Research Article
Nobiletin Inhibits PDGF-BB-Induced Vascular Smooth Muscle Cell Proliferation and Migration and Attenuates Neointimal Hyperplasia in a Rat Carotid Artery Injury Model
DDR
DRUG DEVELOPMENT RESEARCH 75 : 489–496 (2014)
Siyu Guan1,2, Qizhu Tang1*, Wenwei Liu2, Rui Zhu,2 and Bin Li2 Department of Cardiology, Renmin Hospital of Wuhan University,Wuhan 430060, China 2 Department of Cardiology, Xiangyang Central Hospital, Hospital Affiliated to Hubei University of Arts and Science, Xiangyang 441021, China 1
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics
Preclinical Research
Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics
Clinical Development Phases I-III Regulatory, Quality, Manufacturing
Postmarketing Phase IV
ABSTRACT The abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the development of neointimal hyperplasia after vascular injury. Nobiletin, a citrus bioflavonoid, exhibits anti-inflammatory and anti-oxidative activities. The present study evalutaed whether nobiletin could inhibit platelet-derived growth factor (PDGF)-BB- stimulated VSMC proliferation and migration and decrease neointimal hyperplasia in a rat carotid artery injury model. Cultured VSMCs from rat thoracic aortas were treated with nobiletin before being stimulated with 20 ng/ml PDGF-BB, and rats were subjected to carotid artery injury. Nobiletin inhibited PDGF-BB-induced VSMC proliferation and migration, attenuated reactive oxygen species (ROS) production and reduced phosphorylation of ERK1/2 and the expression of nuclear NF-κB p65 in PDGF-BB-stimulated VSMCs. Nobiletin decreased the intima area and the ratio of neointima to media in balloon-injured rat carotid arteries. Serum levels of TNF-α and IL-6 in nobiletin-treated rats were decreased. These results indicated that nobiletin could be a potential protective agent for the prevention and treatment of restenosis after angioplasty. Drug Dev Res 75 : 489–496, 2014. © 2014 Wiley Periodicals, Inc. Key words: nobiletin; vascular smooth muscle cells; neointimal hyperplasia; artery injury
INTRODUCTION
Although percutaneous coronary intervention has improved the survival rate of patients with coronary heart disease, restenosis after angioplasty remains a critical clinical problem in approximately 10% of patients [Moses et al., 2003; Pursnani et al., 2012]. The abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) play pivotal roles in the development of neointimal hyperplasia in the process of restenosis after angioplasty and atherosclerosis [Ross, 1993; Costa and Simon, 2005; Orr et al., 2010]. Inflammation is a key mediator in the migration and prolifera© 2014 Wiley Periodicals, Inc.
tion of VSMCs and inhibition of inflammation could significantly attenuate both processes, resulting in decreases in neointimal hyperplasia in vivo [Kim et al., 2009; Wang et al., 2013; Won et al., 2013]. *Correspondence to: Qizhu Tang, Department of Cardiology, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan 430060, Hubei Province, China. E-mail: [email protected] Received 23 July 2014; Accepted 05 September 2014 Published online in Wiley Online Library (wileyonlinelibrary .com). DOI: 10.1002/ddr.21230
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Nobiletin (5,6,7,8,3',4'-hexamethoxy flavone), a citrus polymethoxyflavone found in citrus fruits, has anti-inflammatory and anti-oxidative activities and antitumor effect in many tumor cells [Murakami et al., 2000b, 2005; Cui et al., 2010]. Nobiletin also inhibited cell migration and metastasis of liver cancer HepG2 cells [Shi et al., 2013]. Recently, Zhou et al. [2009] reported that treatment of nobiletin could inhibit bovine VSMC proliferation via a calcium-mediated c-Jun N-terminal kinase pathway. However, whether nobiletin could inhibit VSMC migration and attenuate the neointimal hyperplasia after vascular injury is unknown. The present study investigated the effect of nobiletin on platelet-derived growth factor (PDGF)BB-stimulated VSMC proliferation and migration in vitro and neointimal hyperplasia in a rat carotid artery injury model.
MATERIALS AND METHODS
After that, 20 μl of the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) was added and the absorbance at 450 nm was measured with a microplate reader (Perkin Elmer, Waltham, MA). Cell viability was assessed by trypan blue exclusion microscopy. Cell migration assay Cell migration activity was investigated with Transwell chamber system (Corning, Corning, NY) following the manufacturer’s instruction. Briefly, 105 cells were seeded into the upper chamber in the serum-free medium with or without 20 μM nobiletin, and the lower chamber was filled with DMEM containing 20 ng/ml PDGF-BB. After incubation for 8 h, cells on the top of the membrane were moved and the cells on the bottom of the membrane were fixed with methanol, stained with 0.5% crystal violet and manually counted in nine independent, randomly chosen visual fields by an investigator blinded to the assignment.
VSMCs Culture and Nobiletin Treatment The protocols for animal care and all the experiments in this study were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University and complied with the guideline for the care and use of laboratory animals published by the US National Institutes of Health (Institute of Laboratory Animal Research et al., 1996). VSMCs were isolated from the thoracic aorta of 6-week-old Sprague-Dawley rats as described previously [Tokunou et al., 2003], and then cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA). The purity of VSMCs was assessed to be more than 95% by fluorescent immunostaining with anti-smooth muscle α-actin antibody (Sigma Aldrich, St. Louis, MO). VSMCs were passaged, and their 3rd to 5th passages were used in the present study. Nobiletin was purchased from Sigma Aldrich and dissolved in dimethyl sulfoxide (DMSO), and stored at −20°C. The final concentration of DMSO in culture media was below 0.1%, and DMSO treatment alone did not alter cell viability, proliferation, and migration in this study.
Cell proliferation assay VSMCs were seeded in 96-well plates at a density of 104 cell per well in 200 μl culture medium. After synchronization by DMEM with 0.1% FBS overnight, cells were treated with normal various final concentrations of nobiletin (10, 20, 30, 50, and 100 μM) for 30 min, and then the cells were stimulated with 20 ng/ml PDGF-BB and incubated for another 24 h. Drug Dev. Res.
Reactive oxygen species (ROS) detection ROS assay kit (Beyotime, Nanjing, China) was used to measure the ROS generation by VSMCs [Zhang et al., 2012]. After synchronized by DMEM with 0.1% FBS overnight, cells in 6-well plates were treated with 50 μM nobiletin or DMSO for 30 min and then exposed to 20 ng/ml PDGF-BB for 1 h. After that, the cells were incubated with 10 μM 2',7'-dichloro dihydrofluorescein diacetate (DCFH-DA) for 30 min in dark at room temperature as instructed by the manufacturer. Fluorescent images were acquired at an excitatory wavelength of 495 nm and the mean densities of the fluorescent images were quantified with Image-Pro Plus 6.0 software, by an investigator blinded to the assignment. Western blot analysis The phosphorylation of ERK1/2 and nuclear translocation of NF-κB p65 were explored by Western blot. For detection of the phosphorylation of ERK1/2, total protein was collected 10 min after stimulation of PDGF-BB. For detection of the expression of NF-κB p65, nuclear protein was obtained 24 h after treatment of PDGF-BB with or without pretreatment of nobiletin. Protein concentration was determined with the bicinchoninic acid protein assay kit (Beyotime). Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. For immunoblotting, the membranes were blocked with non-fat milk and incubated with antibodies against p-ERK1/2 (Thr202/
NOBILETIN AND PDGF-BB-INDUCED VSMCs PROLIFERATION AND MIGRATION
Tyr204) (Cell Signaling, Danvers, MA), ERK1/2 (Cell Signaling), NF-κB p65 (Santa Cruz Technology, Santa Cruz, CA), β-actin (Santa Cruz), and Histone H3 (Abcam, Cambridge, UK) overnight at 4°C. After three washes with PBS containing 0.2% Tween-20, the blots were incubated with horseradish peroxidaseconjugated secondary antibodies (Bio-Rad, Hercules, CA) for 1 h on a shaker at room temperature. After three washes, the blots were detected with enhanced chemiluminescence solution and subsequently analyzed with the ChemiDoc™ MP system (Bio-Rad). Rat carotid artery balloon injury model Thirty male Sprague-Dawley rats weighing (450– 500 g) were purchased from Wuhan University Center for Animal Experiment and randomly assigned into three groups, including sham, control and nobiletin tretaed (n = 10 each). The rats in nobiletin group and control group were anesthetized with 30 mg/kg pentobarbital and subjected to balloon injury of the left common carotid artery as described previously [Xu et al., 2013]. Briefly, after intravenous injection 100 U/kg heparin sodium, the left common, external and internal carotid arteries were exposed, and then the blood flow of the common and internal carotid arteries was temporarily interrupted. After that, the external carotid artery was partially cut at about 2 mm from the arterial bifurcation with micro-scissors. Then, the balloon angioplasty catheter (Medtronic, Fridley, MN) was inserted through the opening of the external carotid artery into the common carotid artery. The balloon was inflated to produce moderate resistance, and gradually withdrawn. After repeating the injury procedure for three times, the catheter was removed, and then the external carotid branch was ligated. Finally, the blood flow of the common and internal carotid arteries was restored, and the wound was sutured. The rats in the sham group underwent the same procedure without the step of balloon insertion. Nobiletin (25 mg/kg/d) by gavage was fed in the nobiletin group 2 days before the surgery and until the day before sacrifice. The other two groups were treated with saline as control. At the end of treatment, rats were euthanized and the venous blood collected. Injured arteries were harvested and fixed with 4% paraformaldehyde for morphometric analysis. Morphometric analysis To determine the intimal and medial crosssectional areas of the carotid arteries, three round crosssections were cut from the approximate middle of the injured artery, stained with hematoxylin–eosin and ana-
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lyzed using Image-Pro Plus 6.0 software. The intimal and medial cross-sectional areas of the carotid arteries were measured by the same investigator blind to the assignment, and the intima/media ratio was calculated.
Detection of serum levels of TNF-α and IL-6 When the rats were killed, blood was collected using Vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ) and centrifuged immediately at 3,000 g for 10 min at 4°C. Then the supernatant was allocated and stored at −80°C until used for the assay. The levels of TNF-α and IL-6 were analyzed using enzyme-linked immunosorbent assay kits (Biosource, Camarillo, CA) according to the manufacturer’s instructions.
Statistical analysis Data are presented as mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA) Data were analyzed using one-way analysis of variance followed by a Newman–Keuls test. A value of P < 0.05 was considered statistically significant.
RESULTS
Nobiletin Inhibits PDGF-BB-Induced Proliferation and Migration of VSMCs As shown in Figure 1A, treatment of nobiletin at low concentrations did not affect cell viability. However, 100 μM nobiletin slightly increased cell death (P < 0.05). While nobiletin (50 μM) did not affect cell growth, nobiletin dose-dependently reduced the proliferation of VSMCs induced by PDGF-BB (all P < 0.05) (Fig. 1B). In addition, 50 μM nobiletin inhibited VSM migration induced by PDGF-BB in a Transwell chamber system (P < 0.05) (Fig. 1C).
Nobiletin Suppresses PDGF-BB-Stimulated ROS Generation of VSMCs ROS plays a crucial role in the proliferation of VSMCs and is responsible for the production of inflammation in the vessel wall [Paravicini and Touyz, 2006]. Compared with untreated cells, PDGF-BB increased the intracellular ROS generation of VSMCs (0.480 ± 0.041 vs. 0.114 ± 0.009, P < 0.01; Fig. 2). Nobiletin suppressed the intracellular ROS generation in PDGF-BB-treated VSMCs (0.204 ± 0.011 vs. 0.480 ± 0 .041, P < 0.01). Drug Dev. Res.
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expression of ERK1/2 and NF-κB p65 were explored. PDGF-BB increased ERK1/2 phosphorylation and the expression of nuclear NF-κB p65 (Fig. 3). Pretreatment with 50 μM nobiletin blocked phosphorylation of ERK1/2 and activation of NF-κB signaling induced by PDGF-BB (both P < 0.05). Nobiletin Attenuates Neointimal Hyperplasia in Balloon Injured Carotid Arteries As shown in Figure 4A, the injured common carotid arteries of the control group developed serious stenosis 14 days after surgery. Compared with the control group, nobiletin reduced the intima area (0.244 ± 0.026 vs. 0.625 ± 0.029, P < 0.01, Fig. 4B) and the ratio of intima to media area (0.990 ± 0.100 vs. 1.615 ± 0.152, P < 0.01, Fig. 4C). Nobiletin Decreases the Serum Levels of TNF-α and IL-6 in Balloon-Injured Rats As shown in Figure 5A, carotid artery injury enhanced serum level of TNF-α 14 days after surgery (129.80 ± 5.65 pg/ml vs. 76.60 ± 4.30 pg/ml, P < 0.01). Compared with controls, nobiletin decreased serum TNF-α (98.18 ± 6.49 pg/mL vs. 129.80 ± 5.65 pg/mL, P < 0.01). Similarly, the serum level of IL-6 was increased in the control group, and nobiletin reduced it in carotid artery injured rats (73.78 ± 4.14 pg/mL vs. 86.93 ± 3.13 pg/mL, P < 0.05) (Fig. 5B). DISCUSSION
Fig. 1. Nobiletin inhibits PDGF-BB-induced proliferation and migration of VSMCs. (A) Treatment of nobiletin at low concentrations did not affect the cell viability. However, 100 μM nobiletin slightly increased cell death. (B) Treatment of nobiletin dosedependently reduced the proliferation of VSMCs induced by PDGFBB. (C) 50 μM nobiletin inhibited the migration of VSMCs induced by PDGF-BB. *P > 0.05 compared with untreated group; #P < 0.05 compared with untreated group; ##P < 0.05 compared with PDGFBB-treated group.
Nobiletin Blocksphosphorylation of ERK1/2 and Activation of NF-κB Signaling Induced by PDGF-BB To explore the potential mechanism for the effect of nobiletin on VSMC proliferation, effects on the Drug Dev. Res.
Neointimal hyperplasia and vessel remodeling are the two major processes for the development of restenosis after angioplasty [Costa and Simon, 2005]. With the wide use of coronary artery stents, vessel remodeling has less impact because of minimal negative remodeling. However, prevention and treatment of neointimal hyperplasia after angioplasty is still a critical challenge [Pendyala et al., 2008; Robertson et al., 2012]. In the present study nobiletin was found to decrease the area of intima and the ratio of intima area to media area at 14 days after surgery in a rat carotid artery injury model, suggesting that nobiletin is a potential treatment for the treatment of neointimal hyperplasia induced by vessel injury. Abnormal proliferation and migration of VSMCs can be induced by cytokines and growth factors, such as angiotensin II and PDGF, after vascular intimal injury [Louis and Zahradka, 2010]. PDGF is crucial regulators of VSMCs biological function with welldefined activities as a mitogen and chemoattractants
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Fig. 2. Nobiletin suppresses PDGF-BB-stimulated ROS generation of VSMCs. (A) Representative immunofluorescence photos of intracellular ROS (×100). VSMCs were treated with or without 50 μM nobiletin for 30 min and then exposed to 20 ng/ml PDGF-BB for 1 h. The ROS were detected by incubating with DCFH-DA. (B) Quantification of fluorescent intensity showed that PDGF-BB remarkably increased the intracellular ROS and pretreatment of nobiletin decreased intracellular ROS in PDGF-BB-treated VSMCs. *P > 0.05 compared with untreated group; #P < 0.05 compared with untreated group; ##P < 0.05 compared with PDGF-BB-treated group.
Fig. 3. Treatment of nobiletin blocks phosphorylation of ERK1/2 and activation of NF-κB signaling induced by PDGF-BB. (A) Representative immunoblots of p-ERK1/2 and total ERK1/2. (B) Densitometric quantification of the phosphorylation of ERK1/2. VSMCs were treated with or without 50 μM nobiletin for 30 min and then exposed to 20 ng/ml PDGF-BB for 10 min. Pretreatment of nobiletin blocked the phosphorylation of ERK1/2 induced by PDGF-BB. (C) Representative immunoblots of nuclear NF-κB p65 signaling. (D) Densitometric quantification of the expression of nuclear NF-κB p65. VSMCs were treated with or without 50 μM nobiletin for 30 min and then exposed to 20 ng/ml PDGF-BB for 24 h. Pretreatment of nobiletin could effectively decrease the expression of nuclear NF-κB p65 in PDGF-BB-stimulated VSMCs. #P < 0.05 compared to untreated group; ##P < 0.05 compared with PDGF-BB-treated group.
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Fig. 4. Nobiletin attenuates neointimal hyperplasia in the balloon injured carotid arteries. (A) Representative hematoxylin–eosin stained sections of carotid arteries for each group (×100). (B) Quantification showed that nobiletin reduced the intima area. (C) Quantification of the ratio of intima area to media area supported the inhibitory effect of nobiletin on neointimal hyperplasia in the balloon injured carotid arteries.
Fig. 5. Nobiletin decreases the serum levels of TNF-α and IL-6 in the carotid arteries balloon-injured rats. (A) Carotid artery injury could enhance the serum level of TNF-α at 14 days after the surgery. Compared with the control group, treatment of nobiletin could significantly decrease the serum level of TNF-α. (B) The serum level of IL-6 was increased in the control group and treatment of nobiletin dramatically reduced it in carotid artery injured rats. #P < 0.05 compared with sham group; ##P < 0.05 compared with control group.
[Sachinidis et al., 1990; Park et al., 2013]. PDGF activates the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways leading to the initiation of multiple biologiDrug Dev. Res.
cal effects [Liu et al., 1997]. PDGF-BB, a subtype of PDGF, potently stimulates VSMC proliferation and migration and participates in the processes of atherosclerosis and restenosis after angioplasty [Cospedal
NOBILETIN AND PDGF-BB-INDUCED VSMCs PROLIFERATION AND MIGRATION
et al., 1999; Kingsley et al., 2002; Zhan et al., 2003]. Since the prevention of pathological VSMC proliferation and migration remains as a major clinical challenge, new therapeutic strategies are needed. In this study, the data demonstrated that nobiletin could inhibit PDGF-BB-induced VSMC proliferation and migration. Nobiletin (10–50 μM) dose-dependently inhibited PDGF-BB-induced VSMC proliferation without impairing cell viability, although a higher dose (100 μM) decreased the cell survival rate. Similarly, Zhou et al. [2009] reported that nobiletin inhibited angiotensin-induced bovine VSMC proliferation and DNA synthesis. However, they found that 100 μM nobiletin alone did not affect cell growth. Interestingly, Shi et al. [2013] showed that 2.5 μM nobiletin decreased cell viability in liver cancer HepG2 cells and treatment of 25 μM nobiletin resulted in less than 40% cell viability. In C6 rat glioma cells, 30 and 100 μM nobiletin significantly decrease cell viability while 10 μM nobiletin did not affect cell viability [Aoki et al., 2013]. The reasons for these discrepancies are unclear, and we suspect that they may be due to the different species origins of VSMCs and different cell types. This means that treatment of nobiletin in vivo may cause unexpected complications and further study is essential before any clinical application. It should be noted that in the animal study we did not observe any obvious behavior and physical abnormalities in rats chronically treated with 25 mg/kg/d nobiletin. Clinical and experimental data have indicated that inflammation may be central to intimal growth after mechanical arterial injury [Costa and Simon, 2005]. Nobiletin decreased serum TNF-α and IL-6 in the balloon-injured rats, indicating that it reduced the inflammatory state in vivo. Lin et al. [2003] reported that nobiletin effectively suppressed expression of proinflammatory cytokines including IL-6 and TNF-α in mouse macrophage J774A cells. In fact, the antioxidative and anti-inflammatory activities of nobiletin have been widely evaluated [Murakami et al., 2000a; Ho and Lin, 2008; Cui et al., 2010; Guo et al., 2012]. In agreement with these studies, our results showed that PDGF-BB could increase ROS generation of VSMCs and subsequently activate ERK1/2 and NF-κB signaling, and pretreatment of nobiletin potently inhibit these changes. In summary, the present study demonstrated that nobiletin could inhibit VSMC proliferation and migration in vitro and neointimal hyperplasia following carotid balloon injury in vivo by regulating ROS/NF-κB pathway and inflammation suggesting that nobiletin is a putative treatment for the prevention of restenosis after angioplasty.
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ACKNOWLEDGMENT
We thank Dr. Xiaolin Wu for assisting the preparation of the manuscript and critical advice. REFERENCES Aoki K, Yokosuka A, Mimaki Y, Fukunaga K, Yamakuni T. 2013. Nobiletin induces inhibitions of Ras activity and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling to suppress cell proliferation in C6 rat glioma cells. Biol Pharm Bull 36:540–547. Cospedal R, Abedi H, Zachary I. 1999. Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases. Cardiovasc Res 41:708–721. Costa MA, Simon DI. 2005. Molecular basis of restenosis and drugeluting stents. Circulation 111:2257–2273. Cui Y, Wu J, Jung SC, Park DB, Maeng YH, Hong JY, Kim SJ, Lee SR, Kim SJ, Kim SJ, et al. 2010. Anti-neuroinflammatory activity of nobiletin on suppression of microglial activation. Biol Pharm Bull 33:1814–1821. Guo S, Qiu P, Xu G, Wu X, Dong P, Yang G, Zheng J, McClements DJ, Xiao H. 2012. Synergistic anti-inflammatory effects of nobiletin and sulforaphane in lipopolysaccharide-stimulated RAW 264.7 cells. J Agric Food Chem 60:2157–2164. Ho SC, Lin CC. 2008. Investigation of heat treating conditions for enhancing the anti-inflammatory activity of citrus fruit (Citrus reticulata) peels. J Agric Food Chem 56:7976–7982. Kim HJ, Yoo EK, Kim JY, Choi YK, Lee HJ, Kim JK, Jeoung NH, Lee KU, Park IS, Min BH, et al. 2009. Protective role of clusterin/apolipoprotein J against neointimal hyperplasia via antiproliferative effect on vascular smooth muscle cells and cytoprotective effect on endothelial cells. Arterioscler Thromb Vasc Biol 29:1558–1564. Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, D.C. National Academy Press. Kingsley K, Huff JL, Rust WL, Carroll K, Martinez AM, Fitchmun M, Plopper GE. 2002. ERK1/2 mediates PDGF-BB stimulated vascular smooth muscle cell proliferation and migration on laminin-5. Biochem Biophys Res Commun 293:1000–1006. Lin N, Sato T, Takayama Y, Mimaki Y, Sashida Y, Yano M, Ito A. 2003. Novel anti-inflammatory actions of nobiletin, a citrus polymethoxy flavonoid, on human synovial fibroblasts and mouse macrophages. Biochem Pharmacol 65:2065–2071. Liu P, Ying Y, Anderson RG. 1997. Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae. Proc Natl Acad Sci USA 94:13666–13670. Louis SF, Zahradka P. 2010. Vascular smooth muscle cell motility: from migration to invasion. Exp Clin Cardiol 15:e75–e85. Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O’Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS, et al. 2003. Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med 349:1315–1323. Murakami A, Nakamura Y, Ohto Y, Yano M, Koshiba T, Koshimizu K, Tokuda H, Nishino H, Ohigashi H. 2000a. Suppressive effects
Drug Dev. Res.
496
GUAN ET AL.
of citrus fruits on free radical generation and nobiletin, an antiinflammatory polymethoxyflavonoid. Biofactors 12:187–192. Murakami A, Nakamura Y, Torikai K, Tanaka T, Koshiba T, Koshimizu K, Kuwahara S, Takahashi Y, Ogawa K, Yano M, et al. 2000b. Inhibitory effect of citrus nobiletin on phorbol esterinduced skin inflammation, oxidative stress, and tumor promotion in mice. Cancer Res 60:5059–5066. Murakami A, Shigemori T, Ohigashi H. 2005. Zingiberaceous and citrus constituents, 1'-acetoxychavicol acetate, zerumbone, auraptene, and nobiletin, suppress lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 murine macrophages through different modes of action. J Nutr 135 (12 Suppl.):2987S– 2992S. Orr AW, Hastings NE, Blackman BR, Wamhoff BR. 2010. Complex regulation and function of the inflammatory smooth muscle cell phenotype in atherosclerosis. J Vasc Res 47:168–180. Paravicini TM, Touyz RM. 2006. Redox signaling in hypertension. Cardiovasc Res 71:247–258. Park ES, Lee KP, Jung SH, Lee DY, Won KJ, Yun YP, Kim B. 2013. Compound K, an intestinal metabolite of ginsenosides, inhibits PDGF-BB-induced VSMC proliferation and migration through G1 arrest and attenuates neointimal hyperplasia after arterial injury. Atherosclerosis 228:53–60. Pendyala L, Jabara R, Shinke T, Chronos N, Robinson K, Li J, Hou D. 2008. Drug-eluting stents: present and future. Cardiovasc Hematol Agents Med Chem 6:105–115. Pursnani S, Korley F, Gopaul R, Kanade P, Chandra N, Shaw RE, Bangalore S. 2012. Percutaneous coronary intervention versus optimal medical therapy in stable coronary artery disease: a systematic review and meta-analysis of randomized clinical trials. Circ Cardiovasc Interv 5:476–490. Robertson KE, McDonald RA, Oldroyd KG, Nicklin SA, Baker AH. 2012. Prevention of coronary in-stent restenosis and vein graft failure: does vascular gene therapy have a role? Pharmacol Ther 136:23–34. Ross R. 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801–809.
Drug Dev. Res.
Sachinidis A, Locher R, Vetter W, Tatje D, Hoppe J. 1990. Different effects of platelet-derived growth factor isoforms on rat vascular smooth muscle cells. J Biol Chem 265:10238–10243. Shi MD, Liao YC, Shih YW, Tsai LY. 2013. Nobiletin attenuates metastasis via both ERK and PI3K/Akt pathways in HGF-treated liver cancer HepG2 cells. Phytomedicine 20:743–752. Tokunou T, Shibata R, Kai H, Ichiki T, Morisaki T, Fukuyama K, Ono H, Iino N, Masuda S, Shimokawa H, et al. 2003. Apoptosis induced by inhibition of cyclic AMP response element-binding protein in vascular smooth muscle cells. Circulation 108:1246– 1252. Wang Z, Zhang X, Chen S, Wang D, Wu J, Liang T, Liu C. 2013. Lithium chloride inhibits vascular smooth muscle cell proliferation and migration and alleviates injury-induced neointimal hyperplasia via induction of PGC-1alpha. PLoS ONE 8: e55471. Won KJ, Jung SH, Lee CK, Na HR, Lee KP, Lee DY, Park ES, Choi WS, Shim SB, Kim B. 2013. DJ-1/park7 protects against neointimal formation via the inhibition of vascular smooth muscle cell growth. Cardiovasc Res 97:553–561. Xu C, Chen J, Zhang J, Hu X, Zhou X, Lu Z, Jiang H. 2013. Naringenin inhibits angiotensin II-induced vascular smooth muscle cells proliferation and migration and decreases neointimal hyperplasia in balloon injured rat carotid arteries through suppressing oxidative stress. Biol Pharm Bull 36:1549–1555. Zhan Y, Kim S, Izumi Y, Izumiya Y, Nakao T, Miyazaki H, Iwao H. 2003. Role of JNK, p38, and ERK in platelet-derived growth factor-induced vascular proliferation, migration, and gene expression. Arterioscler Thromb Vasc Biol 23:795–801. Zhang JK, Yang L, Meng GL, Fan J, Chen JZ, He QZ, Chen S, Fan JZ, Luo ZJ, Liu J. 2012. Protective effect of tetrahydroxystilbene glucoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells. Eur J Pharmacol 689:31–37. Zhou CH, Wu XH, Wu YQ. 2009. Nobiletin, a dietary phytochemical, inhibits vascular smooth muscle cells proliferation via calcium-mediated c-Jun N-terminal kinases pathway. Eur J Pharmacol 615:55–60.
Nobiletin Inhibits PDGF-BB-Induced Vascular Smooth Muscle Cell Proliferation and Migration and Attenuates Neointimal Hyperplasia in a Rat Carotid Artery Injury Model
DDR
DRUG DEVELOPMENT RESEARCH 75 : 489–496 (2014)
Siyu Guan1,2, Qizhu Tang1*, Wenwei Liu2, Rui Zhu,2 and Bin Li2 Department of Cardiology, Renmin Hospital of Wuhan University,Wuhan 430060, China 2 Department of Cardiology, Xiangyang Central Hospital, Hospital Affiliated to Hubei University of Arts and Science, Xiangyang 441021, China 1
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics
Preclinical Research
Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics
Clinical Development Phases I-III Regulatory, Quality, Manufacturing
Postmarketing Phase IV
ABSTRACT The abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the development of neointimal hyperplasia after vascular injury. Nobiletin, a citrus bioflavonoid, exhibits anti-inflammatory and anti-oxidative activities. The present study evalutaed whether nobiletin could inhibit platelet-derived growth factor (PDGF)-BB- stimulated VSMC proliferation and migration and decrease neointimal hyperplasia in a rat carotid artery injury model. Cultured VSMCs from rat thoracic aortas were treated with nobiletin before being stimulated with 20 ng/ml PDGF-BB, and rats were subjected to carotid artery injury. Nobiletin inhibited PDGF-BB-induced VSMC proliferation and migration, attenuated reactive oxygen species (ROS) production and reduced phosphorylation of ERK1/2 and the expression of nuclear NF-κB p65 in PDGF-BB-stimulated VSMCs. Nobiletin decreased the intima area and the ratio of neointima to media in balloon-injured rat carotid arteries. Serum levels of TNF-α and IL-6 in nobiletin-treated rats were decreased. These results indicated that nobiletin could be a potential protective agent for the prevention and treatment of restenosis after angioplasty. Drug Dev Res 75 : 489–496, 2014. © 2014 Wiley Periodicals, Inc. Key words: nobiletin; vascular smooth muscle cells; neointimal hyperplasia; artery injury
INTRODUCTION
Although percutaneous coronary intervention has improved the survival rate of patients with coronary heart disease, restenosis after angioplasty remains a critical clinical problem in approximately 10% of patients [Moses et al., 2003; Pursnani et al., 2012]. The abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) play pivotal roles in the development of neointimal hyperplasia in the process of restenosis after angioplasty and atherosclerosis [Ross, 1993; Costa and Simon, 2005; Orr et al., 2010]. Inflammation is a key mediator in the migration and prolifera© 2014 Wiley Periodicals, Inc.
tion of VSMCs and inhibition of inflammation could significantly attenuate both processes, resulting in decreases in neointimal hyperplasia in vivo [Kim et al., 2009; Wang et al., 2013; Won et al., 2013]. *Correspondence to: Qizhu Tang, Department of Cardiology, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan 430060, Hubei Province, China. E-mail: [email protected] Received 23 July 2014; Accepted 05 September 2014 Published online in Wiley Online Library (wileyonlinelibrary .com). DOI: 10.1002/ddr.21230
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Nobiletin (5,6,7,8,3',4'-hexamethoxy flavone), a citrus polymethoxyflavone found in citrus fruits, has anti-inflammatory and anti-oxidative activities and antitumor effect in many tumor cells [Murakami et al., 2000b, 2005; Cui et al., 2010]. Nobiletin also inhibited cell migration and metastasis of liver cancer HepG2 cells [Shi et al., 2013]. Recently, Zhou et al. [2009] reported that treatment of nobiletin could inhibit bovine VSMC proliferation via a calcium-mediated c-Jun N-terminal kinase pathway. However, whether nobiletin could inhibit VSMC migration and attenuate the neointimal hyperplasia after vascular injury is unknown. The present study investigated the effect of nobiletin on platelet-derived growth factor (PDGF)BB-stimulated VSMC proliferation and migration in vitro and neointimal hyperplasia in a rat carotid artery injury model.
MATERIALS AND METHODS
After that, 20 μl of the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) was added and the absorbance at 450 nm was measured with a microplate reader (Perkin Elmer, Waltham, MA). Cell viability was assessed by trypan blue exclusion microscopy. Cell migration assay Cell migration activity was investigated with Transwell chamber system (Corning, Corning, NY) following the manufacturer’s instruction. Briefly, 105 cells were seeded into the upper chamber in the serum-free medium with or without 20 μM nobiletin, and the lower chamber was filled with DMEM containing 20 ng/ml PDGF-BB. After incubation for 8 h, cells on the top of the membrane were moved and the cells on the bottom of the membrane were fixed with methanol, stained with 0.5% crystal violet and manually counted in nine independent, randomly chosen visual fields by an investigator blinded to the assignment.
VSMCs Culture and Nobiletin Treatment The protocols for animal care and all the experiments in this study were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University and complied with the guideline for the care and use of laboratory animals published by the US National Institutes of Health (Institute of Laboratory Animal Research et al., 1996). VSMCs were isolated from the thoracic aorta of 6-week-old Sprague-Dawley rats as described previously [Tokunou et al., 2003], and then cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA). The purity of VSMCs was assessed to be more than 95% by fluorescent immunostaining with anti-smooth muscle α-actin antibody (Sigma Aldrich, St. Louis, MO). VSMCs were passaged, and their 3rd to 5th passages were used in the present study. Nobiletin was purchased from Sigma Aldrich and dissolved in dimethyl sulfoxide (DMSO), and stored at −20°C. The final concentration of DMSO in culture media was below 0.1%, and DMSO treatment alone did not alter cell viability, proliferation, and migration in this study.
Cell proliferation assay VSMCs were seeded in 96-well plates at a density of 104 cell per well in 200 μl culture medium. After synchronization by DMEM with 0.1% FBS overnight, cells were treated with normal various final concentrations of nobiletin (10, 20, 30, 50, and 100 μM) for 30 min, and then the cells were stimulated with 20 ng/ml PDGF-BB and incubated for another 24 h. Drug Dev. Res.
Reactive oxygen species (ROS) detection ROS assay kit (Beyotime, Nanjing, China) was used to measure the ROS generation by VSMCs [Zhang et al., 2012]. After synchronized by DMEM with 0.1% FBS overnight, cells in 6-well plates were treated with 50 μM nobiletin or DMSO for 30 min and then exposed to 20 ng/ml PDGF-BB for 1 h. After that, the cells were incubated with 10 μM 2',7'-dichloro dihydrofluorescein diacetate (DCFH-DA) for 30 min in dark at room temperature as instructed by the manufacturer. Fluorescent images were acquired at an excitatory wavelength of 495 nm and the mean densities of the fluorescent images were quantified with Image-Pro Plus 6.0 software, by an investigator blinded to the assignment. Western blot analysis The phosphorylation of ERK1/2 and nuclear translocation of NF-κB p65 were explored by Western blot. For detection of the phosphorylation of ERK1/2, total protein was collected 10 min after stimulation of PDGF-BB. For detection of the expression of NF-κB p65, nuclear protein was obtained 24 h after treatment of PDGF-BB with or without pretreatment of nobiletin. Protein concentration was determined with the bicinchoninic acid protein assay kit (Beyotime). Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. For immunoblotting, the membranes were blocked with non-fat milk and incubated with antibodies against p-ERK1/2 (Thr202/
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Tyr204) (Cell Signaling, Danvers, MA), ERK1/2 (Cell Signaling), NF-κB p65 (Santa Cruz Technology, Santa Cruz, CA), β-actin (Santa Cruz), and Histone H3 (Abcam, Cambridge, UK) overnight at 4°C. After three washes with PBS containing 0.2% Tween-20, the blots were incubated with horseradish peroxidaseconjugated secondary antibodies (Bio-Rad, Hercules, CA) for 1 h on a shaker at room temperature. After three washes, the blots were detected with enhanced chemiluminescence solution and subsequently analyzed with the ChemiDoc™ MP system (Bio-Rad). Rat carotid artery balloon injury model Thirty male Sprague-Dawley rats weighing (450– 500 g) were purchased from Wuhan University Center for Animal Experiment and randomly assigned into three groups, including sham, control and nobiletin tretaed (n = 10 each). The rats in nobiletin group and control group were anesthetized with 30 mg/kg pentobarbital and subjected to balloon injury of the left common carotid artery as described previously [Xu et al., 2013]. Briefly, after intravenous injection 100 U/kg heparin sodium, the left common, external and internal carotid arteries were exposed, and then the blood flow of the common and internal carotid arteries was temporarily interrupted. After that, the external carotid artery was partially cut at about 2 mm from the arterial bifurcation with micro-scissors. Then, the balloon angioplasty catheter (Medtronic, Fridley, MN) was inserted through the opening of the external carotid artery into the common carotid artery. The balloon was inflated to produce moderate resistance, and gradually withdrawn. After repeating the injury procedure for three times, the catheter was removed, and then the external carotid branch was ligated. Finally, the blood flow of the common and internal carotid arteries was restored, and the wound was sutured. The rats in the sham group underwent the same procedure without the step of balloon insertion. Nobiletin (25 mg/kg/d) by gavage was fed in the nobiletin group 2 days before the surgery and until the day before sacrifice. The other two groups were treated with saline as control. At the end of treatment, rats were euthanized and the venous blood collected. Injured arteries were harvested and fixed with 4% paraformaldehyde for morphometric analysis. Morphometric analysis To determine the intimal and medial crosssectional areas of the carotid arteries, three round crosssections were cut from the approximate middle of the injured artery, stained with hematoxylin–eosin and ana-
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lyzed using Image-Pro Plus 6.0 software. The intimal and medial cross-sectional areas of the carotid arteries were measured by the same investigator blind to the assignment, and the intima/media ratio was calculated.
Detection of serum levels of TNF-α and IL-6 When the rats were killed, blood was collected using Vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ) and centrifuged immediately at 3,000 g for 10 min at 4°C. Then the supernatant was allocated and stored at −80°C until used for the assay. The levels of TNF-α and IL-6 were analyzed using enzyme-linked immunosorbent assay kits (Biosource, Camarillo, CA) according to the manufacturer’s instructions.
Statistical analysis Data are presented as mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA) Data were analyzed using one-way analysis of variance followed by a Newman–Keuls test. A value of P < 0.05 was considered statistically significant.
RESULTS
Nobiletin Inhibits PDGF-BB-Induced Proliferation and Migration of VSMCs As shown in Figure 1A, treatment of nobiletin at low concentrations did not affect cell viability. However, 100 μM nobiletin slightly increased cell death (P < 0.05). While nobiletin (50 μM) did not affect cell growth, nobiletin dose-dependently reduced the proliferation of VSMCs induced by PDGF-BB (all P < 0.05) (Fig. 1B). In addition, 50 μM nobiletin inhibited VSM migration induced by PDGF-BB in a Transwell chamber system (P < 0.05) (Fig. 1C).
Nobiletin Suppresses PDGF-BB-Stimulated ROS Generation of VSMCs ROS plays a crucial role in the proliferation of VSMCs and is responsible for the production of inflammation in the vessel wall [Paravicini and Touyz, 2006]. Compared with untreated cells, PDGF-BB increased the intracellular ROS generation of VSMCs (0.480 ± 0.041 vs. 0.114 ± 0.009, P < 0.01; Fig. 2). Nobiletin suppressed the intracellular ROS generation in PDGF-BB-treated VSMCs (0.204 ± 0.011 vs. 0.480 ± 0 .041, P < 0.01). Drug Dev. Res.
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expression of ERK1/2 and NF-κB p65 were explored. PDGF-BB increased ERK1/2 phosphorylation and the expression of nuclear NF-κB p65 (Fig. 3). Pretreatment with 50 μM nobiletin blocked phosphorylation of ERK1/2 and activation of NF-κB signaling induced by PDGF-BB (both P < 0.05). Nobiletin Attenuates Neointimal Hyperplasia in Balloon Injured Carotid Arteries As shown in Figure 4A, the injured common carotid arteries of the control group developed serious stenosis 14 days after surgery. Compared with the control group, nobiletin reduced the intima area (0.244 ± 0.026 vs. 0.625 ± 0.029, P < 0.01, Fig. 4B) and the ratio of intima to media area (0.990 ± 0.100 vs. 1.615 ± 0.152, P < 0.01, Fig. 4C). Nobiletin Decreases the Serum Levels of TNF-α and IL-6 in Balloon-Injured Rats As shown in Figure 5A, carotid artery injury enhanced serum level of TNF-α 14 days after surgery (129.80 ± 5.65 pg/ml vs. 76.60 ± 4.30 pg/ml, P < 0.01). Compared with controls, nobiletin decreased serum TNF-α (98.18 ± 6.49 pg/mL vs. 129.80 ± 5.65 pg/mL, P < 0.01). Similarly, the serum level of IL-6 was increased in the control group, and nobiletin reduced it in carotid artery injured rats (73.78 ± 4.14 pg/mL vs. 86.93 ± 3.13 pg/mL, P < 0.05) (Fig. 5B). DISCUSSION
Fig. 1. Nobiletin inhibits PDGF-BB-induced proliferation and migration of VSMCs. (A) Treatment of nobiletin at low concentrations did not affect the cell viability. However, 100 μM nobiletin slightly increased cell death. (B) Treatment of nobiletin dosedependently reduced the proliferation of VSMCs induced by PDGFBB. (C) 50 μM nobiletin inhibited the migration of VSMCs induced by PDGF-BB. *P > 0.05 compared with untreated group; #P < 0.05 compared with untreated group; ##P < 0.05 compared with PDGFBB-treated group.
Nobiletin Blocksphosphorylation of ERK1/2 and Activation of NF-κB Signaling Induced by PDGF-BB To explore the potential mechanism for the effect of nobiletin on VSMC proliferation, effects on the Drug Dev. Res.
Neointimal hyperplasia and vessel remodeling are the two major processes for the development of restenosis after angioplasty [Costa and Simon, 2005]. With the wide use of coronary artery stents, vessel remodeling has less impact because of minimal negative remodeling. However, prevention and treatment of neointimal hyperplasia after angioplasty is still a critical challenge [Pendyala et al., 2008; Robertson et al., 2012]. In the present study nobiletin was found to decrease the area of intima and the ratio of intima area to media area at 14 days after surgery in a rat carotid artery injury model, suggesting that nobiletin is a potential treatment for the treatment of neointimal hyperplasia induced by vessel injury. Abnormal proliferation and migration of VSMCs can be induced by cytokines and growth factors, such as angiotensin II and PDGF, after vascular intimal injury [Louis and Zahradka, 2010]. PDGF is crucial regulators of VSMCs biological function with welldefined activities as a mitogen and chemoattractants
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Fig. 2. Nobiletin suppresses PDGF-BB-stimulated ROS generation of VSMCs. (A) Representative immunofluorescence photos of intracellular ROS (×100). VSMCs were treated with or without 50 μM nobiletin for 30 min and then exposed to 20 ng/ml PDGF-BB for 1 h. The ROS were detected by incubating with DCFH-DA. (B) Quantification of fluorescent intensity showed that PDGF-BB remarkably increased the intracellular ROS and pretreatment of nobiletin decreased intracellular ROS in PDGF-BB-treated VSMCs. *P > 0.05 compared with untreated group; #P < 0.05 compared with untreated group; ##P < 0.05 compared with PDGF-BB-treated group.
Fig. 3. Treatment of nobiletin blocks phosphorylation of ERK1/2 and activation of NF-κB signaling induced by PDGF-BB. (A) Representative immunoblots of p-ERK1/2 and total ERK1/2. (B) Densitometric quantification of the phosphorylation of ERK1/2. VSMCs were treated with or without 50 μM nobiletin for 30 min and then exposed to 20 ng/ml PDGF-BB for 10 min. Pretreatment of nobiletin blocked the phosphorylation of ERK1/2 induced by PDGF-BB. (C) Representative immunoblots of nuclear NF-κB p65 signaling. (D) Densitometric quantification of the expression of nuclear NF-κB p65. VSMCs were treated with or without 50 μM nobiletin for 30 min and then exposed to 20 ng/ml PDGF-BB for 24 h. Pretreatment of nobiletin could effectively decrease the expression of nuclear NF-κB p65 in PDGF-BB-stimulated VSMCs. #P < 0.05 compared to untreated group; ##P < 0.05 compared with PDGF-BB-treated group.
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Fig. 4. Nobiletin attenuates neointimal hyperplasia in the balloon injured carotid arteries. (A) Representative hematoxylin–eosin stained sections of carotid arteries for each group (×100). (B) Quantification showed that nobiletin reduced the intima area. (C) Quantification of the ratio of intima area to media area supported the inhibitory effect of nobiletin on neointimal hyperplasia in the balloon injured carotid arteries.
Fig. 5. Nobiletin decreases the serum levels of TNF-α and IL-6 in the carotid arteries balloon-injured rats. (A) Carotid artery injury could enhance the serum level of TNF-α at 14 days after the surgery. Compared with the control group, treatment of nobiletin could significantly decrease the serum level of TNF-α. (B) The serum level of IL-6 was increased in the control group and treatment of nobiletin dramatically reduced it in carotid artery injured rats. #P < 0.05 compared with sham group; ##P < 0.05 compared with control group.
[Sachinidis et al., 1990; Park et al., 2013]. PDGF activates the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways leading to the initiation of multiple biologiDrug Dev. Res.
cal effects [Liu et al., 1997]. PDGF-BB, a subtype of PDGF, potently stimulates VSMC proliferation and migration and participates in the processes of atherosclerosis and restenosis after angioplasty [Cospedal
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et al., 1999; Kingsley et al., 2002; Zhan et al., 2003]. Since the prevention of pathological VSMC proliferation and migration remains as a major clinical challenge, new therapeutic strategies are needed. In this study, the data demonstrated that nobiletin could inhibit PDGF-BB-induced VSMC proliferation and migration. Nobiletin (10–50 μM) dose-dependently inhibited PDGF-BB-induced VSMC proliferation without impairing cell viability, although a higher dose (100 μM) decreased the cell survival rate. Similarly, Zhou et al. [2009] reported that nobiletin inhibited angiotensin-induced bovine VSMC proliferation and DNA synthesis. However, they found that 100 μM nobiletin alone did not affect cell growth. Interestingly, Shi et al. [2013] showed that 2.5 μM nobiletin decreased cell viability in liver cancer HepG2 cells and treatment of 25 μM nobiletin resulted in less than 40% cell viability. In C6 rat glioma cells, 30 and 100 μM nobiletin significantly decrease cell viability while 10 μM nobiletin did not affect cell viability [Aoki et al., 2013]. The reasons for these discrepancies are unclear, and we suspect that they may be due to the different species origins of VSMCs and different cell types. This means that treatment of nobiletin in vivo may cause unexpected complications and further study is essential before any clinical application. It should be noted that in the animal study we did not observe any obvious behavior and physical abnormalities in rats chronically treated with 25 mg/kg/d nobiletin. Clinical and experimental data have indicated that inflammation may be central to intimal growth after mechanical arterial injury [Costa and Simon, 2005]. Nobiletin decreased serum TNF-α and IL-6 in the balloon-injured rats, indicating that it reduced the inflammatory state in vivo. Lin et al. [2003] reported that nobiletin effectively suppressed expression of proinflammatory cytokines including IL-6 and TNF-α in mouse macrophage J774A cells. In fact, the antioxidative and anti-inflammatory activities of nobiletin have been widely evaluated [Murakami et al., 2000a; Ho and Lin, 2008; Cui et al., 2010; Guo et al., 2012]. In agreement with these studies, our results showed that PDGF-BB could increase ROS generation of VSMCs and subsequently activate ERK1/2 and NF-κB signaling, and pretreatment of nobiletin potently inhibit these changes. In summary, the present study demonstrated that nobiletin could inhibit VSMC proliferation and migration in vitro and neointimal hyperplasia following carotid balloon injury in vivo by regulating ROS/NF-κB pathway and inflammation suggesting that nobiletin is a putative treatment for the prevention of restenosis after angioplasty.
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ACKNOWLEDGMENT
We thank Dr. Xiaolin Wu for assisting the preparation of the manuscript and critical advice. REFERENCES Aoki K, Yokosuka A, Mimaki Y, Fukunaga K, Yamakuni T. 2013. Nobiletin induces inhibitions of Ras activity and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling to suppress cell proliferation in C6 rat glioma cells. Biol Pharm Bull 36:540–547. Cospedal R, Abedi H, Zachary I. 1999. Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases. Cardiovasc Res 41:708–721. Costa MA, Simon DI. 2005. Molecular basis of restenosis and drugeluting stents. Circulation 111:2257–2273. Cui Y, Wu J, Jung SC, Park DB, Maeng YH, Hong JY, Kim SJ, Lee SR, Kim SJ, Kim SJ, et al. 2010. Anti-neuroinflammatory activity of nobiletin on suppression of microglial activation. Biol Pharm Bull 33:1814–1821. Guo S, Qiu P, Xu G, Wu X, Dong P, Yang G, Zheng J, McClements DJ, Xiao H. 2012. Synergistic anti-inflammatory effects of nobiletin and sulforaphane in lipopolysaccharide-stimulated RAW 264.7 cells. J Agric Food Chem 60:2157–2164. Ho SC, Lin CC. 2008. Investigation of heat treating conditions for enhancing the anti-inflammatory activity of citrus fruit (Citrus reticulata) peels. J Agric Food Chem 56:7976–7982. Kim HJ, Yoo EK, Kim JY, Choi YK, Lee HJ, Kim JK, Jeoung NH, Lee KU, Park IS, Min BH, et al. 2009. Protective role of clusterin/apolipoprotein J against neointimal hyperplasia via antiproliferative effect on vascular smooth muscle cells and cytoprotective effect on endothelial cells. Arterioscler Thromb Vasc Biol 29:1558–1564. Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, D.C. National Academy Press. Kingsley K, Huff JL, Rust WL, Carroll K, Martinez AM, Fitchmun M, Plopper GE. 2002. ERK1/2 mediates PDGF-BB stimulated vascular smooth muscle cell proliferation and migration on laminin-5. Biochem Biophys Res Commun 293:1000–1006. Lin N, Sato T, Takayama Y, Mimaki Y, Sashida Y, Yano M, Ito A. 2003. Novel anti-inflammatory actions of nobiletin, a citrus polymethoxy flavonoid, on human synovial fibroblasts and mouse macrophages. Biochem Pharmacol 65:2065–2071. Liu P, Ying Y, Anderson RG. 1997. Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae. Proc Natl Acad Sci USA 94:13666–13670. Louis SF, Zahradka P. 2010. Vascular smooth muscle cell motility: from migration to invasion. Exp Clin Cardiol 15:e75–e85. Moses JW, Leon MB, Popma JJ, Fitzgerald PJ, Holmes DR, O’Shaughnessy C, Caputo RP, Kereiakes DJ, Williams DO, Teirstein PS, et al. 2003. Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med 349:1315–1323. Murakami A, Nakamura Y, Ohto Y, Yano M, Koshiba T, Koshimizu K, Tokuda H, Nishino H, Ohigashi H. 2000a. Suppressive effects
Drug Dev. Res.
496
GUAN ET AL.
of citrus fruits on free radical generation and nobiletin, an antiinflammatory polymethoxyflavonoid. Biofactors 12:187–192. Murakami A, Nakamura Y, Torikai K, Tanaka T, Koshiba T, Koshimizu K, Kuwahara S, Takahashi Y, Ogawa K, Yano M, et al. 2000b. Inhibitory effect of citrus nobiletin on phorbol esterinduced skin inflammation, oxidative stress, and tumor promotion in mice. Cancer Res 60:5059–5066. Murakami A, Shigemori T, Ohigashi H. 2005. Zingiberaceous and citrus constituents, 1'-acetoxychavicol acetate, zerumbone, auraptene, and nobiletin, suppress lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 murine macrophages through different modes of action. J Nutr 135 (12 Suppl.):2987S– 2992S. Orr AW, Hastings NE, Blackman BR, Wamhoff BR. 2010. Complex regulation and function of the inflammatory smooth muscle cell phenotype in atherosclerosis. J Vasc Res 47:168–180. Paravicini TM, Touyz RM. 2006. Redox signaling in hypertension. Cardiovasc Res 71:247–258. Park ES, Lee KP, Jung SH, Lee DY, Won KJ, Yun YP, Kim B. 2013. Compound K, an intestinal metabolite of ginsenosides, inhibits PDGF-BB-induced VSMC proliferation and migration through G1 arrest and attenuates neointimal hyperplasia after arterial injury. Atherosclerosis 228:53–60. Pendyala L, Jabara R, Shinke T, Chronos N, Robinson K, Li J, Hou D. 2008. Drug-eluting stents: present and future. Cardiovasc Hematol Agents Med Chem 6:105–115. Pursnani S, Korley F, Gopaul R, Kanade P, Chandra N, Shaw RE, Bangalore S. 2012. Percutaneous coronary intervention versus optimal medical therapy in stable coronary artery disease: a systematic review and meta-analysis of randomized clinical trials. Circ Cardiovasc Interv 5:476–490. Robertson KE, McDonald RA, Oldroyd KG, Nicklin SA, Baker AH. 2012. Prevention of coronary in-stent restenosis and vein graft failure: does vascular gene therapy have a role? Pharmacol Ther 136:23–34. Ross R. 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801–809.
Drug Dev. Res.
Sachinidis A, Locher R, Vetter W, Tatje D, Hoppe J. 1990. Different effects of platelet-derived growth factor isoforms on rat vascular smooth muscle cells. J Biol Chem 265:10238–10243. Shi MD, Liao YC, Shih YW, Tsai LY. 2013. Nobiletin attenuates metastasis via both ERK and PI3K/Akt pathways in HGF-treated liver cancer HepG2 cells. Phytomedicine 20:743–752. Tokunou T, Shibata R, Kai H, Ichiki T, Morisaki T, Fukuyama K, Ono H, Iino N, Masuda S, Shimokawa H, et al. 2003. Apoptosis induced by inhibition of cyclic AMP response element-binding protein in vascular smooth muscle cells. Circulation 108:1246– 1252. Wang Z, Zhang X, Chen S, Wang D, Wu J, Liang T, Liu C. 2013. Lithium chloride inhibits vascular smooth muscle cell proliferation and migration and alleviates injury-induced neointimal hyperplasia via induction of PGC-1alpha. PLoS ONE 8: e55471. Won KJ, Jung SH, Lee CK, Na HR, Lee KP, Lee DY, Park ES, Choi WS, Shim SB, Kim B. 2013. DJ-1/park7 protects against neointimal formation via the inhibition of vascular smooth muscle cell growth. Cardiovasc Res 97:553–561. Xu C, Chen J, Zhang J, Hu X, Zhou X, Lu Z, Jiang H. 2013. Naringenin inhibits angiotensin II-induced vascular smooth muscle cells proliferation and migration and decreases neointimal hyperplasia in balloon injured rat carotid arteries through suppressing oxidative stress. Biol Pharm Bull 36:1549–1555. Zhan Y, Kim S, Izumi Y, Izumiya Y, Nakao T, Miyazaki H, Iwao H. 2003. Role of JNK, p38, and ERK in platelet-derived growth factor-induced vascular proliferation, migration, and gene expression. Arterioscler Thromb Vasc Biol 23:795–801. Zhang JK, Yang L, Meng GL, Fan J, Chen JZ, He QZ, Chen S, Fan JZ, Luo ZJ, Liu J. 2012. Protective effect of tetrahydroxystilbene glucoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells. Eur J Pharmacol 689:31–37. Zhou CH, Wu XH, Wu YQ. 2009. Nobiletin, a dietary phytochemical, inhibits vascular smooth muscle cells proliferation via calcium-mediated c-Jun N-terminal kinases pathway. Eur J Pharmacol 615:55–60.
