Original Article
Resveratrol Caus Antiatherogenic Effects in an Animal Model of Atherosclerosis
Rossane Serafim Matos, Liz Andréa Villela Baroncini, Leonardo Brandão Précoma, Guilherme Winter, Pedro Henrique Lambach Caron, Flávia Kaiber, Dalton Bertolim Précoma
Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil
Abstract
Background: Resveratrol protects the cardiovascular system by a number of mechanisms, including an
tioxidant and anti-platelet activities.
Objective: T o asss the potential anti-inflammatory and antiatherogenic effects of resveratrol using rabbits fed a hypercholesterolemic diet (1% cholesterol).
Methods: T wenty white male rabbits were lected and divided into two groups: control group (CG), 10 rabbits; and resveratrol group (RG), 10 rabbits. The animals were fed a hypercholesterolemic diet for 56 days. For the RG diet, resveratrol (2mg/kg weight/day) was added from days 33 - 56.
Results: There was no significant difference in the total rum cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides between the groups. Of the CG, 70% had advanced aortic atherosclerotic lesions (types III, IV, V, or VI). All animals from the RG had mild aortic atherosclerotic lesions (types I or II, or no lesions). The intima area and the intima/media layer area ratio was significantly lower in the RG as compared to the CG (p<0.001). Positive areas for VCAM-1 molecules were lower in the RG (p=0.007). The MCP-1 and IL-6 concentrations were lower in the RG than the CG (p=0.039 and p=0.015, respectively).
Conclusion: Resveratrol had significant anti-atherogenic and anti-inflammatory effects in an animal model with rabbits fed a hypercholesterolemic diet (1% cholesterol). (Arq Bras Cardiol 2012;98(2):13
6-142)
Keywords: Revesratrol; antioxidants; atherosclerosis; dyslipidemias.
Mailing Address: Liz Andréa Villela Baroncini •
Rua Buenos Aires, 764 / 601, Batel - 80250-070 – Curitiba, PR, Brazil
E-mail: lizavb@cardiol.br; Manuscript received May 26, 2011, revid manuscript received July 20, 2011; accepted August 09, 2011.Methods
Animals
T wenty white adult New Zealand male rabbits at a mean age of 30 days were lected. Animals were handled in compliance with the Guiding Principles in the Care and U of Animals. Protocol approval was obtained from the Animal Rearch Committee of the Pontifícia Universidade Católica. The animals were divided into 2 groups, as follows: control group (CG), 10 rabbits; and resveratrol group (RG), 10 rabbits. During the 56 days of the study, the animals were fed a specific diet for the species (Nuvilab®) plus 1% cholesterol from lyophilized eggs. From days 29 – 56, resveratol (Resveratrol®; extracted from the Galena® Laboratory) was added to the RG diet (2mg/kg/day) and
administered by oral gavage. On day 56, the animals underwent disction of the aortic arch and descending aorta. Anesthesia was induced with ketamine (Vetanarcol®, 3.5 mg/kg; König) and intramuscular xylazine (Coopazine®, 5 mg/kg; Coopers). After the procedure, the rabbits were sacrificed by a lethal do of barbiturate. The sample size was calculated bad on the study by Zou et al6.
Blood chemistry
Blood samples were obtained on the first day of the experiment and immediately before sacrifice by cardiac puncture. Clinical
Introduction
Trans-resveratrol (resveratrol) is a polyphenolic compound found in fresh grapes, grape juice, and wine.
Resveratrol is produced by the skin of grapes in respon to
fungal exposure. Resveratrol has been implicated in many,
but not all, studies as a mediator of alcohol-independent
cardiovascular protection that is allegedly conferred by
drinking red wine1. Resveratrol protects the cardiovascular
医保报销额度system by a number of mechanisms, including resveratrol-
mediated inhibition of low-density lipoprotein oxidation,
inhibition of platelet aggregation, synthesis of pro-
atherogenic eicosanoids, inhibition of cell proliferation, and
incread vasorelaxation. Recent studies have also shown
that resveratrol suppress the induction of procoagulant
tissue factor, one of the key components thought to be
responsible for high mortality from cardiovascular dia2-5.
The objective of this study was to asss the potential anti-
inflammatory and anti-atherogenic effects of resveratrol
using an experimental animal model with rabbits fed a
hypercholesterolemic diet (1% cholesterol).
136
Original Article
Arq Bras Cardiol 2012;98(2):136-142
Matos et al
Resveratrol and atherosclerosis警花出更
laboratory asssment included total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TGC). Measurements were taken using an automated system (Abbott Architect ci8200; Abbott Laboratories, Abbott Park, IL, USA).Histologic analysis
The arteries were removed and washed with 10% formaldehyde buffered with phosphate (ph=7.6) and waxed. For evaluation of this experimental model, qualitative and quantitative histologic measurements were adopted. The hematoxylin and eosin (HE) stained slices were analyzed blindly in a microscopy jellyfish five-head Olympus ® BX 40. The atherosclerotic lesions were graded from 0 - VI according to the qualitative criteria propod by Stary et al 7-9. The morphometric analysis was performed on stained orcein (elastic) to determine the area of the intima and the medial layers of the aortic arch and descending aorta, as well as to determine the intima/media layer area ratio (IMR - the area of the intimal layer divided by the area of the medial layer). For this asssment, the more injured gment or the gment with more advanced stage of atherosclerosis was previously lected in the HE-stained slice. The analys were performed microscopically in conjunction with Image Pro-plus ® 4.5 software (Media Cybernetics Inc., Silver Spring, MD, USA).Immunohistochemistry
We evaluated the concentration of the vascular cell adhesion molecule (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and interleucin-6 (IL-6) in the intima of the arteries using primary
monoclonal antibodies. The reading was performed with an Olympus ® BX50 microscope, with an objective of 20 times.Statistical analysis
Categorical variables were expresd as percentages and continuous variables were expresd as the mean ± SD and medians. The Shapiro-Wilks test was ud for testing sample normality. For symmetry conditions, some variables were submitted to logarithmic transformation. For dichotomic nominal variables, the Fisher exact test was ud to compare the groups. For quantitative parameters, Student’s t -test and Mann-Whitney non-parametric test were ud for the comparison between CG and RG. Statistical significance was indicated by p < 0.05.
Results
T wo animals from the CG died in the middle of the experiment and were removed from the final analysis. The initial mean body weight of animals in the CG (1.78 kg) and RG (1.82 kg) were similar (p=0.274). At the end of the study , CG animals exhibited a mean body weight of 2.58 kg, while RG animals had a mean body weight of 2.39 kg (p=0.537). Lipid profiles
Baline TC, HDL-C, LDL-C, and TGC levels were relatively equivalent in all groups before initiation of the diet. At the end of the experiment, an analysis of TC, HDL-C, LDL-C, and rum TG indicated no significant difference between the groups (Table 1).
Table 1 - Lipid profile between control group (CG) and Resveratrol group (RG)
Variable Group Mean Minimum Maximum SD (±)p
TC (basal)
CG 70.3047.00115.0021.79RG
44.5030.0062.0010.980.008TC (euthanasia)
CG 1676.70347.002885.00768.67RG
2173.751626.002820.00353.840.112TGC (basal)
CG 196.4096.00295.0071.94RG
114.0078.00181.0037.110.010TGC (euthanasia)
冷冻的饺子怎么煮
CG 220.2060.00669.00204.32RG
158.14114226.0075.240.762LDL-C (basal)
CG 11.80 2.0049.0015.17RG
2.08 1.37 2.750.400.346LDL-C (euthanasia)
CG 1596.12298.002719.20743.03 RG
2084.631373.002748.00399.960.114HDL-C (basal)
CG 19.3812.0029.00 6.33RG
16.3811.0025.00 4.240.280HDL-C (euthanasia)
CG 37.4029.0050.007.29RG
怎样腌制鱼干34.25
25.00
55.00
9.97
0.449TC means total cholesterol; TGC means triglycerides; LDL-C means low density cholesterol
偏偏喜欢你歌词
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Arq Bras Cardiol 2012;98(2):136-142
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Resveratrol and atherosclerosis
Histologic analysis
At the end of the experiments, it was shown that there was a statistically significant difference between the animal groups with respect to the probability of type I, II, III, IV , V , and VI lesions or abnce of lesions. Of tho animals in the CG, 70% had advanced atherosclerotic lesions (types III, IV , and V) in the aortic arch and descending aorta, while 100% of the RG animals had mild atherosclerotic lesions (types I and II, or no lesions) in the aortic arch and descending aorta (Figure 1). Morphometric analysis
The intimal area was significantly lower in the RG than the CG (p = 0.001; Figure 1). There was a si
gnificantly greater reduction in the intima/media layer area ratio (IMR) in the RG than in the CG (p = 0.001). There was no statistically significant difference between the medial layer area between the groups in the aortic arch and descending aorta (Table 2). Immunohistochemistry
An analysis of positive areas for VCAM-1 revealed a significant difference between groups, with a higher concentration in CG
(p=0.007). Analysis of MCP-1 and IL-6 showed significantly higher values in the CG compared with the RG (p=0.039 and p=0.015, respectively; T able 3; Figure 2).
瓜熟蒂落的近义词Discussion
Resveratrol, prent in the grape juice and wine, is considered the major polyphenol responsible for cardiovascular benefits becau of its antioxidant and anti-platelet activities 10,11. Resveratrol has anti-inflammatory properties which are manifested as inhibition of ICAM-1 and VCAM-1expression and the attachment of monocytes to endothelial cells, inhibition of lipopolysaccharide (LPS)-induced synthesis of TNF-a and IL-1-b , and relea of IL-6 from monocytes 12. Resveratrol also inhibits the migration and proliferation of vascular smooth muscle cells (SMCs) to the intima layer, which is considered the sine qua non of atherogenesis 13,14. Collectively, the effects are thought to be res
ponsible for decread incidence of heart dia in the French population, and is thus termed the French Paradox (a very low mortality rate due to cardiovascular
dia, despite a high-fat diet), becau of the population’s
Figure 1 - Histologic slices from the descending aorta. A – Resveratrol group with atherosclerotic lesion (type I). Black arrow indicates intima layer. B – Control group
with atherosclerotic lesion (type III). Black arrow indicates intima layer.
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preference for red wine 15. In this study, we induced atherosclerotic lesions with the addition of lyophilized egg to the normal diet of rabbits during 4 weeks. This experimental model of inducing atherosclerosis using lyophilized egg is rarely described in the literature, though it is proven to be effi
cient and less costly when compared to the u of industrialized cholesterol. Resveratrol was added in the diet of RG after 4 weeks to test its capacity to interfere in established atherosclerotic lesions. We demonstrated that resveratrol inhibits the progression of atherosclerotic lesions by reducing the intima area and the IMR, and showing no advanced lesions in the aortic arch and descending aorta in the RG. In a similar study using rabbits, Castro et al 16 concluded that resveratrol worked as a preventive agent in the development of atherosclerotic lesions as they obrved a low-degree of foam cell invasion in the tunica media of animals treated with resveratrol. The mechanisms by which resveratrol inhibits the formation of advanced atherosclerotic lesions appears to be through its inhibitory effect on LDL oxidation, platelet aggregation, and vascular proliferation of smooth muscle cells. LDL oxidation is a main cau of endothelial injury and induction of the expression of pro-inflammatory molecules in endothelial cells. Resveratrol has been shown to protect lipids from peroxidative degradation and to stop the uptake of oxidized LDLs in the vascular wall in a concentration-dependent manner 17-19. However, there are conflicting results regarding the effects of resveratrol on lipid levels, becau some studies have failed to show a reduction in plasma lipids levels induced by such a substance 20. In this study, we did not uncover any evidence of a significant difference in lipid profiles between groups, which may have reflected the short period of the experiment. Daugherty et al 21, in a similar study in hypercholesterolemic rabbits, showed that th
e reduction in atherosclerotic lesions condary to the reduction in LDL-C levels is a process that takes months, or even years. In contrast, in this study, we showed that a hyphercholesterolemic diet caus the development of atherosclerotic lesions associated with an inflammatory process, and resveratrol reduced the concentration of VCAM-1, MCP-1, and IL-6 in the intima layer of hypercholesterolemic rabbits. It is known that inflammation mediates all stages of atherosclerosis from initiation to progression, and eventually plaque rupture 17. Indeed, VCAM-1, MCP-1, and IL-6 facilitate the adherence of monocytes to the endothelial surface and facilitate leukocyte tranndothelial migration 22-25. This process is exacerbated when blood LDL levels are high from poor dietary habits or an inherited genetic pre-disposition, and signals the endothelium to increa adhesion molecule expression and potentiate the inflammatory respon 26. Even though we did not make the correlation between blood lipid levels and the inflammatory process, we believe that such a possible correlation should be the subject of future studies. Also, we did not analyze the monocyte/macrophage concentration
Table 2 - Quantitative histopathological parameters between control group (CG) and resveratrol group (RG) expresd in square micrometer.
Variable Group n Mean Median SD (±)p*Intima
CG 1075.1068.2551.66< 0.001
RG 8 4.76 1.38 6.75Media
CG 10176.59196.9952.690.315RG 8209.70205.4939.77IMR
CG 100.410.370.25< 0.001RG
8
0.03
0.01
0.04
IMR reprents intima/media area ratio. *Mann- Whitney.
Table 3 - Immunohistochemical variables in descending aorta between control group (CG) and resveratrol group (RG) expresd in square micrometer
Variable Group n Mean Median SD (±)p*VCAM-1
CG 105339.075209.0337910.007
RG 81452.961035.591137MCP-1
CG 10117721100379470.039RG 8550947812745IL-6
孤单背影
CG 103852362326940.015RG
8
1273
1128
580
VCAM-1 means vascular cell adhesion molecule; MCP-1 means monocyte chemotactic protein-1; IL-6 means interleucin-6. * Student’s t-test.
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that appears to have a crucial role in the development of atherosclerosis. This cell appears to be involved in all stages of atherosclerotic plaque development and is increasingly regarded as a candidate for therapeutic intervention and as a potential biomarker of dia progression and respon to therapy 27-29.
Conclusions
Resveratrol had significant anti-atherogenic and anti-inflammatory effects in an animal model of rabbits fed a
hypercholesterolemic diet (1% cholesterol).
Figure 2 - Immunohistochemistry showing positive areas for MCP-1, VCAM-1, and IL-6. A – Control group. Black arrow indicates intima layer with positive areas for
MCP-1 (brown-speckled areas). B – Resveratrol group. Black arrow indicates intima layer without positive areas for MCP-1. C – Control group. Positive areas for VCAM-1 (brown-speckled areas). D – Resveratrol group. Positive areas for VCAM-1. E- Control group. Positive areas for IL-6 (brown-speckled areas). F- Resveratrol group. Positive areas for IL-6.
Potential Conflict of Interest
No potential conflict of interest relevant to this article was reported.
Sources of Funding
There were no external funding sources for this study.
Study Association
This article is part of the thesis of master submitted by Rossane Serafim Matos, from Pontifícia Universidade Católica do Paraná.八年级历史思维导图
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