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Bioactive Constituents, Metabolites, and Functions
Melatonin inhibits in vitro smooth muscle cell inflammation and proliferation and atherosclerosis in apolipoprotein E-deficient mice Hung-Yuan Li, Yann-Lii Leu, Ya-Chieh Wu, and shu-huei Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06217 • Publication Date (Web): 20 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019
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Journal of Agricultural and Food Chemistry
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Melatonin inhibits in vitro smooth muscle cell inflammation and proliferation and atherosclerosis
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in apolipoprotein E-deficient mice
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Running title: Melatonin prevents atherosclerosis
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Hung-Yuan Li1#, Yann-Lii Leu2-4#, Ya-Chieh Wu5, Shu-Huei Wang6
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1Department
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2Graduate
of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan;
Institute of Natural Products, Chang Gung University, Taoyuan, Taiwan;
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3Chinese
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Taoyuan, Taiwan;
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4Center
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5Department
of Nursing, Ching-Kuo Institute of Management and Health, Keelung, Taiwan;
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6Department
of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei,
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Taiwan
Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University,
for Traditional Chinese Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan
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#These
authors contributed equally to this work.
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Corresponding author: Dr. Shu-Huei Wang, Department of Anatomy and Cell Biology, College of
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Medicine, National Taiwan University, No. 1, Section 1, Ren-Ai Rd, Taipei, 100, Taiwan. Telephone:
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+886-2-23123456-88180, Fax: +886-2-23915292, E-mail:
[email protected] ACS Paragon Plus Environment
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Abstract
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Chronic inflammation and proliferation play important roles in atherosclerosis progression. This study
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aimed to identify the mechanisms responsible for the anti-inflammatory and antiproliferative effects of
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melatonin on tumor necrosis factor-α (TNF-α)- and platelet-derived growth factor-BB
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(PDGF-BB)-treated rat aortic smooth muscle cells (RASMCs). Melatonin reduced TNF-α-induced
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RASMC inflammation by decreasing vascular cell adhesion molecule-1 (VCAM-1) expression and
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nuclear factor-kappa B (NF-κB) P65 activity by inhibiting P38 mitogen-activated protein kinase
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phosphorylation (P < 0.05). Additionally, melatonin inhibited PDGF-BB-induced RASMC
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proliferation by reducing mammalian target of rapamycin (mTOR) phosphorylation (P < 0.05), but not
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migration in vitro. Melatonin also reduced TNF-α- and PDGF-BB-induced reactive oxygen species
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(ROS) production (P < 0.05). Furthermore, melatonin treatment (prevention and treatment groups)
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significantly repressed high cholesterol diet-stimulated atherosclerotic lesions in vivo (19.59±4.11%,
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20.28±5.63%, 32.26±12.06%, respectively, P < 0.05). Taken together, the present study demonstrated
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that melatonin attenuated TNF-α-induced RASMC inflammation and PDGF-BB-induced RASMC
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proliferation in cells and reduced atherosclerotic lesions in mice. These results showed that melatonin
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has anti-inflammatory and antiproliferative properties and may be a novel therapeutic target in
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atherosclerosis.
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Key words: atherosclerosis, smooth muscle cells, inflammation, VCAM-1, proliferation
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Journal of Agricultural and Food Chemistry
Introduction
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The characters of atherosclerosis, a chronic vascular disease, is inflammation and vascular smooth
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muscle cell (VSMC) proliferation1. Monocyte recruitment and adhesion to vessels are crucial events in
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the early stage of atherosclerotic progression. Vascular cell adhesion molecule-1 (VCAM-1), an
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inflammatory and atherosclerotic marker, is an adhesion molecule that is abundantly expressed by
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smooth muscle cells in atherosclerotic lesions and injured arteries2. VCAM-1 facilitates monocyte
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infiltration into atherosclerotic vascular walls and increases VSMC migration, contributing to further
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atherosclerosis exacerbation3. Therefore, modulating VSMC inflammation, proliferation, and migration
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may have therapeutic effects on atherosclerosis.
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Melatonin (N-acetyl-5-methoxytrypamine), an indolamine, has multiple functions in the regulation
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of circadian rhythms4, the inhibition of tumor growth and metastasis5, 6, and the inhibition of
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inflammation7, 8. In cardiovascular disease, melatonin exerts multiple protective effects by preventing
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endothelial cell pyroptosis9, acting as a free radical scavenger10, inhibiting myeloperoxidase11, exerting
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antioxidant effects10,
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neutrophil transmigration14, reducing fatty acid levels, neutralizing free radicals15, reducing lipid
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peroxidation16, modulating cholesterol clearance, inhibiting low-density lipoprotein (LDL) oxidation17,
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18
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effects of melatonin on VSMCs remain unknown. Thus, it is important to elucidate the function and
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regulation of melatonin on atherosclerotic VSMCs for the clinical therapeutic application of melatonin.
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The aim of this study was to elucidate the anti-inflammatory and antiproliferative effects and
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regulatory mechanisms of melatonin on TNF-α- and PDGF-BB-stimulated RASMCs. The present
12,
reducing endothelium-derived adhesion molecule formation13, inhibiting
and reducing neointimal formation19. But the anti-inflammatory, antimigratory and antiproliferative
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study, melatonin reduced TNF-α-induced RASMC inflammation by decreasing VCAM-1 expression
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and monocyte adhesion via decreasing P38 phosphorylation and NF-κB P65 activation. Furthermore,
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melatonin reduced foam cell formation in oxidized LDL (oxLDL)-induced RAW264.7 macrophages.
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In addition, melatonin also repressed PDGF-BB-stimulated RASMC proliferation by arresting cells in
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the G0/G1 phase and regulating the expression of cell cycle regulator proteins. And these regulatory
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effects were regulated by the mTOR pathway. Furthermore, melatonin pretreatment significantly
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reduced in vivo atherosclerotic plaque areas in apolipoprotein E (ApoE)-deficient mice.
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Materials and methods
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Chemicals
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Antibodies against α-smooth muscle actin (α-SMA), P21Cip1, P27Kip1, phospho-/total c-Jun N-terminal
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kinase (JNK), phospho-/total P38, phospho-/total extracellular signal-related kinases (ERK)1/2,
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phospho-/total Akt, phospho-/total P65, β-actin, GAPDH, 5-bromo-2´-deoxyuridine (BrdU) were
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purchased from GeneTex (Irvine, CA, USA). Antibodies against phospho-/total mTOR, CDK4, cyclin
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D1, CDK2, and cyclin E were purchased from Cell Signaling Technology (Beverly, MA, USA).
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Anti-VCAM-1 monoclonal antibody was purchased from Santa Cruz Biotechnology (CA, USA).
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Anti-Iba-1 polyclonal antibody was purchased from Wako (Osaka, Japan). Fluorescent-dye conjugated
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secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).
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SB203580 was purchased from Biomol (Plymouth Meeting, PA, USA). Sirolimus (Rapamycin) was
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purchased from Selleck Chemicals (Houston, TX, USA). oxLDL was purchased from Biomedical
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Technologies (Alfa Aesar, LLC, MA, USA). TNF-α and PDGF-BB were purchased from PeproTech ACS Paragon Plus Environment
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(Co, Rocky Hill, NJ, USA). Melatonin (≥99.5%), crystal violet, BrdU, N-acetyl cysteine (NAC), DAPI
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(4',6-diamidino-2-phenylindole) and propidium iodide (PI) were purchased from Sigma-Aldrich (St.
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Louis, MO, USA). BCECF-AM, dihydroethidium (DHE), and dichloro-dihydro-fluorescein diacetate
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(DCFH-DA) were purchased from Molecular Probes (Invitrogen, Carlsbad, CA, USA).
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Cell cultures
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RASMCs and RAW264.7 macrophages were purchased from Bioresource Collection and Research
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Center (BCRC) and cultured in Dulbecco's modification of Eagle medium (DMEM). Before
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conducting the experiments, RASMCs were precultured in a serum-starved medium for 24 h. U937
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cells obtained from the BCRC were cultured in RPMI-1640. All media contained 10% FBS and 1%
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antibiotic solution.
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Cell viability
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The treated cells were fixed, washed and then stained with crystal violet solution for 30 min. After
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washing the cells with PBS, 10% glacial acetic acid solution was added to elute the dye, and read by
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ELISA reader.
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RNA preparation and quantitative PCR
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RASMCs pretreated with melatonin and then coincubated with 10 ng/ml TNF-α were harvested, and
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total cellular RNA was extracted using TRIzol solution (Sigma) according to the manufacturer's
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specifications. RNA concentrations were determined by spectrophotometric analysis. Contaminated
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genomic DNA was removed from 2 μg of total RNA by using a DNase digestion kit (Promega,
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Madison, WI, USA). Total RNA (0.8 μg) was reverse-transcribed using an RT kit (Yeastern).
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VCAM-1 expression was analyzed using quantitative PCR with the following VCAM-1 primers: ACS Paragon Plus Environment
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5’-ATCTTCTGCTCGGCAAGTC-3’
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(reverse). SYBR Green PCR premix (Thermo, Wilmington, DE) and a Light Cycler 480 (Roche) were
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used to detect the real-time quantitative PCR products of the cDNA samples produced by reverse
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transcription according to the manufacturer's instructions. The β-actin gene was used for normalization.
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PCR was conducted in triplicate for each experimental condition tested.
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Western blot analysis
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RASMCs were pretreated with melatonin, P38 inhibitor (SB203580), sirolimus or NAC and then
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coincubated with 10 ng/ml TNF-α or 20 ng/ml PDGF-BB for the indicated time. The treated cells were
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lysed and subjected to immunoblotting analysis. The primary antibodies (all at 1:1000) were incubated
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overnight and then incubated with secondary antibodies (all at 1:6000) for 1 h. Immunoreactivity was
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detected with ECL (GE Healthcare Bioscience). Gel-Pro software were used for band intensities
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quantification. The band density was normalized to the expression of the internal control.
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Luciferase promoter assay
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RASMCs were cultured in a 12-well plate and transfected with P65 luciferase reporter constructs (1 μg,
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Promega) using Genjet transfection reagent. After treatment, the cells were lysed and processed with a
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Bright-GloTM Luciferase Assay System (Promega). Luciferase activity was measured with an Infinite
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200 PRO NanoQuant plate reader (TECAN). All transfection experiments were repeated independently
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at least three times.
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Immunofluorescence
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Cells or tissues were fixed with 4% paraformaldehyde and then incubated with primary antibodies or
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normal IgG antibody overnight. Then, the cells or tissues were incubated with FITC-conjugated
(forward)
and
5’-GTTCTGACCTACATCTGGAGTG-3’
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secondary antibody. DAPI staining was used to labeled nuclei. The fluorescent images were examined
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by a fluorescence microscope.
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Determination of intracellular reactive oxygen species (ROS) levels
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Intracellular ROS levels were monitored by flow cytometry using fluorescent probes, namely,
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DCFH-DA and DHE. Briefly, treated cells were subsequently mixed with 10 μM DCFH-DA or DHE
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for 30 min. After being washed with phosphate buffered solution, the cells were analyzed or observed
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by immunofluorescence microscopy or flow cytometry.
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Cell cycle analysis
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The treated cells were subsequently trypsinized and stained with PI. Cell cycle progression was
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assessed, and the data were analyzed by ModFit software.
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Foam cell formation & staining
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Cells were pretreated with 0.1 mM melatonin for 1 h and then coincubated with 25 µg/ml oxidized
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low-density lipoprotein (OxLDL) for another 48 h. The cells were subsequently stained with 1 μM
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BODIPY for 30 min. After being washed with phosphate buffered solution, the cells were observed by
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immunofluorescence microscopy.
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Lactate dehydrogenase assay
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Cytotoxicity was analyzed by measuring lactate dehydrogenase (LDH) activity in the culture
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supernatants of vehicle control-, PDGF-BB- and PDGF-BB/melatonin-treated RASMCs. Cell-free
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supernatants were obtained by centrifugation for 10 min at 4°C, and LDH activity was measured by an
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LDH assay kit according to the manufacturer's instructions (Randox Laboratories. Ltd., UK).
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Scratch assay ACS Paragon Plus Environment
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After the RASMCs were treated with 0.1 mM melatonin or 5 nM sirolimus, wounds were created.
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Then, the cells were incubated with vehicle control or 20 ng/ml PDGF-BB for 24 h. The cell migration
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rate and wound closure were measured by MetaMorph software.
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Cell proliferation assay
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Treated cells were fixed with a 95% ethanol/5% acetic acid solution and then treated with 1 N
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hydrochloric acid (HCl) and sodium borate. The cells were subsequently incubated with an anti-BrdU
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antibody overnight and then incubated with FITC-conjugated antibody. DAPI was stained for labeling
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cell nuclei. Cells in six fields were counted to evaluate BrdU-positive cell number.
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Adhesion assay
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RASMCs were pretreated with 0.1 mM melatonin and subsequently coincubated with TNF-α for 24 h.
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The BCECF-AM-labeled U937 cells were co-incubated with the aorta or treated RASMCs for 60 min.
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Non-adherent U937 cells were washed, and the aorta and RASMCs were observed by fluorescence
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microscopy. Cells in six fields were counted to determine the number of BCECF-AM-labeled U937
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cells.
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Animal studies
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ApoE-deficient Male mice (8 w) were obtained from the National Laboratory Animal Center (Taipei,
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Taiwan). All animal experiments were performed according to the protocols approved by the
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Institutional Animal Care and Use Committees (IACUC) at National Taiwan University College of
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Medicine. The mice were divided into four groups (N=6-9 per group): Control group, which was fed a
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standard chow diet; Cholesterol-fed group, which was fed a 0.15% cholesterol-enriched diet (Purina
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Mills, Inc., USA) for 15 weeks; Prevention group (cholesterol diet/melatonin), which was fed a 0.15% ACS Paragon Plus Environment
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cholesterol-enriched diet and received melatonin (10 mg/kg/day) orally for 15 weeks; and Treatment
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group (cholesterol diet/melatonin 7W), which was fed a 0.15% cholesterol-enriched diet for 15 weeks
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and received melatonin (10 mg/kg/day) orally from weeks 9-15. Fasting blood samples were collected
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to measure triglyceride, total cholesterol, LDL, high-density lipoprotein (HDL), glucose (Randox
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Laboratories. Ltd., UK), creatinine, alanine transaminase (ALT), and aspartate aminotransferase (AST)
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(Fortress, Antrim, UK) levels. At the end of experiment, the mice were euthanized with sodium
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pentobarbital (120 mg/kg i.p.), the thoracic aorta and aortic lesion were removed and then fixed with
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4% paraformaldehyde solution for embedding and section.
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ORO staining and immunohistochemical staining
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The 8-μm-thick sections were collected from the each thoracic aorta and aortic lesion for
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pathomorphological examination. Aortic lesion sections stained with trichrome and Oil Red O were
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also analyzed microscopically to determine collagen and plaque areas. Image-Pro Plus was used for
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analyzing images. The thoracic aorta sections were immunohistochemically stained for α-SMA (SMC
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marker), Iba-1 (macrophage marker), and VCAM-1, reacted with FITC-conjugated antibodies, and
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then examined by fluorescence microscopy. Omission of primary antibodies or use of normal IgG
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instead of primary antibodies were routinely employed as controls.
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Statistical analysis
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All values are reported as the means ± SD. Statistical comparisons were performed using a two-tailed
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Student’s t-test or one-way analysis of variance (ANOVA) by Tukey's post hoc test. Nonparametric
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tests were used. Significance differences between two groups were determined with the Mann-Whitney
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test. Statistical significance was considered at P-values
