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Fucosylated Chondroitin Sulfate from Sea Cucumber Apostichopus japonicus Retards Atherosclerosis in Apolipoprotein E-deficient Mice Ying Zhang, Haihong Sun, Shucun Qin, Yuanyuan Song, Yanhong Si, Pengbo Hou, Nana Yang, and Shoudong Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03479 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 7, 2017
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Journal of Agricultural and Food Chemistry
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Fucosylated Chondroitin Sulfate from Sea Cucumber Apostichopus japonicus
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Retards Atherosclerosis in Apolipoprotein E-deficient Mice
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Ying Zhang†,*, Haihong Sun‡, Shucun Qin†, Yuanyuan Song†, Yanhong Si†, Pengbo
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Hou†, NaNa Yang†, Shoudong Guo†,*
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†
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Atherosclerosis, Taishan Medical University, Taian, Shandong 271000, China.
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‡
Key Laboratory of Atherosclerosis in Universities of Shandong Province, Institute of
Marine Chemical Research Institute, Qingdao, Shandong 266071, China.
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*
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Shoudong Guo; Tel: +86-0538-6229517; Fax: +86-0538-6225275; E-mail: SD-GUO
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@hotmail.com;
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+86-0538-6118246; Fax: +86-0538- 6225275; E-mail: sdyrx@163. com.
Corresponding author
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ABSTRACT: Fucosylated chondroitin sulfate (FucCS) is exclusively found at sea
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cucumbers, and it has been demonstrated to bear lipid-lowering effect in rodent
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models. The aim of this study is to investigate whether FucCS could attenuate
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atherosclerosis. FucCS was successfully obtained from sea cucumber A. japonicas,
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and its effect was investigated in apolipoprotein E-deficient (apoE−/−) mice. Results
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showed that high-dose FucCS (H-FucCS, 150 mg/kg) significantly decreased the
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plasma levels of LDL-C, TG, TNF-α and IL-6; increased the level of HDL-C and
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reduced the lipids accumulation in the aortic sinus and liver compared to the model
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group. Western blotting results showed that H-FucCS could down-regulate the
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proteins expression of MCP-1, VCAM-1, ICAM-1 and the phosphorylation of NF-κB
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p65 compared to the model group. Further study on the proteins expression of MAPK
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subfamilies indicated that FucCS administration had no influence on the
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phosphorylation of JNK1/2 and Erk1/2, but it significantly reduced the
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phosphorylation of p-38. These results suggest that p38/NF-κB pathway may partially
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contribute to the anti-inflammatory activity of FucCS. This study indicated that
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FucCS ameliorates atherosclerosis, at least in part, by its lipid-lowering and
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anti-inflammatory effects in apoE−/− mice. Furthermore, food intake of sea cucumbers
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may benefit the primary prevention of atherosclerosis.
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KEYWORDS: sea cucumber, atherosclerosis, inflammation, lipid-lowering, MAPK
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pathway
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INTRODUCTION
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Atherosclerosis mainly induced by hyperlipideamia and inflammation is the basic
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pathological change of cardiovascular disease (CVD), which is currently the leading
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cause of death worldwide.1 Although there are many therapeutic strategies, the
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mortality of urban and rural residents induced by CVD increases progressively year
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by year. This fact indicates that daily prevention is important for CVD, and the
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traditional food therapy in China and other eastern countries maybe a useful way to
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attenuate the onset and progression of atherosclerosis.
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Sea cucumbers belong to Holothuroidea in biological classification. They are
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nutritional foods and medicinal resources extensively consumed in China, Japan,
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Korea, and other Asian countries.2 Sea cucumbers are rich in sulfated polysaccharides,
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such as fucosylated chondroitin sulfate (FucCS) and fucoidan.2-4 FucCS is a unique
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glycosaminoglycan (GAG) found exclusively at sea cucumbers,5-8 and its structural
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characteristics have been clarified by depolymerization.9-11 FucCS from A. japonicas
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is generally composed of alternating 3-linked N-acetyl β-D-galactosamine (GalNAc)
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and 4-linked β-D-glucuronic acid (GlcUA) units with sulfated fucose branches either
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at the O-3 position of GlcUA or O-6 and O-4 positions of the GalNAc.2,12 Its
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conformational and physicochemical properties have been elucidated by high
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performance size exclusion chromatography combined with multi-angle laser
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scattering, viscometry, and differential refractive index detection and atomic force
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microscopy.7,13
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Previous publications suggested that FucCS from the sea cucumbers have
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antithrombotic14-15
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anti-adipogenic activity by regulating the Wnt/β-catenin pathway.18 Furthermore, a
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effects.11,13,16-17
hypolipidemic
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FucCS
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previous publication demonstrated that chondroitin sulfate (CS) can retard the
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progression of atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice via its
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lipid-lowering and anti-inflammatory effect.19 More importantly, the presence of
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fucose (Fuc) at the O-3 position of the GlcUA is reported to benefit the medical
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properties of CS.20 However, the effect of the FucCS on atherosclerosis is rarely
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known. The aim of this study is to investigate whether FucCS can attenuate
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atherosclerosis and the underlying mechanisms.
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MATERIALS AND METHODS
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Materials
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Dry body wall of sea cucumber Apostichopus japonicus was purchased from a local
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market (Qingdao, Shandong, China). Q-sepharoseTM Fast Flow and Sephacryl
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S200HR were the products of GE healthcare (Piscataway, NJ, USA). Monosaccharide
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standards, dextran standards (Mw: 5.0, 11.6, 23.8, 48.6, 147.6, 273.0, 409.8, 667.8,
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1100.0 kDa) and 1-phenyl-3-methyl-5-pyrazolone (PMP) were bought from
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Sigma-Aldrich (St. Louis, MO, USA). Dialysis membrane (molecular weight cut off
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3500) was purchased from Lvniao (Yantai, Shandong, China). Simvastatin was the
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product of Sine Wanxiang Pharmaceutical CO. LTD (Shanghai, China). Rat
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monoclonal antibody against intercellular adhesion molecule-1 (ICAM-1), rabbit
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monoclonal antibodies against vascular cell adhesion molecule-1 (VCAM-1), Jun
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N-terminal protein kinase (JNK), phospho-JNK, nuclear factor of kappa protein 65
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(NF-κB p65) and phosphor-NF-κB p65, and rabbit polyclonal antibodies against p38
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mitogen-activated protein kinase (MAPK), monocyte chemoattractant protein-1
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(MCP-1), extracellular signal regulated kinase 1 and 2 (Erk1/2), and phospho-Erk1/2
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were obtained from Abcam (Cambridge, MA, USA). Mouse monoclonal antibody 4
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against β-actin and rabbit monoclonal antibody against phospho-p38 MAPK were
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obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Secondary
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antibodies were from Beijing ComWin Biotech Co., Ltd. (Beijing, China). Assay kits
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for triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol
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(HDL-C) and low density lipoprotein cholesterol (LDL-C) were the products of
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Biosino Bio-technology and Science Incorporation (Beijing, China). Enzyme-linked
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immunosorbent assay (ELISA) kits for TNF-α and IL-6 were BlueGene products
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(Shanghai, China). Bicinchoninic acid (BCA) protein quantitative kit was the product
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of Solarbio (Beijing, China). High-fat diet (15 % fat and 1.25 % cholesterol) was
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provided by Keaoxieli Feed Co., Ltd. (Beijing, China). All other chemicals and
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reagents used were analytical purity grade.
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Preparation and Chemical Analysis of FucCS
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FucCS from the sea cucumber A. japonicus was prepared according to a previous
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publication.21 Briefly, the dried body walls (100 g) were cut into small pieces,
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suspended in 2 L of 0.1 mol/L sodium acetate buffer (pH=6) containing 10 g of
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papain, 5 mM EDTA, and 5 mM cysteine, and then incubated at 60 °C for 24 h. The
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mixture was centrifugated at 2000 × g for 10 min at 4 °C to obtain the supernatant,
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which was then precipitated with 2 volumes of 95 % ethanol. The precipitate was
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collected by centrifugation at 2000 × g for 20 min at 4 °C, dialyzed against distilled
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water, condensed and lyophilized. The crude extract was used for anion exchange
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separation with ÄKTA-FPLC. The Q-sepharoseTM Fast Flow column was eluted with
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a linear gradient of NaCl (0~2.0 mol/L) according to the previous publications.21-22
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The flow rate was 2.0 mL/min, and the eluent was collected at 5.0 mL/tub. The
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FucCS containing fractions were collected, dialyzed and lyophilized. The dried
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FucCS was further purified by a Sephacryl S200 HR column (2.6 × 90 cm) taking 0.2 5
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mol/L ammonium bicarbonate as eluent. The fractions of the major peak were
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collected and used for the following studies.
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The purity and molecular weight of the FucCS was determined by size-exclusion
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chromatographic analysis (TOSOH TSKgel G4000PWXL) according to the
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publication.22 Sulfate ester content was detected according to the method reported by
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Therho and Hartiala.23 The monosaccharide composition was determined by
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pre-column PMP derivatization.24
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Anti-atherosclerosis Experiment in ApoE-/- Mice
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Male apoE–/– mice (20.0 ± 2.0 g) were purchased from Beijing HFK Bioscience Co.,
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LTD (Beijing, China; license number: SCXK2009-0004). All care and handling of
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animals were approved by the Animal Care and Use Committee of Taishan Medical
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University. After 10 days of adaptation to the facilities, the mice were randomly
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allocated to four groups (n = 10 each): High-fat diet group (model group), simvastatin
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group (Simv. 20 mg/kg/d), low-dose FucCS group (L-FucCS, 50 mg/kg/d) and
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high-dose FucCS group (H-FucCS, 150 mg/kg/d). Mice in each group were fed a
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high-fat diet (15 % fat and 1.25 % cholesterol) for 8 weeks. Then, the mice were
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anaesthetized with 10 % chloral hydrate, and blood was sampled. Plasma was
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obtained by centrifugation at 1100 × g for 5 min at 4 °C. The mice were perfused with
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5 mL of PBS through left ventricle, and then heart and aortas were removed. 5 hearts
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and aortas in each group were perfusion-fixed with 4 % paraformaldehyde for
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histological and morphological staining. The rest of the aortas were used for Western
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blotting analysis.
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Plasma Analysis
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Plasma TG, TC, HDL-C and LDL-C were measured by assay kits from Biosino 6
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Bio-technology and Science Incorporation (Beijing, China) according to the
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manufacturers’ instructions. Plasma levels of TNF-α and IL-6 were determined by
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ELISA kits from BlueGene (Shanghai, China) according to the manufacturers’
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instructions.
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Histopathologic Analysis
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Heart samples were embedded in optimal cutting temperature compound, and
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5.5-µm-thick cryosections were cut from the aorta to left ventricle. Cryosections were
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stained with hematoxylin and eosin (H & E). The plaques were identified by Oil-Red
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O staining. All images were captured with an Olympus BX51 microscope equipped
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with a video camera. The extent of atherosclerosis was quantified using an Image
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Pro-Plus software.25
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Western Blot Analysis
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This experiment was performed according to our previous publications.25-26
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Data Analysis
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All of the bioassay results were expressed as the mean ± standard deviation (SD) for
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at least three independent experiments. Statistical analysis was performed using
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one-way analysis of variance followed by Student-Newmann-Keuls multiple
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comparison tests with SPSS 13.0 software. Differences were considered to be
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significant at a P
