studies revealed the underlying mechanisms of polyphenol-rich MLE in the prevention of atherosclerosis via inhibiting proliferation and migration of VSMCs by way of up-regulating p53, inhibiting cyclindependent kinase, and blocking small GTPase, Akt/NF-κB signals. The inhibitory effect of MLE on the proliferation and migration of VSMCs has been attributed to the polyphenol component of MLE.30,31 MWEs are rich in phenolic acids and 5097

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Figure 6. Effect of a p53 inhibitor on MWEs-induced p53 activation and expressions of related proteins in A7r5 cells. A7r5 cells were pretreated with or without 20 nM pifithrin-α for 30 min before the addition of MWEs at the indicated concentrations for 24 h. Fifty micrograms of total cell extracts was loaded per lane onto a 10% SDS-polyacrylamide gel and electro-blotted onto a nitrocellulose membrane. (A) p53 and Bax (B) p53 in nuclei and mitochondria extracts, (C) MAPK activation, (D) nuclear proteins, (E) Bcl-2 family protein, and (F) caspase 3, Fas-L, and cytochrome c. β-actin, C23, and Tom20 were used as internal controls. #p < 0.05 compared MWEs only and MWEs with pifithrin-α.

flavonoids, including gallic acid, caffeic acid, protocatechuic acid, catechin, epigallocatechin gallate, epicatechin, rutin, quercetin, and naringenin. Gallic acid has been reported to have a potential hypolipidemic effect on mice fed with a high-fat diet, as well as an ability to intervene in platelet−leukocyte interactions, thereby preventing atherosclerosis. Protocatechuic acid has also been reported to have an anti-inflammatory effect by inhibiting LDL oxidation. Flavones and catechins are the most powerful flavonoids that can protect against free radicals. Quercetin has been reported to inhibit the activity of xanthine oxidase, thereby reducing oxidative injury. Similarly, catechin, epicatechin, and epigallocatechin gallate have also been reported to have inhibitory effects on free radical production by inhibiting xanthine oxidase.31 The supplementation of rutin was reported to promote the excretion of fecal sterols, thereby leading to a decreased absorption of dietary cholesterol as well as lower plasma and hepatic cholesterol.41 Caffeic acid has been reported to inhibit PDGF-induced proliferation of vascular smooth muscle cells, thus to prevent atherosclerosis and restenosis.42 In the present in vivo and in vitro studies, we found that MWEs could effectively induce apoptosis of A7r5 VSMCs in a

dose-dependent manner. As shown in Figure 3A, the MWEsinduced apoptosis was significantly increased after treatment with inhibitors of PI3K and ERK. This indicated that the mechanism of MWEs-induced apoptosis was less related to PI3K and ERK signaling. MWEs may inhibit VSMCs proliferation by way of inhibiting PI3K and ERK signaling with another unknown mechanism but not apoptosis. After treatment with inhibitors of p38 and JNK, the MWEs-induced increase in apoptosis was reduced, indicating that MWEs induce apoptosis via these two pathways. After treatment with DN-JNK and DN-p38, the increased expressions of JNK-p and p38-p by MWEs were reduced, which confirmed MWEs stimulate apoptosis via the JNK and p38 pathways. The increased expressions of phosphorylated-p53 and p21 after treatment with MWEs were significantly reduced after treatment with DN-JNK and DN-p38, indicating that JNK and p38 affect p53. The MWEs-induced increased expression of Bax was significantly reduced after treatment with DN-JNK but not changed after treatment with DN-p38, which indicates that JNK but not p38 affects the expression of Bax. The decreased expressions of the antiapoptosis proteins Bcl-2 and Mcl-1 after 5098

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Figure 7. Effect of caspase inhibitors on MWEs-induced apoptosis in A7r5 cells. Cultured cells were treated with or without MWEs (3.0 mg/mL) for 48 h in the absence or presence of 5 μg/mL NOK-1 or 50 μM Z-VAD-FMK for 30 min. (A) Apoptosis was analyzed by flow cytometry. (B) Quantitative assessment of the percentage of A7r5 cells in the sub-G1 phase was done as indicated by propidium iodide (PI) and represents the average of three independent experiments. #p < 0.00001 compared with control, *p < 0.005, and **p < 0.0005 compared with MWEs only.

treatment with MWEs were only partially recovered after treatment with DN-JNK and DN-p38, suggesting that JNK and p38 affect the expressions of Bcl-2 and Mcl-1, but that there may still be other pathways regulating Bcl-2 and Mcl-1. Only the MWEs-induced enhanced expression of Jun-p, but not Fos, was decreased after treatment with DN-JNK. However, the MWEs-induced enhanced expressions of Jun-p and Fos were decreased after treatment with DN-p38, indicating that p38 is the key factor affecting transcription factors Jun and Fos and that JNK affects Jun. The MWEs-induced increased expressions of cytochrome c, cleavage caspase-3, and Fas-L in cytosol were all significantly reduced after treatment with DN-JNK and DN-p38, which confirmed the pathway by which MWEs activate p38 and JNK and then affect Fas-L and apoptosis. After treatment with a p53 inhibitor, the MWEs-induced increased apoptosis decreased significantly, indicating that MWEs induce apoptosis by way of p53. The MWEs-induced increased expression of Bax was decreased after treatment with a p53 inhibitor, indicating that the MWEs-induced apoptosis in mitochondria is

related to p53. As shown in Figure 6B, after treatment with MWEs, the expressions of p53 in nuclei and mitochondria were enhanced, and these increased expressions were reduced again after treatment with a p53 inhibitor, indicating that MWEs affect the expressions of p53 in both nuclei and mitochondria. The MWEs-induced increased expression of phosphorylated JNK was decreased significantly after treatment with a p53 inhibitor. However, the MWEs-induced increased expression of phosphorylated p38 was not affected by treatment with a p53 inhibitor. These results indicate that p53 also affects MWEsinduced phosphorylation of JNK but not p38 phosphorylation. The MWEs-induced increased expressions of p-Jun, AIF, and ENDOG were reduced significantly after treatment with a p53 inhibitor; however, the MWEs-induced increased expression of Fos was not affected after treatment with a p53 inhibitor, indicating that p53 is upstream of Jun, AIF, and ENDOG but not Fos. The MWEs-induced decreased expressions of the antiapoptotic proteins Bcl-2 and Mcl-1 were recovered after treatment with a p53 inhibitor, however the MWEs-induced 5099

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decreased expression of phosphorylated Bad was not affected by treatment with a p53 inhibitor. The MWEs-induced increased expressions of cytochrome c and activated caspase 3 from mitochondria were decreased after treatment with a p53 inhibitor. The MWEs-induced increased expression of Fas-L was also decreased after treatment with a p53 inhibitor. This indicates that MWEs affect apoptosis both in intrinsic mitochondria and extrinsic Fas-L pathways by way of p53. The increased number of apoptotic cells in the sub-G1 phase induced by the MWEs was reduced after treatment with the inhibitors of Fas ligand and caspase, indicating that MWEs induce apoptosis by way of both Fas-L and caspase pathways. In summary, MWEs first activate JNK/p38 and p53, which in turn activate the Fas-L and mitochondria pathways. This then causes mitochondria translocation of Bax as well as the reduction of Bcl-2, which triggers the cleavage of procaspases and finally results in apoptosis of VSMCs. It is known that intimal hyperplasia is the main cause of restenosis after PCI. Stimulating apoptosis of VSMCs and thereby inhibiting VSMCs proliferation and migration is important for the prevention of atherosclerosis in the early stage, and this may be able to reduce the restenosis rate of PCI. The present study shows that MWEs can effectively stimulate the apoptosis of VSMCs by regulating both extrinsic and intrinsic pathways, thereby preventing atherosclerosis and intimal hyperplasia with restenosis after PCI. Our findings show that 0.5−1.0% of MWEs is effective to reduce HCD-fed-induced intimal hyperplasia in rabbits. Therefore, 2.5−5.0 g/day of MWEs for humans may be enough to prevent atherosclerosis.

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AUTHOR INFORMATION

Corresponding Author

*E- mail: [email protected]. Fax: 886-4- 23248167. Tel.: 886-424730022 ext. 11670. Author Contributions #

These authors contributed equally to this work and therefore share first authorship. Funding

This study was supported by a grant (no. NSC99-2320-B-040013-MY3) from the Ministry of Science and Technology, Taiwan. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ATF-2: activating transcription factor-2; AP-1: activator protein 1; cyto c: cytochrome c; DAPI: 4′, 6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; Fas-L: Fas ligand; HPLC: highperformance liquid chromatography; HCD-fed: high cholesterol diet fed; JNK: c-Jun N-terminal kinase; LDL: low-density lipoprotein; MLE: mulberry leaf extract; MWEs: mulberry water extracts; PCI: percutaneous coronary intervention; VSMCs: vascular smooth muscle cells

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