Article pubs.acs.org/jnp
Hispolon Attenuates Balloon-Injured Neointimal Formation and Modulates Vascular Smooth Muscle Cell Migration via AKT and ERK Phosphorylation Yi-Chung Chien,† Guang-Jhong Huang,‡,§ Hsu-Chen Cheng,† Chieh-Hsi Wu,⊥ and Ming-Jyh Sheu*,⊥ †
Department of Life Science and Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan § Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung 404, Taiwan ⊥ School of Pharmacy, China Medical University, Taichung 404, Taiwan ‡
ABSTRACT: The pathological mechanism of restenosis is attributed primarily to excessive proliferation and migration of vascular smooth muscle cells (VSMC). The preventive effects of hispolon (1) on balloon injury-induced neointimal formation were investigated, and 1 showed potent activity in inhibiting fetal bovine serum-induced VSMC outgrowth. Hispolon (1) significantly inhibited VSMC migration, as shown by trans-well assays. Compound 1 decreased the expression and secretion of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The expression of the endogenous inhibitors of these proteins, namely, tissue inhibitors of MMP (TIMP-1 and TIMP-2), increased. The inhibition by noncytotoxic doses of 1 of VSMC migration was through its negative regulatory effects on FAK phosphorylation, ERK1/2 phosphorylation, and PI3K/AKT. These results demonstrate that 1 can inhibit the migration of VSMC by reduced expression of MMP-9 through the suppression of the FAK signaling pathway and of the activity of PI3K/AKT. The data obtained suggest that 1 might block balloon injuryinduced neointimal hyperplasia via the inhibition of VSMC proliferation and migration, without inducing apoptosis. “Sanghwang” in Taiwan. It is popular in oriental countries and has been used traditionally as a food and medicine. This organism contains many bioactive compounds and is known for improving health and preventing and alleviating lymphatic diseases and is also used to treat cancer.6,7 An ethanol extract of P. merrillii showed potent α-glucosidase and aldose reductase activities. These α-glucosidase and aldose reductase inhibitors were identified as hispidin, hispolon (1), and inotilone, which were isolated from an EtOAc-soluble fraction of an ethanol extract of P. merrillii.8 Our group reported recently that 1 inhibits foot paw edema in mice.9 Hispolon (1) inhibits the metastasis of SK-Hep1 cells by reduced expression of matrix metalloproteinase-2 (MMP-2), MMP-9, and protease urokinase-type plasminogen activator (uPA), suggesting that nontoxic doses of 1 may be used as an antimetastatic agent.10 Others have also shown that 1 has antiproliferative and immunomodulatory activities.11 An ethanol extract of P. merrillii was found to exhibit antioxidant and free-
T
he main obstacle of percutaneous transluminal coronary angiography (PTCA) with placement of a non-drug-eluting stent is in-stent restenosis. Restenosis indicates an array of healing response of injured vessels. Endothelial disruption, inflammation, cell proliferation, and migration of vascular smooth muscle cells (VSMC) have been carefully described.1 Currently, the prevention of migration and proliferation of VSMC becomes more prominent during the processes of drugeluting stents in clinical trials or applications.2,3 Mushrooms are both functional foods and important sources of physiologically beneficial medicines. They produce various classes of secondary metabolites with interesting biological activities and thus have the potential to be used as valuable chemical resources for drug discovery.4 Several mushrooms belonging to the genera Inonotus and Phellinus have been used as traditional medicines for the treatment of gastrointestinal cancer, cardiovascular disease, heart disease, stomach ailments, and diabetes.5 Interestingly, these mushrooms commonly produce a number of yellow antioxidant pigments that comprise hispidin derivatives and phenols. Phellinus merrillii (Murrill) Rydvarden (Hymenochaetaceae) is a mushroom that is called commonly © 2012 American Chemical Society and American Society of Pharmacognosy
Received: March 20, 2012 Published: September 11, 2012 1524
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radical-scavenging activities.12 Previous studies have shown that pretreatment with antioxidants can reduce significantly balloon injury-induced neointimal formation.13 Accordingly, in the investigation, the inhibitory effect of the fruiting body of P. merrillii on balloon injury-induced restenosis was evaluated for its potential in treating neointimal formation. Since earlier studies have shown that pretreatment with antioxidants can significantly reduce balloon injury-induced neointimal formation,13 an ethanol extract of P. merrillii containing antioxidants inclusive of 1 may have potential to prevent restenosis. In an antiinflammatory assay, 1 decreased paw edema 4−5 h after λcarrageenin administration and increased activities of superoxide dismutase, glutathione peroxidase, and glutathione reductase in the liver tissue.9 It was demonstrated also that 1 significantly attenuated malondialdehyde levels in the edematous paw at 5 h after Carr injection.12 Therefore, 1 was investigated to determine if it contributes to the attenuation of VSMC proliferation and migration. The present study provides a general insight into the pharmacological mechanism of 1 in preventing the outgrowth of smooth muscle cells, which is a potential intervention for balloon injury-induced neointimal formation.
expression, suggesting that this compound may affect neointimal formation (Figures 1 and 2). Determination of a Noncytotoxic Concentration of Hispolon (1). Since the outgrowth of VSMC has been regarded as the major factor leading to restenosis, the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the inhibitory effects of 1 on cell viability of VSMC. Compound 1 exhibited potency against VSMC viability with IC50 values of 37.05 ± 0.13 μg/mL. As shown in Figure 3, 1 inhibited the viability of VSMC in a concentration-dependent manner. Hispolon (1) (5−25 μg/mL) demonstrated noncytotoxic effects on VSMC with a cytotoxicity of less than 10%. The inhibitory effect of 1 on cell viability became significant at 30, 35, and 40 μg/mL (p < 0.05) after 24 h incubation. Since the MTT assay showed that 1 at 30, 35, and 40 μg/mL significantly suppressed cell viability, it was postulated that the inhibitory effects of 1 on cell viability might be mediated by apoptosis. Therefore, 1 was evaluated at 10, 20, 30, 40, 50, and 60 μg/mL to determine its effects on the cell cycle and on apoptosis (Figure 4A). The results demonstrated that treatment for 24 h with 1 at 10, 20, 30, and 40 μg/mL had no effects on apoptosis in the sub-G1-phase (Figure 4B). Therefore, the concentrations 10, 15, and 20 μg/mL were selected for subsequent studies. Hispolon (1) Inhibited FBS-Stimulated VSMC Motility in a Wound-Healing Assay. The effect of 1 was assessed on the migration of VSMC using a wound-healing assay in which the confluent monolayer was scraped with a sterile micropipet tip to create a scratch wound (Figure 5A). It was found that 1 at 10, 15, and 20 μg/mL inhibited the migration of VSMC by 12 ± 0.25%, 6.51 ± 0.78%, and 43.21 ± 1.23% after incubation for 18 h (Figure 5B). Hispolon (1) Inhibited FBS-Stimulated VSMC Migration. A trans-well assay was used to investigate the migration and invasion of VSMC at 18 h after treatment with 1 (Figure 6A). It was found that 1 at 15 and 20 μg/mL decreased migration significantly (Figure 6B) of VSMC. Hispolon (1) Inhibit the Release of MMP-9 in VSMC. Previous studies have shown that the expression or activation of MMPs is upregulated in human atherosclerotic lesions and in rat arteries after balloon catheter injury.17 Increased pro-MMP-9, MMP-2, and pro-MMP-2 activities were detected after arterial injury in a rabbit model of intimal hyperplasia.17 To examine the possible mechanisms of migration with 1, MMP-2 and MMP-9 activities were determined in a culture medium of VSMC by zymographic analysis (Figure 7A). In the absence of treatment, VSMC constitutively secreted high levels of MMP-9 and MMP2. Hispolon (1) inhibited MMP-9 and MMP-2 activities after incubation for 24 h (Figure 7B). Hispolon (1) Activated MMP and TIMP Expression in VSMC. The physiological activities of MMPs are also related closely to that of their specific endogenous tissue inhibitors of metalloproteinase (TIMPs).18 In general, activation of MMP-9 is regulated primarily by the balance between proenzyme activation and the inhibition by TIMP-119. MMP-2 activation occurs on the cell membrane through the formation of a trimolecular complex composed of the membrane-type MMP (MT1-MMP), TIMP-2, and pro-MMP-2.20 Inhibition of invasion mediated by MMPs plays a key feature in the prevention of the metastasis of VSMC. The control of MMP synthesis and activation can therefore be regarded as an important target for the prevention of VSMC progression. Moreover, there is substantial evidence for the inhibition of MMPs and suppression of invasiveness and
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RESULTS AND DISCUSSION Hispolon (1) Inhibits Balloon Injury-Induced Neointimal Formation on the Rat Carotid Artery. Restenosis of the artery shortly following PTCA is a major limitation to the success of the procedure and is primarily due to smooth muscle cell accumulation in the artery wall at the site of balloon injury. VSMC migration and proliferation contribute to the intimal hyperplasia.14 Therefore, modulation of the growth of VSMC has critical therapeutic implications.15 Intimal hyperplasia induced by balloon injury was evident as compared with the normal control (Figure 1A). The present results showed that both doses of 1 (10 and 20 mg/mL) were effective in preventing neointimal formation (Figure 1A). Using computerized image analysis, area ratios of intimal and media layers were calculated, and reductions of about 25% and 58% in the area ratio of 1-treated groups were found at 10 and 20 mg/mL, respectively, as compared with the balloon-injured control group (Figure 1B). Hispolon (1) Inhibits Balloon Injury-Induced Proliferating Cell Nuclear Antigen (PCNA) Immunostaining. PCNA is synthesized in the early G1- and S-phases of the cell cycle and is required for cells to progress from the G1-phase to the S-phase. PCNA therefore can be used as a marker for proliferating cells in both normal and disease states.16 Here, the effects of two different doses of 1 (10 and 20 mg/mL) were shown on PCNA immunostaining after balloon injury. PCNA-positive cells were abundant in the balloon injury group (Figure 2). However, the PCNA immunostaining of cells treated with 1 at 10 and 20 mg/ mL (Figure 2) was less evident. These data provide evidence that 1 suppressed the level of PCNA, suggesting that the antiproliferative effect of 1 results from its ability to block entry of the cells into the S-phase due to interference in the early G0/G1 transition phase. Hispolon (1) suppressed PCNA 1525
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Figure 1. Preventive effect of 1 on balloon injury-induced neointima formation. (A) The left panel represents low-power (100×) observations from a balloon-injured vessel (a), a balloon-injured vessel treated with 1 at 10 mg/mL (b), a balloon-injured vessel treated with 1 at 20 mg/mL (c), and a sham control (d). The right panel represents the high-power (400×) observations from a balloon-injured vessel (e), a balloon-injured vessel treated with 1 at 10 mg/mL (f), a balloon-injured vessel treated with 1 at 20 mg/mL (g), and a sham control (h). L, lumen; N, neointimal; E, elastic fiber; M, medium. Black arrowhead indicates elastic fiber; bidirection arrowhead indicates neointimal formation. (B) Neointimal:medium area ratio in balloon-injured rat carotid arteries. The control group (control) shows a significantly higher area ratio as compared with the groups treated with 1 at a lower concentration (10 mg/mL) or a higher concentration (20 mg/mL). Values are means of two separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group: *p < 0.05, **p < 0.01. 1526
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Figure 2. Cross sections from Sprague−Dawley rat coronary arteries after balloon injury and staining with proliferating cell nuclear antigen (PCNA): a balloon-injured vessel (a); a balloon-injured vessel treated with a solution of 1 at 10 mg/mL (b); a balloon-injured vessel treated with 1 at 20 mg/mL (c). Each tissue sample of the rat artery was cut into 10 μm thick sections and mounted on glass slides for immunohistochemistry. The antibodies were monoclonal mouse antibody PCNA (1:2000 dilution). Red arrow means positive cells. The control group (control) shows a significantly higher PCNA positive as compared with the groups treated with 1 at a lower concentration (10 mg/mL) or a higher concentration (20 mg/mL) (d). Values are means of three separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group: *p < 0.05, **p < 0.01.
Figure 3. The viability suppression effects of 1 treatment on VSMC. Cells were incubated for 24 h with 10% fetal bovine serum alone (control) or with different concentrations of 1: 5, 10, 15, 20, 25, 30, 35, and 40 μg/mL. Values are means of three separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group at 0 h: *p < 0.05, **p < 0.01, ***p < 0.001.
metastases of VSMC using low molecular weight fucoidan.21 To explore further the modulation of pro-MMP and MMP activation by 1, MMP-2/9 and TIMP-1/2 protein expression levels were determined. Hispolon (1) strongly increased TIMP-1 and TIMP-2 activity in a concentration-dependent manner (Figure 8A and B). Hispolon (1) Inhibited Phosphorylated Focal Adhesion Kinase (FAK), Phosphorylated Extracellular Signal-Regulated Kinase (ERK), and PI3K/AKT Signaling in VSMC. Indolfi suggests that the mitogen-activated protein kinases
(MAPK) is majorly involved in the regulation of VSMC proliferation.22 During vascular injury, the MAPK signaling pathway is activated, and the inhibition of activated ERK pathway by drugs or gene therapy can reduce neointima formation.23 Ras gene has been reported from our laboratory that it might play an important role in regulating neointima formation.24 The downstream targets of the Ras signaling pathway include ERK1/2, which is involved in the proliferation of vascular smooth muscle cells.25 The abovementioned pathways are to be activated during integrin binding to extracellular matrix (ECM), 1527
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Figure 4. Hispolon (1) affects the percentage of cell cycle distribution in VSMC. (A) All the cells were treated with 15% fetal bovine serum with the addition of 1 at 10, 20, 30, 40, 50, and 60 μg/mL for 24 h. The value on the x axis represents the DNA content, while the shaded area indicates the percentage of cells at the S-phase. PE-A, phycoerythrin-A. (B) Percentage of sub-G1 content in vascular smooth muscle cells treated with 1 at 10, 20, 30, 40, 50, and 60 μg/mL for 24 h. Values are means of three separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group (0 μg/mL 1 with 10% FBS-treated group). *p < 0.05, **p < 0.01.
PI3K, indicating that the AKT protein is another potential target for hispolon (1) (Figure 9A). AKT mediates cell survival and growth signals by phosphorylating and inactivating proapoptotic proteins.30 To investigate further the involvement of PI3K/Akt, a series of experiments were performed to measure the expression of candidate signaling molecules on the stimulation of 1. The results showed that incubation of VSMC cells with 1 (10, 15, and 20 μg/mL) led to a concentrationdependent decrease of PI3K and p-AKT levels (Figure 9). In conclusion, our results imply that the ERK1/2-MAPK together with PI3K/AKT pathways might be potential targets to regulate fetal bovine serum-induced VSMC migration.
bringing about the transduction of those external stimuli from the ECM to nucleus.26 To evaluate the effect of 1 on FAK and ERK1/2 protein phosphorylation, VSMC were treated with 1 at 10, 15, and 20 μg/mL for 24 h. As shown in Figure 9A, 1 at 10, 15, and 20 μg/mL suppressed FAK phosphorylation. The phosphorylation of MAPKs in VSMC was analyzed after treatment with 1 (10, 15, and 20 μg/mL) for 30 min, and the data showed that 1 significantly affects the phosphorylation of ERK (p-ERK1/2) in VSMC cells (Figure 9B). Treatment with the MEK inhibitor PD98059 successfully inhibited ERK 1/2 MAPK activities and VSMC proliferation.27 The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays a pivotal role in the regulation of cellular growth, apoptosis, and metabolism.28 PI3K/AKT signaling is required reportedly for VSMC migration and proliferation, while the absence of AKT impairs VSMC proliferation and migration.29 The level of AKT kinase phosphorylation was increased by serum in the present study, and 1 inhibited serum-induced phosphorylation of AKT and
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EXPERIMENTAL SECTION
Chemicals. Hispolon (1) was purchased from BJYM Pharmaceutical. & Chemical Co., Ltd. (Beijing, People’s Republic of China). The purity of 1 used in the present study was >95%. Dulbecco’s modified Eagle’s medium (DMEM), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenylte1528
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Figure 5. Inhibitory effect of 1 on wound-healing migration of VSMC. VSMC were treated with 5, 10 and 20 μg/mL of 1 for 18 h, and the migration distances of cells were calculated. (A) Cell migration was examined in a wound-healing assay. Representative photographs of invading cells that received either control or 1 treatment. (B) Migrated cells across the black lines were counted in six random fields from each treatment. The mean number of cells in the denuded zone is quantified by three independent experiments. Values are means of three separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group: *p < 0.05. trazolium bromide (MTT), RNase A, Pluronic F-127, and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Trypsin−EDTA, fetal bovine serum (FBS), and penicillin/ streptomycin were purchased from Gibco Life Technologies, Inc. (Paisley, UK). Hematoxylin and eosin Y were obtained from Merck (Whitehouse Station, NJ, USA). Cell culture supplies were purchased from Costar (Corning, Inc., Cypress, CA, USA). The antibody against PCNA, Akt, MAPK/ERK 1/2 protein, and phosphorylated proteins were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-ERK1/2, anti-PI3K, anti-FAK, anti-pFAK, MMP-2, MMP-9, and horseradish peroxidase-conjugated goat anti-mouse IgG antibody were purchased from Santa Cruz Biotechnology Co. (Santa Cruz, CA, USA). Cell Culture. A10 VSMC derived from rat thoracic aorta were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units of penicillin, and 100 mg of streptomycin per mL. The cells were kept in a humidified 5% CO2−95% air incubator at 37 °C. Cells were cultured in DMEM medium containing various concentrations of 1 with 10% serum. Cells cultured in medium containing 0.5% serum without 1
to maintain the minimal physiologic condition served as a negative control. Cells cultured in medium containing 15% serum without 1 served as a positive control. Cytotoxicity Assay. According to the method reported by Huang et al. with some modifications, the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was performed to measure the cytotoxicity of 1 on VSMC. Cells were seeded in 96-well plates with 1 × 104 cells/well in DMEM supplemented with 10% FBS.10 After 24 h, cells were washed with PBS and then exposed to either 10% FBS alone or serial dilutions (5, 10, 15, 20, 25, 30, 35, and 40 μg/mL) of 1. After 24 h, the number of viable cells was determined. In brief, MTT (3 mg/mL in PBS) was added to each well (25 μL per 200 mL of medium), and the plate was incubated at 37 °C for 2 h. Cells were then spun at 300g for 5 min, and the medium was carefully aspirated. A 50 μL sample of dimethylsulfoxide was added, and the absorbance at 570 nm was measured for each well on an ELISA reader (Anthos 2001; Anthos Labtec, Salzburg, Austria). Flow Cytometric Analysis. Cellular total DNA contents of the treated cells were assessed using flow cytometry following propidium iodide staining.31 Cells were harvested with trypsin−EDTA, washed 1529
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Figure 6. Down-regulatory effects of 1 on a trans-well migration assay of VSMC. (A) Cell migration was examined in a trans-well with polycarbonate filters (pore size, 8 μm); (B) Migration abilities of VSMC were quantified by counting the number of cells in the outer polycarbonate membrane under microscopy and represented the average of three independent experiments with duplicate samples. Values are means of three separate experiments, with standard errors represented by vertical bars. Mean value was significantly different from that of the control group: *p < 0.05, **p < 0.01.
Figure 7. Down-regulatory effects of 1 on MMP-2 and MMP-9 release. To examine the possible mechanisms of migrations with 1, the activities of MMP-2 and MMP-9 in culture medium of VSMC were determined by zymographic analysis. (A) In the absence of treatment, VSMC constitutively secreted high levels of MMP-9 and MMP-2. (B) Compound 1 inhibited MMP-9 and MMP-2 activities in a concentration-dependent manner for 24 h. Mean value was significantly different from that of the control group: *p < 0.05, **p < 0.01. twice with 10 mL of ice-cold PBS, fixed in 70% ethanol, and kept at 4 °C before fluorescence activated cell sorting (FACS) analysis. For DNA content analysis, cells were centrifuged and resuspended in 0.3 mL of DNA staining solution (100 Ag/mL PI, 0.2% Nonidet P-40, and 1 mg/ mL RNase A (DNase-free) in PBS lacking Ca2+ and Mg2+; at a 1:1:1
ratio by vol). The cell suspension was stored on ice in a dark room for a minimum of 30 min and analyzed within 2 h. Cells were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). PI fluorescence was amplified linearly, and both the area and width of the fluorescence pulse were measured. Ten thousand events were acquired, 1530
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Figure 8. Hispolon (1) affects the levels of TIMP-1 and TIMP-2 protein expressions. (A) VSMC were treated with 10, 15, and 20 μg/mL 1 for 24 h, and cell lysates were subjected to SDS-PAGE followed by Western blotting and subsequently quantified by densitometric analysis (using control as 100%). (B) Mean value was significantly different from that of the control group (15% FBS): *p < 0.05, **p < 0.01. and the percentages of hypodiploid (apoptotic, sub-G1) events and percentages of cells in the G0/G1, S, and G2/M phases were determined using DNA analysis software, ModFitLT, version 2.0 (Verity Software, Topsham, ME, USA). Wound-Healing Assay. This assay is according to the method reported by Huang et al. with some modifications.10 For cell motility determination, VSMC (3 × 104) were seeded in a six-well tissue culture plate and grown to 80−90% confluence. After aspiration of the medium, the center of the cell monolayers was scraped with a sterile micropipet tip to create a denuded zone (gap) of constant width. Subsequently, cellular debris was washed with PBS, and VSMC cells were exposed to various concentrations of 1 (10, 15, and 20 μg/mL). Wound closure was monitored and photographed at 18 h with a Nikon inverted microscope. To quantify the migrated cells, pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation. Artificial lines fitting the cutting edges were drawn on pictures of the original wounds and overlaid on the pictures of cultures after incubation. Cells that had migrated across the white lines were counted in six random fields from each triplicate treatment. Cell Migration Assay. VSMC migration was assayed in trans-well chambers (Millipore, CA, USA) according to the method reported by Huang et al. with some modifications.10 Briefly, trans-well chambers with 6.5 mm polycarbonate filters of 8 μm pore size were used. VSMC (4 × 105 mL−1) and 0, 10, 15, and 20 μg/mL 1 were suspended in DMEM (100 μL, serum-free), placed in the upper trans-well chamber, and incubated for 18 h at 37 °C. Then, the cells on the upper surface of the filter were completely wiped away with a cotton swab, and the lower surface of the filter was fixed in methanol, stained with Giemsa, and counted under a microscope at a magnification of 200×. For each replicate, the tumor cells in 10 randomly selected fields were determined and the counts were averaged. Determination of MMP-2 and MMP-9 by Zymography. MMP in the medium released from VSMC was assayed using gelatin zymography (7.5% zymogram gelatin gels), according to a method reported by Huang et al. with some modification.10 Briefly, the culture medium was electrophoresed (120 V for 90 min) in a 10% SDS-PAGE gel containing 0.1% gelatin. The gel was then washed at room temperature in a solution containing 2.5% (v/v) Triton X-100 with two
changes and subsequently transferred to a reaction buffer for enzymatic reaction containing 1% NaN3, 10 mM CaCl2, and 40 mM Tris-HCl, pH 8.0, maintained at 37 °C with shaking overnight (for 12−15 h). Finally, the MMP gel was stained for 30 min with 0.25% (w/v) Coomassie blue in 10% acetic acid (v/v) and 20% methanol (v/v) and destained in 10% acetic acid (v/v) and 20% methanol (v/v). Western Blotting Analysis. This analysis is according to the method reported by Huang et al. with some modifications.10 VSMC cultured in six-well plates were incubated with 1 at 10, 15, and 20 μg/mL in DMEM containing 15% FBS for 24 h. The cells were then lysed in a buffer containing 2% SDS, 50 mM dithiothreitol, and 62.5 mM TrisHCl, at pH 6.8, followed by incubation at 95 °C for 5 min. Samples were separated using SDS-PAGE, transferred to polyvinylidene fluoride membranes, blocked with 5% nonfat dry milk in PBS−Tween for 1 h, and then probed with the desired antibodies (anti-PCNA, Akt, MAPK/ ERK 1/2, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38 MAPK proteins and phosphorylated proteins, anti-ERK1/2, anti-PI3K, anti-FAK, anti-pFAK, MMP-2, MMP-9, and horseradish peroxidase-conjugated goat anti-mouse IgG antibody) overnight at 4 °C. The blots were then incubated with horseradish peroxidase-linked secondary antibody for 1 h followed by development with the electrochemical luminescence reagent and exposure to Hyperfilm (Amersham, Arlington Heights, IL, USA). Balloon Angioplasty. Twenty-four male Sprague−Dawley rats weighing 350−400 g were purchased from BioLASCO (Taipei, Taiwan). Twenty-four rats were used and divided into four groups including total injury control without 1 (n = 8) and 10 mg/mL (n = 8) and 20 mg/mL (n = 8) 1-treated groups. Animals were housed in a 12 h light−dark cycle with free access to food and water. All experimental procedures involving animals were approved by the ethics committee of the Institutional Animal Care and Use Committee of China Medical University (IACUC; protocol number 100-105-N). Angioplasty of the carotid artery was performed using a balloon embolectomy catheter, as described previously.31 In brief, the balloon catheter (2F Fogarty) (Becton-Dickinson, Franklin Lakes, NJ, USA) was introduced through the right external carotid artery into the aorta, and the balloon was inflated at 1.3 kg/cm2 using an inflation device. An inflated balloon was pushed and pulled through the lumen three times to damage the vessel. 1531
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Figure 9. Dose-dependent effects of 1 on the protein expression and phosphorylation level of PI3K, pFAK, pAKT, and p-ERK1/2. (A) After activation with 15% FBS, VSMC were treated with 10, 15, and 20 μg/mL 1, respectively. The expression of proteins was analyzed by Western blotting. β-Actin was used as a loading control. (B) Mean value was significantly different from that of the control group (15% FBS): *p < 0.05, **p < 0.01, ***p < 0.001.
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Two concentrations of 1 (10 and 20 mg/mL) suspended in 200 μL of 30% pluronic-F127 gel were coated onto arterial adventitia of the balloon-injured carotid artery.32 Two weeks after balloon injury, rats were sacrificed. For morphological examination, right common carotid arteries were collected and then fixed in 4% paraformaldehyde and embedded in a Parafilm block. Embedded vessel tissues were cut into 10 μm thick slices, and then, slices were stained with hematoxylin and eosin Y (Merck, Whitehouse Station, NJ, USA). The manifestation of vessel restenosis was presented as the ratio of the neointimal to media areas. Immunohistochemistry Demonstration of Proliferating Cell Nuclear Antigen. Each tissue sample of the rat artery was cut into 10 μm thick sections and mounted on glass slides for immunohistochemistry. The antibody used was monoclonal mouse antibody PCNA (1:2000 dilution; Novus Biologicals, Littleton, CO, USA). Statistical Analysis. Values are expressed as means ± SD and analyzed using one-way ANOVA followed by LSD test for comparisons of group means. All statistical analyses were performed using SPSS for Windows, version 10 (SPSS, Inc.). The level of significance was set at p value < 0.05.
AUTHOR INFORMATION
Corresponding Author
*Tel: +886-2205-3366, ext 5158. Fax: +886-4-22078083. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
The study was supported by grants from the National Science Council (NSC 100-2320-B-039-014), China Medical University (CMU99-TC-29, CMU100-ASIA-18), and Department of Health Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004 and DOH101-TD-B-111-005). This research was also supported by the National Science Council and by the Ministry of Education, Taiwan, Republic of China, under the ATU plan (NSC 101-2311-B-005-006-MY3). We are grateful to J. Conrad for English writing assistance. 1532
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dx.doi.org/10.1021/np3002145 | J. Nat. Prod. 2012, 75, 1524−1533
