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WIN55212-2 ameliorates atherosclerosis associated with suppression of pro-inflammatory responses in ApoE-knockout mice

Yan Zhao^a, Yan Liu^a, Weiping Zhang^a, Jiahong Xue^a, Yue Z. Wu^a, Wei Xu^a, Xiao Liang^a, Tao Chen^a, Chiharu Kishimoto^c and Zuyi Yuan^a^,^b^,^,

^a Department of Cardiovascular Medicine, the First Affiliated Hospital of Xi'an Jiaotong University College of Medicine, Xi'an, Shaanxi 710061, China

^b Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education, Xi'an, Shaanxi 710061, China

^c Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606–8507, Japan

Received 26 January 2010; revised 27 August 2010; accepted 15 September 2010. Available online 21 September 2010.

Abstract

The role of inflammation in all stages of atherosclerosis has been actively investigated, with an emphasis on the discovery of novel and innovative drugs for treatment and prevention. The anti-inflammatory and immunomodulatory capacity of cannabinoids are well established, and these agents have a broad therapeutic potential in various inflammatory diseases, including cardiovascular diseases. The aim of this study was to investigate the effect of WIN55212-2, a synthetic cannabinoid, on atherosclerosis using the apolipoprotein E-knockout (ApoE^−/−) mouse on a cholate-containing high-fat diet. Our results showed that WIN55212-2 reduced the size of atherosclerotic lesions in the aorta root, and did not affect serum lipid levels significantly. Furthermore, alleviation of atherosclerosis by WIN55212-2 was associated with a smaller content of macrophages in plaque lesion as well as decreasing pro-inflammatory gene and NF-κB activation in aortic tissues. Oxidized LDL (ox-LDL) dramatically induced NF-κB activation, and enhanced pro-inflammatory mRNA and protein in peritoneal macrophages isolated from ApoE^−/− mice. It is noteworthy that all of the above-mentioned effects of ox-LDL were attenuated by WIN55212-2. Moreover, WIN55212-2 also attenuated the inflammatory response that LPS induced. AM630, a cannabinoid receptor 2 (CB₂) special antagonist completely abolished the protective effects of WIN55212-2 both in vivo and in vitro. Our data provide strong evidence that WIN55212-2 can potentially inhibit atherosclerosis in ApoE^−/− mice. Importantly, all the beneficial effects of WIN55212-2 in our model were closely associated with the suppression of pro-inflammatory responses and were mediated by the CB₂ receptor.

Keywords: WIN55212-2; Atherosclerosis; Inflammation; Macrophage

Article Outline

Introduction

Materials and methods: 2.1. Experimental atherosclerosis; 2.2. Drugs and treatment protocols; 2.3. Lipid measurements; 2.4. Quantification of atherosclerotic lesions by immunohistochemistry; 2.5. LDL isolation and oxidation; 2.6. Cell culture; 2.7. RNA isolation and quantitative RT-PCR; 2.8. Protein preparation; 2.9. Western blotting; 2.10. Measurement of MCP-1, IL-6 and TNF-α concentration; 2.11. Electrophoretic mobility-shift assay (EMSA); 2.12. Statistical analysis

Results: 3.1. CB₂ receptors are expressed in plaque lesion and peritoneal macrophages; 3.2. Effects of WIN55212-2 on physiological parameters; 3.3. WIN55212-2 reduces atherosclerotic lesion size in ApoE^−/− mice; 3.4. Immunohistochemical analysis of macrophage accumulation in plaque lesion; 3.5. WIN55212-2 reduced inflammatory gene in plaque lesions; 3.6. WIN55212-2 reduced NF-κB activation in advanced plaque lesions; 3.7. WIN55212-2 reduced thioglycolate-elicited peritoneal macrophage inflammatory gene; 3.8. WIN55212-2 reduced thioglycolate-elicited peritoneal macrophage NF-κB activation and IκB phosphorylation

Discussion

Acknowledgements

References

1. Introduction

Atherosclerosis is a chronic inflammatory disease characterized by intense immunological activity, which increasingly threatens human health worldwide. The identification and development of anti-inflammatory therapies has thus become an active area of investigation in recent years. Numerous studies suggest that cannabinoids or cannabinoid receptor agonists suppress the production of cytokines in innate and adaptive immune responses in both animal models and human cell cultures([Yuan et al., 2002], [Sacerdote et al., 2005], [Mestre et al., 2005] and [Mormina et al., 2006]) suggesting that these drugs might be useful in the treatment of chronic inflammatory diseases, including atherosclerosis.

To date, two major cannabinoid receptors, CB₁ and CB₂, have been identified and characterized. CB₁ is expressed predominantly in the central and peripheral nervous system. CB₂ is present primarily on immune and hematopoietic cells ([Mackie, 2006] and [Pacher et al., 2006]) and recent studies have shown that CB₂ is expressed in the brain (Van Sickle et al., 2005), myocardium (Mukhopadhyay et al., 2007) cardiomyoblasts (Shmist et al., 2006) and endothelial cells of various origins ([Ashton et al., 2006], [Golech et al., 2004] and [Maccarrone et al., 2006]). Several studies suggest that the type 2 cannabinoid receptor CB₂ is the main peripheral molecular target, which is responsible for the inhibitory properties of the cannabinoids ([Buckley et al., 2000], [Ni et al., 2004], [Ihenetu et al., 2003] and [Ghosh et al., 2006a]) and for the cardioprotection afforded by cannabinoid receptor agonists in models of inflammation-mediated tissue damage ([Lagneux and Lamontagne, 2001], [Krylatov et al., 2002], [Hajrasouliha et al., 2008] and [Joyeux et al., 2002]).

WIN55212-2, a cannabinoid receptor agonist, has a 7-fold higher affinity for CB₂ than for CB₁ (Showalter et al., 1996), which showed immunosuppressive and anti-inflammatory effects in multiple studies both in vivo and in vitro ([Kenneth, 2003], [Ghosh et al., 2006b] and [Marchalant et al., 2007]). Moreover, in an earlier study we observed that WIN55212-2 can inhibit the early atherosclerotic lesion in ApoE^−/− mice through decreasing the of adhesion molecules (Zhao et al., 2010).

In this study, we focus on the advanced stage of atherosclerotic lesion. Our data demonstrate that WIN55212-2 can alleviate atherosclerotic lesions in ApoE^−/− mice, attenuate macrophage infiltration and suppress the inflammatory response of plaque lesions. We provide strong evidence that the inhibitory effects of WIN55212-2 on atherosclerosis can be mediated by activating the CB₂ receptor.

2. Materials and methods

2.1. Experimental atherosclerosis

The ApoE^−/− mice were a generous gift from Dr Edward M. Rubin (University of California, Berkeley, CA). The mice were kept in a temperature-controlled facility on a 12 h light/12 h dark cycle with free access to food and water. After weaning at 4 weeks old, male mice were fed a normal chow diet until 6 weeks old, after which the animals were switched to a diet containing 21% fat and 0.15% cholesterol. For the histological analysis of atherosclerotic plaque development, littermate ApoE^−/− mice were fed a cholate-containing high-fat diet for 16 weeks. All animal experiments were done in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996).

2.2. Drugs and treatment protocols

(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55212-2) was obtained from the Cayman Chemical Company (Ann Arbor, MI). 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1Hindol-3-yl](4-methoxyphenyl)-methanone (AM630) was obtained from Tocris Bioscience (Bristol, UK). Appropriate dilutions of the vehicle (DMSO) were used as controls in all experiments.

Littermate ApoE^−/− mice were divided randomly into 4 groups of 8 animals and administered by daily injection i.p. for 8 weeks starting at 14 weeks old with: (1) vehicle only, as a control; (2) 1 mg ofWIN55212-2/kg; (3) 0.5 mg ofWIN55212-2/kg; and (4) 1 mg ofWIN55212-2/kg plus 0.5 mg of AM630/kg. Littermate controls were injected with the same volume of vehicle.

2.3. Lipid measurements

Overnight fasting blood samples were obtained by right ventricular puncture under anaesthesia using an injection i.p. of 10% chloral hydrate (3 ml/kg) (Shuanghe Limited, Beijing, China). Serum was separated by centrifugation. Plasma was isolated by centrifugation at 10,000 g for 10 min at 4 °C and stored at − 80 °C. Serum total cholesterol (TC), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) were measured with assay kits (Dongou Company, Hangzhou, China) according to the manufacturer's instructions.

2.4. Quantification of atherosclerotic lesions by immunohistochemistry

Mice were killed by bleeding following puncture of the right ventricle and the vasculature was perfused with sterile PBS. The root of the aorta was dissected under a microscope and frozen in OCT embedding medium for serial 10 μm cryosectioning covering 1.0 mm of the root. The first section was harvested when the valve cusps became visible.

Plaques were stained with Oil red O and counterstained with hematoxylin. The macrophage content was analysed by immunostaining with a rat anti-mouse Mac-3 monoclonal antibody (BD PharMingen, San Diego, CA) that was revealed with a biotin-conjugated anti-rat antibody.

At least 4 sections per mouse were inspected for each immunostaining. Section images were captured digitally with an OlympusBX51 imaging system and quantified with Image-Pro Plus 6.0 software. Plaque lesion area and the percentage of the total cross-sectional vessel wall area were quantified as described (Yuan et al., 2003). The content of macrophages in lesions was determined by counting the number of positively hematoxylin-stained cells inside the internal elastic lamina as described (Gotsman et al., 2006). Three to five random microscopic fields were analysed at a magnification of 400×.

2.5. LDL isolation and oxidation

LDL (d = 1.019–1.063 g/ml) was isolated from plasma (donated by healthy human volunteers) by sequential ultracentrifugation and dialysed for 72 h at 4 °C against PBS containing 10% (w/v) EDTA. To prepare ox-LDL, LDL was dialysed extensively to remove EDTA. Isolated LDL was oxidized by 10 μM CuSO₄ for 18 h at 37 °C, and then exposed to 200 μM EDTA at 4 °C for 24 h. Oxidation of LDL was measured by the thiobarbituric acid-reactive substances assay. Malondialdehyde in native LDL was 0.421 nmol/mg protein and 6.693 nmol/mg protein in ox-LDL.

2.6. Cell culture

Thioglycolate-elicited peritoneal macrophages from 6-week-old ApoE^−/− mice were plated at a concentration of 2 × 10⁶ cells/ml in DMEM containing 10% (v/v) fetal bovine serum (FBS), 2 mmol l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from BioWhittaker, Verviers, Belgium). After incubation for 4 h at 37 °C, the cells were washed and incubated for 24 h at 37 °C with DMEM containing 10% FBS. Cells were incubated at 37 °C with 10 μM WIN55212-2 or the same volume of vehicle for 2 h before stimulation with 50 μg/ml of ox-LDL and 10 μg/ml of LPS for 24 h. For the CB₂ receptor antagonist studies, cells were incubated at 37 °C with 1 μM AM630 for 30 min before the addition of the WIN55212-2. Cells were subsequently harvested and used for mRNA and protein analysis as described below.

2.7. RNA isolation and quantitative RT-PCR

Total RNA was isolated from aortic arches and peritoneal macrophage samples using TRIzol® reagent (Invitrogen) according to the manufacturer's instructions. Quantitative RT-PCR analysis was used to evaluate the of investigated genes using an SYBR® PrimeScript TMRT-PCR kit (TaKaRa) according to the manufacturer's instructions. All real-time reactions were performed on the iQ5TM Multicolor real-time PCR detection system (Bio-Rad). The PCR procedure used 40 cycles of 5s at 95 °C, 20s at 58.5 °C and 10s at 72 °C and each sample was analysed in triplicate. The sequences of the primers used are given in Table 1 and the data were analysed as described (Livak and Schmittgen, 2001).

Table 1. Primer sequences. Genes Forward primer Reverse primer GAPDH 5-TCAACGGCACAGTCAAGG-3 5-ACTCCACGACATACTCAGC-3 CB₂ 5-TTGCCCTAGTCCCTGCTCTG-3 5-CCTGCCTGTCCTGGTGCTC-3 MCP-1 5-CTCACCTGCTGCTACTCATTCAC-3 5-ATGTCTGGACCCATTCCTTCTTG-3 TNF-α 5-GCTCTTCTGTCTACTGAACTTCGG-3 5-ATGATCTGAGTGTGAGGGTCTGG-3 IL-6 5-AGCCAGAGTCCTTCAGAGAGATAC-3 5-AATTGGATGGTCTTGGTCCTTAGC-3 Full-size table
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2.8. Protein preparation

Tissue and cell samples were homogenized in lysis buffer (150 mmol/l NaCl, 20 mmol/l Tris (pH 7.5), 1 mmol/l EDTA, 0.1% (v/v) Triton X-100) containing 0.5% Protease Inhibitor Cocktail III (EMD Biosciences Inc, Madison, WI). The lysate was placed on ice for 30 min then centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was recovered and measured using an RCDC Protein Assay Kit (Bio-Rad Laboratories Inc, CA).

2.9. Western blotting

Lysates for Western blot analysis were prepared from cells and aortic tissues. Protein concentrations were determined using BCA (Pierce, Rockford IL, USA) reagents according to the manufacturer's instructions. Radioimmunoprecipitation assay buffer containing freshly added Protease Inhibitor Cocktail was used to generate whole cell lysates. Protein extracts (50 μg) were separated by SDS-PAGE (10% polyacrylamide gel) and transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were blocked at 4 °C in 5% (w/v) non-fat milk powder dissolved in Tris-buffered saline containing Tween-20 for 2 h at room temperature, and then incubated with the following primary polyclonal antibodies: rabbit anti-mouse anti-CB₂ antibody (Cambridge Science Park, Cambridge, UK), rabbit monoclonal antibody NF-κB P65 (Cell Signaling, Danvers, MA) and p-IΚB-α (Cell Signaling, Danvers, MA) at room temperature for 1 h, followed by incubation at room temperature for1 h with the secondary antibodies. The proteins were visualized by enhanced chemiluminescence (Pierce, USA) and exposed to X-ray film (Kodak, USA).

2.10. Measurement of MCP-1, IL-6 and TNF-α concentration

The amounts of MCP-1, IL-6, adiponectin and TNF-α in culture supernatants were assessed by enzyme-linked immunosorbent assay (ELISA) in triplicate. The assay was done with an OptEIA™ mouse MCP-1 set, a mouse TNF-a set (BD Biosciences Pharmingen, CA), a mouse IL-6 set (R&D systems, MN) or a mouse adiponectin set (R& D systems, MN). The sample was thawed, diluted with assay diluent and assayed. The amounts of MCP-1, IL-6, adiponectin and TNF-α were quantified from a standard curve obtained using the SOFTmax curve-fitting program (Molecular Devices, CA).

2.11. Electrophoretic mobility-shift assay (EMSA)

The activation of NF-κB under different experimental conditions was determined by the electromobility-shift assay (EMSA). The nuclear extract from treated macrophages was prepared with a Nuclear Extract kit (Pierce, Rockford IL) and the protein content in the nuclear lysate was determined using a BCA protein kit (Pierce, Rockford IL). Each nuclear extract (10 μg) was incubated with labelled oligonucleotide probes and 2 μg of poly(dI–dC) in 20 μl of binding buffer. The nuclear extract (8 μg) and 50 fmol of oligonucleotides were incubated at room temperature for 20 min in 20 μl of binding buffer (10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 3 mM DTT, 0.5 μg of poly(dI–dC), 5 mM MgCl₂, 0.1% Tween 20) . The specificity of the bands was confirmed by supershifting with specific antibodies of p65 and p50 (Santa Cruz Biotechnology) and by competition with cold oligonucleotides. DNA–protein complexes were separated on native 5% polyacrylamide gels at 100 V with 0.5 × TBE as running buffer. The signals of the gels were scanned and quantified with an Odyssey infrared imaging system (LI-COR).

2.12. Statistical analysis

Values are presented as mean ± S.D. Statistical analysis of the data was done with Student's t-test or by one-way analysis of variance (ANOVA), followed by Fisher's protected least-significant-difference test. Statistical significance was set at P < 0.05.

3. Results

3.1. CB₂ receptors are expressed in plaque lesion and peritoneal macrophages

As shown in Fig. 1, CB₂ receptors are expressed in plaque lesions and peritoneal macrophages as demonstrated by immunohistochemical assays (Fig. 1A), conventional RT-PCR (Fig. 1B) and Western blot (Fig. 1C). It is noteworthy that there was more CB₂ receptor in the aortic root with plaque lesion (left-hand panel) than without (right-hand panel) in Fig. 1A, suggesting that the CB₂ receptor might have a vital role in the development of atherosclerosis.

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Fig. 1.

Cannabinoid CB₂ receptors are expressed in aortic tissue of ApoE^−/− mice and in peritoneal macrophages. A) of CB₂ receptors in a plaque lesion of ApoE^−/− mice demonstrated by staining. B) Analysis of CB₂ using Western blotting in aortic tissue and peritoneal macrophages. C) The detection of CB₂ gene using RT-PCR in aortic tissue and peritoneal macrophages.

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3.2. Effects of WIN55212-2 on physiological parameters

As shown in Table 2, there was no significant difference of body weight, Serum total cholesterol (TC), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) among the groups treated with vehicle, WIN55212-2 or WIN55212-2 plus AM630, respectively.

Table 2. Physiological parameters in ApoE^−/− mice at sacrifice. Group of animals Body weight Total cholesterol
(mg/dl) High-density lipoprotein cholesterol
(mg/dl) Triglycerides
(mg/dl) Vehicle 27.6 ± 0.8 798.2 ± 55.4 142.3 ± 8.9 121 ± 15.5 WIN (0.5 mg/kg/day) 25.7 ± 1.0 821 ± 64.5 147.6 ± 3.9 128 ± 10.5 WIN (1 mg/kg/day) 26.2 ± 1.8 772.2 ± 24.6 138.5 ± 5.4 104 ± 18.7 WIN (1 mg/kg/day) + AM630 (0.5 mg/kg/day) 26.5 ± 0.6 823 ± 66.5 142.3 ± 8.9 120 ± 29.5 Full-size table

Values are means ± SD; treatment with WIN55212-2 or WIN55212-2 plus AM630 did not significantly modify the physiological parameters of ApoE^−/− mice.

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3.3. WIN55212-2 reduces atherosclerotic lesion size in ApoE^−/− mice

ApoE^−/− mice were kept on a cholesterol-rich diet for 16 weeks to allow the formation of fibrofatty plaques. The surface area covered by fibrofatty plaque lesions was quantified in Oil red O-stained samples (Fig. 2), and specimens from mice treated with WIN55212-2 or WIN55212-2 plus AM360 were compared with the vehicle-treated control group. We used two methods to determine atherosclerotic lesion size: one is a relative value of plaque size, expressed as percentage (%), and the other is an absolute value of plaque size, expressed as μm². As shown in Fig. 2, while the results obtained through the two different methods are not entirely consistent, but they both showed WIN55212-2 dramatically reduced the fractional area of lesions.

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Fig. 2.

WIN55212-2 reduces the development of atherosclerotic lesions in ApoE^−/− mice. Littermate male ApoE^−/− mice were given a daily injection i.p. of WIN55212-2, AM630 plus WIN55212-2, or vehicle alone for 8 weeks starting at 14 weeks old. A) Representative photomicrographs of Oil red O-stained fatty streaks (n = 8). B) and C) Quantitative analysis of atherosclerotic lesion sizes in the aortic root. Magnification, 40×. Data are presented as the mean ± S.D.; n = 8 per group; *P < 0.05.

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3.4. Immunohistochemical analysis of macrophage accumulation in plaque lesion

The inflammatory cell infiltrations were assessed immunohistologically by the relative number of macrophages (Mφ). The percentage of Mφ-positive cells was reduced significantly in the WIN55212-2-treated group compared with the control group (Fig. 3). However, treatment of mice with WIN55212-2 plus AM630 did not suppress the frequency of Mφ-positive cells compared with the controls.

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Fig. 3.

Effect of WIN55212-2 on macrophage (Mφ) accumulation in the lesions. Frequency of Mφ in WIN55212-2 mice is decreased dramatically compared with that of the control mice. In contrast, treatment with AM630 plus WIN55212-2 increased the frequency of Mφ. A) Representative photomicrographs of macrophages stained with Mac₃. B) Quantitative analysis of the percentage of lesion area occupied by macrophages. Magnification, 400×. The data are presented as mean ± S.D.; n = 8 per group; in comparison with the control, *P < 0.05.

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3.5. WIN55212-2 reduced inflammatory gene in plaque lesions

To further assess the inflammatory status of mice from the four different groups, we detected the pro-inflammatory genes TNF-α, IL-6 and MCP-1 mRNA in plaque lesions of the aortic tissue (Fig. 4). Compared with the control group, TNF-α was decreased by 59% (P < 0.05), IL-6 was decreased by 80% (P < 0.05) and MCP-1 was decreased by 73% (P < 0.05) in the group treated with WIN55212-2 at 1 mg/kg and the group treated with WIN55212-2 at 0.5 mg/kg showed a similar response. All of these effects of WIN55212-2 were abolished completely by the presence of AM630.

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Fig. 4.

Inhibitory effects of WIN55212-2 on of inflammatory mRNA in the aortic arch isolated from ApoE^−/− mice. The of the inflammatory genes, including IL-6 (A), TNF-α (B) and MCP-1 (C), was measured by real-time RT-PCR. The data are presented as mean ± S.D.; n = 8 per group; in comparison with the control, *P < 0.05.

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3.6. WIN55212-2 reduced NF-κB activation in advanced plaque lesions

The induction of the inflammatory genes TNF-α, IL-6 and MCP-1 is regulated by the transcription factor NF-κB. To examine whether WIN55212-2 action is associated with the NF-κB pathway, we used Western blotting to test the effect of WIN55212-2 on the DNA binding activity of NF-κB subunit p65 in plaque lesion. As shown in Fig. 5, WIN55212-2 dramatically inhibited the DNA binding activity of NF-κB subunit p65 in nuclear protein extract of aortic tissue from mice treated with WIN55212-2.

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Fig. 5.

Inhibitory effect of WIN55212-2 on NF-κB activation in aortic arch tissue isolated from ApoE^−/− mice. Nuclear proteins were extracted from aortic arch tissue. NF-κB activation was determined using p65 Western blot as described in Materials and methods. The data are presented as mean ± S.D.; n = 8 per group; in comparison with the control, *P < 0.05.

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3.7. WIN55212-2 reduced thioglycolate-elicited peritoneal macrophage inflammatory gene

It has been reported that monocyte-derived macrophages abound in plaque lesions and have a vital role in the development of atherosclerosis (Boyle, 2005). Macrophages produce a number of pro-atherosclerotic inflammatory cytokines, such as TNF-α, IL-6 and MCP-1, and cause advanced atherosclerosis. To further explore the possible mechanism underlying the protective effect of WIN55212-2, we analysed the cytokine of ox-LDL and LPS stimulated thioglycolate-elicited peritoneal macrophages by real-time PCR and ELISA. As shown in [Fig. 6] and [Fig. 8], ox-LDL and LPS stimulation upregulated TNF-α, IL-6 and MCP-1 mRNA and protein s. Interestingly, decreased production of TNF-α, IL-6 and MCP-1 was observed after treatment with WIN55212-2. Similarly, the effect of WIN55122-2 was abolished by AM630.

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Fig. 6.

Effect of WIN55212-2 on of inflammatory gene mRNA. IL-6(A), (E), TNF-α(B), (F)and MCP-1(C), (G) mRNA levels were measured by real-time RT-PCR in triplicate. The data are presented as the mean ± S.D.; *P < 0.05 vs. the control, #P < 0.01 vs. the ox-LDL or LPS pre-incubation group.

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3.8. WIN55212-2 reduced thioglycolate-elicited peritoneal macrophage NF-κB activation and IκB phosphorylation

To examine the effects of WIN55212-2 on the activation of NF-κB, the samples prepared from thioglycolate-elicited peritoneal macrophage were treated at 37 °C with WIN55212-2 (10⁻⁵ M) for 2 h before activation with ox-LDL (50 μg/ml) for 1 h. As shown in Fig. 7, NF-κB–DNA complexes formed by nuclear extract from control non-activated cells were detectable, indicating that thioglycolate-elicited peritoneal macrophage had basal NF-κB activity. In contrast, incubation of nuclear extracts from ox-LDL-activated cultures with the ³²P-labelled κB oligonucleotide exhibited increased DNA binding activity. More importantly, ox-LDL-induced NF-κB–DNA binding was inhibited significantly by treatment with WIN55212-2. Treatment with AM630 abolished the inhibitory effect of WIN55212-2. In parallel with the changes in NF-κB–DNA binding activity, ox-LDL-induced degradation of IκB-α was inhibited by WIN55212-2. As shown in Fig. 8, p-IκB-α protein was decreased significantly by treatment with WIN55212-2.

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Fig. 7.

Electrophoretic mobility-shift assay of nuclear extracts and p-IκB-α in peritoneal macrophages isolated from ApoE^−/− mice. Isolated peritoneal macrophages were cultured without (control) or with WIN55212-2 or with WIN55212-2 plus AM630 as described in Materials and methods. EMSA experiments with the consensus sequence for NF-κB and p-IκB-α were done as described in Materials and methods.

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Fig. 8.

Effect of WIN55212-2 on the release of inflammatory cytokines MCP-1, IL-6 and TNF-α in peritoneal macrophage isolated from ApoE^−/− mice. Isolated peritoneal macrophage were cultured without (control) or with WIN55212-2 or with WIN55212-2 plus AM630 as described in Materials and methods. The levels of IL-6 (A), (E), MCP-1(B), (F) and TNF-α (C), (G) were measured by ELISA. Values are presented as mean ± S.D. All results shown are representative of the results of three independent experiments. *P < 0.05 vs. control, # P < 0.05 vs. ox-LDL or LPS pre-incubation group.

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4. Discussion

This study demonstrated that WIN55212-2 significantly alleviated atherosclerosis through inhibiting macrophage accumulation and of inflammatory genes in plaque lesions. The anti-atherosclerotic effects and the immunomodulatory properties of WIN55212-2 were almost completely abrogated by AM630, indicating the involvement of the CB₂ receptors. The plasma lipid profiles in the ApoE^−/− mice revealed that WIN55212-2 had little effect on serum levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) or triglyceride (TG), suggesting that the anti-atherogenic effect of WIN55212-2 is mediated via mechanisms other than an improvement of lipid profiles.

The significant role of macrophages in the development of atherosclerosis has been demonstrated ([Dansky et al., 1997], [Song et al., 2001] and [Zhou et al., 2000]), and less macrophage accumulation could prevent the development of atherosclerosis. Cannabinoids have been reported to modulate the migration of various cell types ([Montecucco et al., 2008], [Rajesh et al., 2008] and [Rajesh et al., 2007]). In addition, it has been reported that treatment with a low dose of delta-9-tetrahydrocannabinol (THC) reduced progression of atherosclerosis in ApoE^−/− mice (Steffens et al., 2005). In this study, we demonstrated that WIN55212-2 can decrease macrophage content in the aortic lesions of ApoE^−/− mice, which is consistent with the results of an earlier study (Steffens et al., 2005). However, there is some limitation in the approach that we used by counting the number of cells positively stained by hematoxylin inside the internal elastic lamina. This represents a rough method to address macrophage number and could lead to an overestimation of macrophages into the lesion.

Recently, important insight into the role of cytokines in atherogenesis has been gained in vivo and in vitro (Kleemann et al., 2008). Among cytokines, we targeted TNF-α, IL-6 and MCP-1, which are implicated in the pathogenesis of atherosclerosis ([McKellar et al., 2009], [Dubinski and Zdrojewicz, 2007] and [Gu et al., 1998]). Interestingly, our data showed that WIN55212-2 dramatically decreased TNF-α, IL-6 and MCP-1 mRNA in plaque lesions of aortic tissue from ApoE^−/− mice, which might be favourable in inhibiting the development of atherosclerosis. Furthermore, WIN55212-2 significantly decreased TNF-α, IL-6 and MCP-1 mRNA and protein in thioglycolate-elicited peritoneal macrophages from the ApoE^−/− mice stimulated by ox-LDL or LPS, which might offer a novel approach to the treatment of the dysregulation of cytokines release in atherosclerosis.

The activation of pro-inflammatory transcription factor NF-κB is one of the major molecular events that involve inflammation. Earlier studies showed that the activation of transcription factor NF-κB inhibited by WIN55212-2 leads to the suppression of the release of pro-inflammatory mediators (De Filippis et al., 2007). Here, we confirmed that NF-κB could be activated by ox-LDL in macrophages, and NF-κB activation is inhibited dramatically by WIN55212-2 in macrophages. Moreover, the decreased levels of p-IκB-α observed in our studies is correlated well with the activation of NF-κB which, once in the nucleus, binds to its corresponding response element on the promoter of its target gene and initiates transcription. Taken together, our findings suggest strongly that the NF-κB pathway is involved in the inhibitory action of WIN55212-2 on the release of inflammatory cytokines, such as TNF-α, IL-6 and MCP-1. Furthermore, the reduced plaque lesions and decreasing TNF-α, IL-6 and MCP-1 mRNA and NF-κB P65 protein in the aortic tissues indicate that WIN55212-2 ameliorated atherosclerosis in ApoE-knockout mice through suppressing activation of the NF-κB pathway.

Another important finding in this study was that blocking the CB₂ receptor, the main cannabinoid receptor on immune cells, abolished the protective effects of WIN55212-2 on atherosclerosis in ApoE^−/− mice in vivo and the anti-inflammatory effect of WIN55212-2 on thioglycolate-elicited peritoneal macrophages in vitro. These results suggest the beneficial effects of WIN55212-2 are mediated by the CB₂ receptor, but not by the CB₁ receptor.

However, the CB₁ receptor has been shown to be expressed in human coronary atheroma (Sugamura et al., 2009); CB₁ inhibition attenuates atherosclerosis and decreases inflammation ([Sugamura et al., 2009], [Pacher, 2009] and [Dol-Gleizes et al., 2009]). These pieces of evidence indicate that CB₁ receptor activation might play an opposing role in atherosclerosis. In this study, AM630 has an effect opposite to that of WIN55212-2, which is frequently significant compared to the control, suggesting: (i) there is basal cannabinoid activation; and (ii) CB₂ receptor block might enhance the endogenous activation of CB₁ receptors, leading to the additional pro-inflammatory effects on atherogenesis, that is, activation of CB₁ receptors might associated with serious atherosclerosis.

In summary, our results suggest that WIN55212-2 might offer a novel approach to the treatment of a variety of inflammatory diseases, including atherosclerosis. All the beneficial effects of WIN55212-2 in our model were associated with the suppression of pro-inflammatory responses and were mediated by the CB₂ receptor.

Acknowledgments

This study was supported in part by Natural Science Foundation of China (30570732, 30871043 to Yuan ZY, and 30971219 to Liu Y).

References

Ashton et al., 2006 J.C. Ashton, D. Friberg, C.L. Darlington and P.F. Smith, of the cannabinoid CB2 receptor in the rat cerebellum: an immunohistochemical study, Neurosci. Lett. 396 (2006), pp. 113–116. Abstract | Article | PDF (255 K) | View Record in Scopus | Cited By in Scopus (61)

Boyle, 2005 J.J. Boyle, Macrophage activation in atherosclerosis: pathogenesis and pharmacology of plaque rupture, Curr. Vasc. Pharmacol. 3 (2005), pp. 63–68. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (107)

Buckley et al., 2000 N.E. Buckley, K.L. McCoy, E. Mezey, T. Bonner, A. Zimmer, C.C. Felder and M. Glass, Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB(2) receptor, Eur. J. Pharmacol. 396 (2000), pp. 141–149. Abstract | Article | PDF (241 K) | View Record in Scopus | Cited By in Scopus (235)

Dansky et al., 1997 H.M. Dansky, S.A. Charlton, M.M. Harper and J.D. Smith, T and B lymphocytes play a minor role in atherosclerotic plaque formation in the apolipoprotein E-deficient mouse, Proc. Natl Acad. Sci. USA 94 (1997), pp. 4642–4646. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (205)

De Filippis et al., 2007 D. De Filippis, A. Russo, D. De Stefano, M.C. Maiuri, G. Esposito, M.P. Cinelli, C. Pietropaolo, R. Carnuccio, G. Russo and T. Iuvone, Local administration of WIN 55, 212–2 reduces chronic granuloma-associated angiogenesis in rat by inhibiting NF-kappaB activation, J. Mol. Med. 85 (2007), pp. 635–645. View Record in Scopus | Cited By in Scopus (17)

Dol-Gleizes et al., 2009 F. Dol-Gleizes, R. Paumelle, V. Visentin, A.M. Mares, P. Desitter, N. Hennuyer, A. Gilde, B. Staels, P. Schaeffer and F. Bono, Rimonabant, a selective cannabinoid CB1 receptor antagonist, inhibits atherosclerosis in LDL receptor-deficient mice, Arterioscler. Thromb. Vasc. Biol. 29 (2009), pp. 12–18. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (28)

Dubinski and Zdrojewicz, 2007 A. Dubinski and Z. Zdrojewicz, The role of interleukin-6 in development and progression of atherosclerosis, Pol. Merkuriusz Lek. 22 (2007), pp. 291–294. View Record in Scopus | Cited By in Scopus (5)

Ghosh et al., 2006a S. Ghosh, P. Azhahianambi and J. de la Fuente, Control of ticks of ruminants, with special emphasis on livestock farming systems in India: present and future possibilities for integrated control — a review, Exp. Appl. Acarol. 40 (2006), pp. 49–66. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (15)

Ghosh et al., 2006b S. Ghosh, A. Preet, J.E. Groopman and R.K. Ganju, Cannabinoid receptor CB2 modulates the CXCL12/CXCR4-mediated chemotaxis of T lymphocytes, Mol. Immunol. 43 (2006), pp. 2169–2179. Abstract | Article | PDF (472 K) | View Record in Scopus | Cited By in Scopus (33)

Golech et al., 2004 S.A. Golech, R.M. McCarron, Y. Chen, J. Bembry, F. Lenz, R. Mechoulam, E. Shohami and M. Spatz, Human brain endothelium: co and function of vanilloid and endocannabinoid receptors, Brain Res. Mol. Brain Res. 132 (2004), pp. 87–92. Abstract | Article | PDF (272 K) | View Record in Scopus | Cited By in Scopus (98)

Gotsman et al., 2006 I. Gotsman, N. Grabie, R. Gupta, R. Dacosta, M. MacConmara, J. Lederer, G. Sukhova, J.L. Witztum, A.H. Sharpe and A.H. Lichtman, Impaired regulatory T-cell response and enhanced atherosclerosis in the absence of inducible costimulatory molecule, Circulation 114 (2006), pp. 2047–2055. View Record in Scopus | Cited By in Scopus (65)

Gu et al., 1998 L. Gu, Y. Okada, S.K. Clinton, C. Gerard, G.K. Sukhova, P. Libby and B.J. Rollins, Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice, Mol. Cell 2 (1998), pp. 275–281. Article | PDF (199 K) | View Record in Scopus | Cited By in Scopus (875)

Hajrasouliha et al., 2008 A.R. Hajrasouliha, S. Tavakoli, M. Ghasemi, P. Jabehdar-Maralani, H. Sadeghipour, F. Ebrahimi and A.R. Dehpour, Endogenous cannabinoids contribute to remote ischemic preconditioning via cannabinoid CB2 receptors in the rat heart, Eur. J. Pharmacol. 579 (2008), pp. 246–252. Abstract | Article | PDF (358 K) | View Record in Scopus | Cited By in Scopus (20)

Ihenetu et al., 2003 K. Ihenetu, A. Molleman, M.E. Parsons and C.J. Whelan, Inhibition of interleukin-8 release in the human colonic epithelial cell line HT-29 by cannabinoids, Eur. J. Pharmacol. 458 (2003), pp. 207–215. Abstract | Article | PDF (291 K) Article | PDF (265 K) | View Record in Scopus | Cited By in Scopus (14)

Joyeux et al., 2002 M. Joyeux, C. Arnaud, D. Godin-Ribuot, P. Demenge, D. Lamontagne and C. Ribuot, Endocannabinoids are implicated in the infarct size-reducing effect conferred by heat stress preconditioning in isolated rat hearts, Cardiovasc. Res. 55 (2002), pp. 619–625. View Record in Scopus | Cited By in Scopus (54)

Kenneth, 2003 J. Kenneth, Severe acute respiratory syndrome (SARS): the new epidemic, Natl Med. J. India 16 (2003), pp. 115–116. View Record in Scopus | Cited By in Scopus (2)

Kleemann et al., 2008 R. Kleemann, S. Zadelaar and T. Kooistra, Cytokines and atherosclerosis: a comprehensive review of studies in mice, Cardiovasc. Res. 79 (2008), pp. 360–376. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (85)

Krylatov et al., 2002 A.V. Krylatov, N.A. Bernatskaia, L.N. Maslov, R.G. Pertwee, R. Mechoulam, G.B. Stefano, M.A. Sharaevskii and O.M. Sal'nikova, Increase of the heart arrhythmogenic resistance and decrease of the myocardial necrosis zone during activation of cannabinoid receptors, Ross. Fiziol. Z. Im. I.M. Sechenova 88 (2002), pp. 560–567. View Record in Scopus | Cited By in Scopus (9)

Lagneux and Lamontagne, 2001 C. Lagneux and D. Lamontagne, Involvement of cannabinoids in the cardioprotection induced by lipopolysaccharide, Br. J. Pharmacol. 132 (2001), pp. 793–796. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (52)

Livak and Schmittgen, 2001 K.J. Livak and T.D. Schmittgen, Analysis of relative gene data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method, Methods 25 (2001), pp. 402–408. Abstract | PDF (713 K) | View Record in Scopus | Cited By in Scopus (14276)

Maccarrone et al., 2006 M. Maccarrone, A. Fiori, M. Bari, F. Granata, V. Gasperi, M.E. De Stefano, A. Finazzi-Agro and R. Strom, Regulation by cannabinoid receptors of anandamide transport across the blood-brain barrier and through other endothelial cells, Thromb. Haemost. 95 (2006), pp. 117–127. View Record in Scopus | Cited By in Scopus (22)

Mackie, 2006 K. Mackie, Cannabinoid receptors as therapeutic targets, Annu. Rev. Pharmacol. Toxicol. 46 (2006), pp. 101–122. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (162)

Marchalant et al., 2007 Y. Marchalant, S. Rosi and G.L. Wenk, Anti-inflammatory property of the cannabinoid agonist WIN-55212-2 in a rodent model of chronic brain inflammation, Neuroscience 144 (2007), pp. 1516–1522. Abstract | Article | PDF (2753 K) | View Record in Scopus | Cited By in Scopus (29)

McKellar et al., 2009 G.E. McKellar, D.W. McCarey, N. Sattar and I.B. McInnes, Role for TNF in atherosclerosis? Lessons from autoimmune disease, Nat. Rev. Cardiol. 6 (2009), pp. 410–417. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (26)

Mestre et al., 2005 L. Mestre, F. Correa, A. Arevalo-Martin, E. Molina-Holgado, M. Valenti, G. Ortar, V. Di Marzo and C. Guaza, Pharmacological modulation of the endocannabinoid system in a viral model of multiple sclerosis, J. Neurochem. 92 (2005), pp. 1327–1339. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (75)

Montecucco et al., 2008 F. Montecucco, F. Burger, F. Mach and S. Steffens, CB2 cannabinoid receptor agonist JWH-015 modulates human monocyte migration through defined intracellular signaling pathways, Am. J. Physiol. Heart Circ. Physiol. 294 (2008), pp. 1145–1155.

Mormina et al., 2006 M.E. Mormina, S. Thakur, A. Molleman, C.J. Whelan and A.R. Baydoun, Cannabinoid signalling in TNF-alpha induced IL-8 release, Eur. J. Pharmacol. 540 (2006), pp. 183–190. Abstract | Article | PDF (218 K) | View Record in Scopus | Cited By in Scopus (8)

Mukhopadhyay et al., 2007 P. Mukhopadhyay, S. Batkai, M. Rajesh, N. Czifra, J. Harvey-White, G. Hasko, Z. Zsengeller, N.P. Gerard, L. Liaudet, G. Kunos and P. Pacher, Pharmacological inhibition of CB1 cannabinoid receptor protects against doxorubicin-induced cardiotoxicity, J. Am. Coll. Cardiol. 50 (2007), pp. 528–536. Article | PDF (2755 K) | View Record in Scopus | Cited By in Scopus (56)

Ni et al., 2004 X. Ni, E.B. Geller, M.J. Eppihimer, T.K. Eisenstein, M.W. Adler and R.F. Tuma, Win 55212-2, a cannabinoid receptor agonist, attenuates leukocyte/endothelial interactions in an experimental autoimmune encephalomyelitis model, Mult. Scler. 10 (2004), pp. 158–164. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (45)

Pacher, 2009 P. Pacher, Cannabinoid CB1 receptor antagonists for atherosclerosis and cardiometabolic disorders: new hopes, old concerns?, Arterioscler. Thromb. Vasc. Biol. 29 (2009), pp. 7–9. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (12)

Pacher et al., 2006 P. Pacher, S. Batkai and G. Kunos, The endocannabinoid system as an emerging target of pharmacotherapy, Pharmacol. Rev. 58 (2006), pp. 389–462. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (455)

Rajesh et al., 2007 M. Rajesh, P. Mukhopadhyay, S. Batkai, G. Hasko, L. Liaudet, J.W. Huffman, A. Csiszar, Z. Ungvari, K. Mackie, S. Chatterjee and P. Pacher, CB2-receptor stimulation attenuates TNF-alpha-induced human endothelial cell activation, transendothelial migration of monocytes, and monocyte–endothelial adhesion, Am. J. Physiol. Heart Circ. Physiol. 293 (2007), pp. 2210–2218.

Rajesh et al., 2008 M. Rajesh, P. Mukhopadhyay, G. Hasko, J.W. Huffman, K. Mackie and P. Pacher, CB2 cannabinoid receptor agonists attenuate TNF-alpha-induced human vascular smooth muscle cell proliferation and migration, Br. J. Pharmacol. 153 (2008), pp. 347–357. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (29)

Sacerdote et al., 2005 P. Sacerdote, C. Martucci, A. Vaccani, F. Bariselli, A.E. Panerai, A. Colombo, D. Parolaro and P. Massi, The nonpsychoactive component of marijuana cannabidiol modulates chemotaxis and IL-10 and IL-12 production of murine macrophages both in vivo and in vitro, J. Neuroimmunol. 159 (2005), pp. 97–105. Abstract | Article | PDF (398 K) | View Record in Scopus | Cited By in Scopus (47)

Shmist et al., 2006 Y.A. Shmist, I. Goncharov, M. Eichler, V. Shneyvays, A. Isaac, Z. Vogel and A. Shainberg, Delta-9-tetrahydrocannabinol protects cardiac cells from hypoxia via CB2 receptor activation and nitric oxide production, Mol. Cell. Biochem. 283 (2006), pp. 75–83. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (23)

Showalter et al., 1996 V.M. Showalter, D.R. Compton, B.R. Martin and M.E. Abood, Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands, J. Pharmacol. Exp. Ther. 278 (1996), pp. 989–999. View Record in Scopus | Cited By in Scopus (311)

Song et al., 2001 L. Song, C. Leung and C. Schindler, Lymphocytes are important in early atherosclerosis, J. Clin. Invest. 108 (2001), pp. 251–259. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (112)

Steffens et al., 2005 S. Steffens, N.R. Veillard, C. Arnaud, G. Pelli, F. Burger, C. Staub, M. Karsak, A. Zimmer, J.L. Frossard and F. Mach, Low dose oral cannabinoid therapy reduces progression of atherosclerosis in mice, Nature 434 (2005), pp. 782–786. View Record in Scopus | Cited By in Scopus (160)

Sugamura et al., 2009 K. Sugamura, S. Sugiyama, T. Nozaki, Y. Matsuzawa, Y. Izumiya, K. Miyata, M. Nakayama, K. Kaikita, T. Obata, M. Takeya and H. Ogawa, Activated endocannabinoid system in coronary artery disease and antiinflammatory effects of cannabinoid 1 receptor blockade on macrophages, Circulation 119 (2009), pp. 28–36. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (31)

Van Sickle et al., 2005 M.D. Van Sickle, M. Duncan, P.J. Kingsley, A. Mouihate, P. Urbani, K. Mackie, N. Stella, A. Makriyannis, D. Piomelli, J.S. Davison, L.J. Marnett, V. Di Marzo, Q.J. Pittman, K.D. Patel and K.A. Sharkey, Identification and functional characterization of brainstem cannabinoid CB2 receptors, Science 310 (2005), pp. 329–332. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (470)

Yuan et al., 2002 M. Yuan, S.M. Kiertscher, Q. Cheng, R. Zoumalan, D.P. Tashkin and M.D. Roth, Delta 9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells, J. Neuroimmunol. 133 (2002), pp. 124–131. Abstract | Article | PDF (236 K) | View Record in Scopus | Cited By in Scopus (78)

Yuan et al., 2003 Z. Yuan, C. Kishimoto, H. Sano, K. Shioji, Y. Xu and M. Yokode, Immunoglobulin treatment suppresses atherosclerosis in apolipoprotein E-deficient mice via the Fc portion, Am. J. Physiol. Heart Circ. Physiol. 285 (2003), pp. 899–906.

Zhao et al., 2010 Y. Zhao, Z. Yuan, Y. Liu, J. Xue, Y. Tian, W. Liu, W. Zhang, Y. Shen, W. Xu, X. Liang and T. Chen, Activation of cannabinoid CB2 receptor ameliorates atherosclerosis associated with suppression of adhesion molecules, J. Cardiovasc. Pharmacol. 55 (2010), pp. 292–298. View Record in Scopus | Cited By in Scopus (6)

Zhou et al., 2000 X. Zhou, A. Nicoletti, R. Elhage and G.K. Hansson, Transfer of CD4(+) T cells aggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice, Circulation 102 (2000), pp. 2919–2922. View Record in Scopus | Cited By in Scopus (231)

Corresponding author. Department of Cardiovascular Medicine, The First Affiliated Hospital of Xi'an Jiaotong University College of Medicine, 277 West Yanta Road, Xi'an, Shaanxi 710061, China. Tel.: + 86 29 8532 3819; fax: + 86 29 8532 3709. Copyright © 2010 Elsevier B.V. All rights reserved.

European Journal of Pharmacology
Volume 649, Issues 1-3, 15 December 2010, Pages 285-292

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Cardiovascular Pharmacology

Abstract

Article Outline

1. Introduction

2. Materials and methods

2.1. Experimental atherosclerosis

2.2. Drugs and treatment protocols

2.3. Lipid measurements

2.4. Quantification of atherosclerotic lesions by immunohistochemistry

2.5. LDL isolation and oxidation

2.6. Cell culture

2.7. RNA isolation and quantitative RT-PCR

2.8. Protein preparation

2.9. Western blotting

2.10. Measurement of MCP-1, IL-6 and TNF-α concentration

2.11. Electrophoretic mobility-shift assay (EMSA)

2.12. Statistical analysis

3. Results

3.1. CB2 receptors are expressed in plaque lesion and peritoneal macrophages

3.2. Effects of WIN55212-2 on physiological parameters

3.3. WIN55212-2 reduces atherosclerotic lesion size in ApoE−/− mice

3.4. Immunohistochemical analysis of macrophage accumulation in plaque lesion

3.5. WIN55212-2 reduced inflammatory gene in plaque lesions

3.6. WIN55212-2 reduced NF-κB activation in advanced plaque lesions

3.7. WIN55212-2 reduced thioglycolate-elicited peritoneal macrophage inflammatory gene

3.8. WIN55212-2 reduced thioglycolate-elicited peritoneal macrophage NF-κB activation and IκB phosphorylation

4. Discussion

Acknowledgments

References

3.1. CB₂ receptors are expressed in plaque lesion and peritoneal macrophages

3.3. WIN55212-2 reduces atherosclerotic lesion size in ApoE^−/− mice