The effects of resveratrol on various conditions have been widely studied previously. This paper aimed to investigate the influence of resveratrol on atherosclerosis (AS). Twenty-four New Zealand male rabbits were randomly and equally assigned to the normal diet group (NDG), fat diet group (FDG), and fat diet with resveratrol group (80 mg/kg/d, RFG). Biochemical indicators from blood samples were analyzed at baseline and 3 months to investigate the effects of resveratrol on blood lipid, lipoprotein-associated phospholipase A2 (Lp-PLA2), liver, and renal function. The indicators including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and Lp-PLA2. At 3 months, arteries were stained with hematoxylin and eosin to study the influence of resveratrol on the aortic intima, smooth muscle layer, and the intima/media ratio. Comparisons of weight, ALT, AST, CREA, TG, TC, HDL-C, LDL-C, and Lp-PLA2 among the three groups showed no significant difference at baseline. However, at the end of 3 months, significant differences were observed in AST, CREA, TC, HDL-C, LDL-C, and Lp-PLA2 between the three groups (
Atherosclerosis (AS) is a disease that seriously endangers human health. Numerous basic and clinical studies indicate that AS is a chronical inflammatory disease, characterized by an abnormal reaction to the damage of blood vessel walls, with symptoms including classic inflammatory degeneration, exudation, and proliferation characteristics [
Resveratrol is a polyphenolic compound that can be naturally produced by numerous plants, including grapes, peanuts, and mulberries [
However, it is still controversial whether resveratrol can improve AS and alter blood lipids and function of liver and kidney. There are also limited data concerning the effect of resveratrol on blood lipoprotein-associated phospholipase A2 (Lp-PLA2). Lp-PLA2, also known as platelet-activating factor acetylhydrolase (PAF-AH), is a 45 kD, calcium-independent enzyme which “belongs to the phospholipase A2 superfamily. Lp-PLA2 can be produced by inflammatory cells in atherosclerotic plaques, such as monocytes, macrophages, T-lymphocytes, and mast cells [
Two main animal AS models have been developed for the study of the molecular mechanisms underlying AS: one is induced by a high-fat diet and another involves intimal injury [
All experiments were approved by the Institutional Animal Care and Use Committee at the China-Japan Union Hospital of Jilin University. All procedures were performed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals.
Three-month-old New Zealand male white rabbits, weighing approximately 2 kg, were obtained from Changchun Yis Laboratory Animal Technology (Jilin, China). The rabbits were given free access to food and water, while housed under a natural day/night cycle.
To test the hypothesis that the fat diet can induce AS, a preliminary experiment was conducted. A control (NDG) rabbit was fed on a normal diet (NDG), while an experimental (FDG) rabbit fed was fed on a fat diet. The normal diet contained carbohydrates 68%, protein 22%, salt 0.5%, and fat 10%, and the fat diet contained 1% cholesterol, 5% lard, 5% yolk powder, and 89% basic rabbit feed (Beijing Botai Hongda Biotechnology Co., Ltd., China). The rabbits were maintained on this diet for 3 months. The aortic arch was harvested from the rabbits after euthanasia by injection with sodium phenobarbital (0.2 g/kg, diluted concentration 3.5%) into the ear vein and put in a test tube containing 4% paraformaldehyde for histological evaluation. Perpendicular to the vascular axis, the aortic arch was cut into approximately 2 mm lengths, embedded in paraffin and stained with hematoxylin and eosin. Atherosclerotic lesions were detected by light microscopy in the rabbit fed on a fat diet. This model has previously been used for studies of AS [
Representative photomicrographs of aortic arch sections stained with hematoxylin-eosin of a normal diet group (NDG) and a fat diet group (FDG) in the preliminary experiment. Magnification: 10x. Bar represents 100
Twenty-four rabbits were randomly assigned to three groups: a normal diet group (NDG) served as a control; a fat diet group (FDG); and an FDG group treated with resveratrol (RFG). The treatment concentrations of resveratrol were determined according to a preliminary experiment and a literature review (Beijing Solarbio Science & Technology Co., Ltd., China). Resveratrol (80 mg/kg/day) was mixed in with the feed (50 g, twice a day) and accounted for about 0.2% of the feed [
Blood samples were collected by heart puncture from the rabbits in the three groups at the baseline, before commencement of the diet in the morning, and after 3 months. The determinations of blood levels of alanine aminotransferase (ALT, IU/L), aspartate aminotransferase (AST, IU/L), creatinine (CREA, µmmol/L), triglycerides (TG, mmol/L), total cholesterol (TC; mmol/L), high-density lipoprotein cholesterol (HDL-C, mmol/L), low-density lipoprotein cholesterol (LDL-C, mmol/L), and Lp-PLA2 (diluted 10-fold, ng/mL) were conducted. A chemiluminescent methodology was used to analyze the levels of ALT, AST, CREA, TG, TC, HDL-C, and LDL-C on the AU5800 autoanalyzer (Beckman Coulter, USA) via serum detection kits (Beckman Coulter, USA). Dry-type fluorescence immunity analysis (Guang Zhou Labsim Biotech Co., Ltd., China) was performed to determinate the levels of Lp-PLA2 using a lipoprotein-related phospholipase A2 kit (Vazyme Biotech Co., Ltd., China), and the methodology was quantum dot fluorescence immunoassay.
After euthanasia of the rabbits by injection with sodium phenobarbital (0.2 g/kg, diluted to a concentration of 3.5%) into the ear vein, perpendicular to the vascular axis, the aortic arch was cut into approximately 2 mm lengths and placed in a test tube containing 4% paraformaldehyde. Samples were embedded in paraffin and stained with hematoxylin and eosin. Measurements of the thickness of the intima and smooth muscle layer were conducted by light microscopy (Olympus Corporation, Japan). The software connected with the microscope screenshot has a measurement tool. Clicking the measure button, the measured value will automatically appeared with dragging from a certain point to zero. After measuring the thickness of the intima and the smooth muscle layer under the microscope, the mean and standard deviations were determined, and the intima/middle layer ratio was subsequently calculated.
All statistical analyses were performed using the SPSS 19.0 software (IBM, Armonk, New York); data from the experiments were analyzed and expressed as the mean ± standard errors of the means (SEM). One-way analysis of variance (One way ANOVA) was used for further analysis. If the variance between groups was homogeneous, a comparison was conducted using the Bonferroni method, and the
The comparison of weight, ALT, AST, CREA, TG, TC, HDL-C, LDL-C, and Lp-PLA2 levels between the three groups showed no significant difference at the baseline. The differences in pairwise comparison between the three groups also showed no significant difference at the baseline (
Biochemical analysis of rabbits from NDG (
NDG ( | FDG ( | RFG ( | ||||
---|---|---|---|---|---|---|
Weight (kg) | 0.96 ± 0.05 | 0.99 ± 0.08 | 1.01 ± 0.08 | 0.903 | 0.420 | |
ALT | 43.20 ± 9.65 | 41.22 ± 8.83 | 44.76 ± 12.80 | 0.226 | 0.800 | |
AST | 36.38 ± 8.61 | 33.73 ± 5.91 | 34.88 ± 4.98 | 0.317 | 0.732 | |
CREA | 67.94 ± 8.39 | 64.72 ± 6.27 | 65.75 ± 9.69 | 0.318 | 0.731 | |
TG | 0.63 ± 0.20 | 0.50 ± 0.16 | 0.48 ± 0.07 | 2.230 | 0.132 | |
TC | 1.94 ± 0.21 | 1.88 ± 0.28 | 1.99 ± 0.43 | 0.240 | 0.789 | |
HDL-C | 0.93 ± 0.19 | 0.94 ± 0.25 | 0.93 ± 0.17 | 0.006 | 0.994 | |
LDL-C | 0.84 ± 0.22 | 0.95 ± 0.12 | 0.80 ± 0.27 | 1.053 | 0.367 | |
Lp-PLA2# | 461.50 ± 89.57 | 525.48 ± 32.50 | 494.88 ± 32.56 | 2.424 | 0.113 |
#The test result of homogeneity of variance was
Intergroup comparison at the end of the 3-month experiment showed that the levels of AST, CREA, TC, HDL-C, LDL-C, and Lp-PLA2 were significantly different among the three groups (
Biochemical analysis of rabbits from NDG (
NDG ( | FDG ( | RFG ( | |||
---|---|---|---|---|---|
Weight (kg)# | 2.90 ± 0.08 | 2.98 ± 0.15 | 2.96 ± 0.07 | 1.160 | 0.333 |
ALT | 44.81 ± 7.95 | 50.86 ± 24.53 | 61.06 ± 20.26 | 1.506 | 0.245 |
AST# | 40.51 ± 6.46 | 84.32 ± 54.94 | 77.91 ± 24.54a | 3.668 | 0.043 |
CREA | 77.38 ± 10.45 | 111.92 ± 11.38a | 127.63 ± 10.84a,b | 44.526 | <0.001 |
TG | 0.62 ± 0.21 | 0.90 ± 0.42 | 0.76 ± 0.28 | 1.567 | 0.232 |
TC# | 2.19 ± 0.30 | 30.32 ± 5.74a | 11.84 ± 2.78a,b | 120.264 | <0.001 |
HDL# | 1.03 ± 0.20 | 3.28 ± 1.61a | 1.58 ± 0.35a | 11.946 | <0.001 |
LDL | 0.91 ± 0.22 | 16.45 ± 3.16a | 6.23 ± 1.53a,b | 120.880 | <0.001 |
Lp-PLA2# | 520.14 ± 51.55 | 1928.88 ± 385.78a | 953.20 ± 96.66a,b | 77.698 | <0.001 |
#The test result of homogeneity of variance was
In addition, intragroup blood index comparisons were performed in the NDG, FDG, and RFG rabbits between the two time points: baseline and at 3 months. There were no significant differences in ALT, TG, LDL-C, and Lp-PLA2 levels between the baseline and after 3 months in NDG rabbits (Table
Biochemical analysis of rabbits from RFG (
Baseline ( | After 3 months ( | |||
---|---|---|---|---|
Weight(kg) (NDG) | 0.96 ± 0.05 | 2.90 ± 0.08 | −73.655 | <0.001 |
Weight (kg) (FDG) | 0.99 ± 0.08 | 2.98 ± 0.15 | −49.925 | <0.001 |
Weight (kg) (RFG) | 1.01 ± 0.08 | 2.96 ± 0.07 | −39.000 | <0.001 |
ALT (NDG) | 43.20 ± 9.65 | 44.81 ± 7.95 | −0.781 | 0.461 |
ALT (FDG) | 41.22 ± 8.83 | 50.86 ± 24.53 | −1.172 | 0.280 |
ALT (RFG) | 44.76 ± 12.80 | 61.06 ± 20.26 | −2.160 | 0.068 |
AST (NDG) | 36.38 ± 8.61 | 40.51 ± 6.46 | −2.490 | 0.042 |
AST (FDG) | 33.73 ± 5.91 | 84.32 ± 54.94 | −2.639 | 0.033 |
AST (RFG) | 34.88 ± 4.98 | 77.91 ± 24.54 | −5.587 | 0.001 |
CREA (NDG) | 67.94 ± 8.39 | 77.38 ± 10.45 | −7.002 | <0.001 |
CREA (FDG) | 64.72 ± 6.27 | 111.92 ± 11.38 | −10.794 | <0.001 |
CREA (RFG) | 65.75 ± 9.69 | 127.63 ± 10.84 | −12.390 | <0.001 |
TG (NDG) | 0.63 ± 0.20 | 0.62 ± 0.21 | 0.086 | 0.934 |
TG (FDG) | 0.50 ± 0.16 | 0.90 ± 0.42 | −2.230 | 0.061 |
TG (RFG) | 0.48 ± 0.07 | 0.76 ± 0.28 | −2.986 | 0.020 |
TC (NDG) | 1.94 ± 0.21 | 2.19 ± 0.30 | −2.919 | 0.022 |
TC (FDG) | 1.88 ± 0.28 | 30.32 ± 5.74 | −14.078 | <0.001 |
TC (RFG) | 1.99 ± 0.43 | 11.84 ± 2.78 | −9.275 | <0.001 |
HDL (NDG) | 0.93 ± 0.19 | 1.03 ± 0.20 | −3.359 | 0.012 |
HDL (FDG) | 0.94 ± 0.25 | 3.28 ± 1.61 | −3.815 | 0.007 |
HDL (RFG) | 0.93 ± 0.17 | 1.58 ± 0.35 | −4.880 | 0.002 |
LDL (NDG) | 0.84 ± 0.22 | 0.91 ± 0.22 | −1.270 | 0.245 |
LDL (FDG) | 0.95 ± 0.12 | 16.45 ± 3.16 | −13.489 | <0.001 |
LDL (RFG) | 0.80 ± 0.27 | 6.23 ± 1.53 | −9.221 | <0.001 |
Lp-PLA2# (NDG) | 461.50 ± 89.57 | 520.14 ± 51.55 | −1.930 | 0.095 |
Lp-PLA2# (FDG) | 525.48 ± 32.50 | 1928.88 ± 385.78 | −10.090 | <0.001 |
Lp-PLA2# (RFG) | 494.88 ± 32.56 | 953.20 ± 96.66 | −12.816 | <0.001 |
#The test result of homogeneity of variance was
Figure
Representative photomicrographs of aortic arch sections stained with hematoxylin-eosin of a normal diet group (NDG), a fat diet group (FDG), and a resveratrol-treated FDG group (RFG). Magnification: 10x. Bar represents 100
Pathological parameters of aortic arch atherosclerosis observed under the microscope of RFG and FDG.
Group | Mean | SD (±) | ||
---|---|---|---|---|
Intima | FDG | 124.76 | 6.83 | 0.001 |
RFG | 52.44 | 14.94 | ||
Smooth muscle layer | FDG | 194.16 | 10.2 | 0.65 |
RFG | 173.48 | 4.05 | ||
Intima/media ratio | FDG | 0.64 | 0.04 | 0.001 |
RFG | 0.30 | 0.09 |
Unit,
The prevention and treatment of cardiovascular and cerebrovascular diseases is an important topic of medical research, and studies have shown that AS participates in the development of many of these diseases [
Some studies had found that Lp-PLA2 was associated with plaque progression and vulnerability [
Lipid disorders are closely associated with the development of AS [
AST and ALT enzymes are mainly concentrated in the liver and are involved in the conversion of sugars and proteins in vivo [
CREA is a product of human muscle metabolism, which is produced by the irreversible nonenzymatic dehydration of creatine [
In summary, the present study suggests that resveratrol could inhibit the incrassation of arterial intima. In addition, resveratrol treatment reduced the levels of TC, LDL-C, Lp-PLA2, and CREA in rabbits fed a high-fat diet. Thus, resveratrol may have a function in preventing AS. However, clinical trials are needed to further investigate this effect.
Resveratrol might have antiatherosclerosis effect on a rabbit model of AS.
The data used to support the findings of this study are included within the article.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This wok was supported by the Jilin Province Natural Science Foundation (no. 20160101073JC).