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Transresveratrol (t-resveratrol; 3,5,4′-trihydroxy-trans-stilbene) is a polyphenolic compound found in fresh grapes, grape juice and wine, and has been found to reduce the total cholesterol level in hypercholesterolemic rats. The objective of the present study was to assess the effects of t-resveratrol on platelet-neutrophil complex formation and neutrophil reactive oxygen species (ROS) status in control and hypercholesterolemic rats using a modified flow cytometric method. Rats (n=80) were divided into five groups (control, ethanol, resveratrol, hypercholesterolemic and resveratrol-administered hypercholesterolemic groups), comprising 16 animals per group. Serum levels of lipids and H2O2 were determined using commercially available kits, while platelet-neutrophil complex formation and neutrophil ROS status were determined using a modified flow cytometric method. Serum total cholesterol and low-density lipoprotein cholesterol levels were found to be increased and the high-density lipoprotein cholesterol level was found to be decreased in the HC group compared with the control group (P<0.001). Treatment of HC rats with t-resveratrol significantly lowered total cholesterol and low-density lipoprotein cholesterol levels (P<0.001). In the hypercholesterolemic group, levels of serum H2O2 platelet-neutrophil complex formation and neutrophil ROS status were significantly increased (P<0.001). On the other hand, in the resveratrol-administered hypercholesterolemic group, serum H2O2 levels, platelet-neutrophil complex formation and neutrophil ROS status were decreased compared with the hypercholesterolemic group (P<0.001). Serum H2O2 levels, platelet-neutrophil complex and neutrophil ROS status were positively correlated with one another. The present study is the first to demonstrate the protective effect of t-resveratrol against hypercholesterolemia-induced platelet-neutrophil complex formation and neutrophil ROS burst. Further investigations on its plausible role in antihypercholesterolemic treatment are warranted.
Under certain conditions, platelets can activate neutrophils via physical contact or via humoral agents. Conversely, activated neutrophils can activate platelets (1). After neutrophil activation in response to various stimuli, different oxygen radicals are produced, which can be assessed according to different methods (2). Cross-talk between neutrophils and platelets contributes to the pathophysiology of cardiovascular disorders such as unstable angina, myocardial infarction and thrombosis (3–5). In animals fed a high-cholesterol diet and in hypercholesterolemic patients, increased lymphocytes (lymphocytosis), neutrophils (neutrophilia) and thrombocytosis as well as increased platelet-neutrophil aggregate formation have been reported (6,7). Platelet-neutrophil aggregate formation and lipid peroxidation occur simultaneously in hypercholesterolemia, and these factors trigger atherothrombotic processes (7). Trans-resveratrol (t-resveratrol; 3,5,4′-trihydroxy-trans-stilbene) is a polyphenolic compound found in fresh grapes, grape juice and wine, and has been demonstrated to reduce the total cholesterol level in hypercholesterolemic rats (8). We have previously shown the effect of t-resveratrol on platelet activation in rats fed a high-cholesterol diet (9), but no information is available on the effects of t-resveratrol on platelet-neutrophil interactions in hypercho-lesterolemia. However, flow cytometry is progressively replacing these methods and has several advantages: it is rapid, sensitive and multiparametric, and allows cell subpopulations to be studied in environments resembling their physiological conditions. Thus, our objective was to assess the effects of t-resveratrol on platelet-neutrophil complex formation and neutrophil reactive oxygen species (ROS) status in control and hypercholesterolemic rats using a modified flow cytometric method.
T-resveratrol and cholesterol were commercial products obtained from Sigma-Aldrich (USA) and Merck (USA). T-resveratrol was dissolved in ethanol (50% v/v, Merck, USA) before administration.
The study protocol was approved by the Research Ethics Committee of Akdeniz University (Antalya, Turkey) and conducted in accordance with the Declaration of Helsinki.
Eighty rats (mean [± SD] body weight 230±20 g; mean age three months) were divided into five groups (16 animals per group): control (C); ethanol (E); t-resveratrol (R); hypercholesterolemic (HC); and t-resveratrol-administered hypercholesterolemic (HCR) groups. Rats in the C, E and R groups were fed with pelleted standard commercial basal rat diet, while the HC and HCR groups received the same basal diet plus 5% pure cholesterol, prepared as previously described (9). Each group underwent a different treatment protocol for the final 20 days of the study: C group were fed the standard diet for the final 20 days; 0.1 mL of ethanol (50% v/v) was administered intraperitoneally to the E group; 0.1 mL of t-resveratrol (20 mg/kg/day) was administered intraperitoneally to the R group; the HC group were fed the standard diet plus 5% cholesterol; and the HCR group was administered 0.1 mL t-resveratrol (20 mg/kg/day) intraperitoneally. The diets were prepared fresh every week and all animals were given tap water ad libitum. Food and water consumption and body weight were measured daily. Hypercholesterolemia was induced and evaluated as previously described (9).
After the experimental period, the rats were fasted overnight and were euthanized in the morning under ether anesthesia following withdrawal of 8 mL to 10 mL blood by cardiac puncture. Serum and plasma samples were separated immediately using low-speed centrifugation (4000 rpm for 10 min at 4°C) and all analytical variables were tested in each rat. Serum H2O2 levels were determined immediately in fresh samples using a commercially available ELISA kit. Serum lipid profiles (serum total cholesterol [TC], low-density lipoprotein-cholesterol [LDL-C] and high-density lipoprotein-cholesterol [HDL-C]) were determined spectrophotometrically using enzymatic assay methods following the protocol outlined in the kit.
Flow cytometric analysis was performed as previously described, with minor modifications (5). Briefly, erythrocytes were removed from blood samples (100 μL) using a lysis solution, then samples were incubated for 20 min at 25°C in the dark with 10 μL allophycocyanin-conjugated anti-CD45 (Becton Dickinson, USA) and 10 μL phycoerythrin-conjugated anti-CD41 (MCA2245-PE, Serotec Ltd, United Kingdom). After incubation, samples were centrifuged two times for 7 min in 400 g, and washed with phosphate-buffered saline. The percentage of positive cells was evaluated using fluorescence intensity, which was analyzed on a Becton-Dickinson FACS-SCAN Cytometer at 488 nm.
Cells were preincubated for 20 min at 25°C in the dark with allophycocyanin-conjugated anti-CD45, further incubated in 5 μL 2,7-dichloro-dihydrofluorescein diacetate (DCF-DA, Sigma, USA) and 50 μL HEPES (AI0511, AMRESCO LLC, Solon, USA) buffer in the dark for 30 min. After activation, cells were fixed in 500 μL formyl saline and the fluorescence was recorded at 488 nm excitation using a FACS-SCAN Cytometer and CellQuest Program (Becton-Dickinson, USA). ROS production was quantified using mean DCF-DA fluorescence intensities.
Results are shown as the mean value ± SEM. All data were analyzed using ANOVA with Tukey’s test as a post hoc test. P<0.05 was considered to be statistically significant. Pearson correlation coefficients were used to determine linear relationships among the studied variables. All calculations were performed using SPSS version 15.0.0 (IBM Corporation, USA).
Baseline characteristics and serum lipid levels in control and treatment groups are presented in Table 1. Because animals were randomly divided into five groups, their initial body weights were not significantly different. After 80 days, rats fed the 5% cholesterol diet showed an increase of 53.45±5.78% in body weight compared with their initial body weights, while the gain in body weight was 40.31±4.20% in the control group. The difference between HC and C groups were statistically significant (P<0.001). On the other hand, the HCR group had an increase in body weight of 44.63±4.02% and this increase was significantly lower than the HC group (P<0.001).
Serum cholesterol levels were more than twofold higher in cholesterol-fed rats compared with rats fed the standard diet for 80 days. Total cholesterol and LDL-C levels were found to be increased and HDL-C level was found to be decreased in the HC group compared with controls. Treatment of HC rats with t-resveratrol significantly lowered TC and LDL-C levels.
Platelet-neutrophil complex formation was expressed as percentage and neutrophil ROS status as mean fluorescence intensity (Figures 1 and and2,2, Table 2) and assayed by flow cytometry. The percentage of neutrophils forming complexes with platelets and neutrophil ROS status in the control rats was considered to be the basal level. While ethanol administration increased serum H2O2 levels, platelet-neutrophil complex formation and neutrophil ROS status (P<0.001) compared with the C group, resveratrol administration significantly decreased the serum H2O2 levels, platelet-neutrophil complex formation and neutrophil ROS status compared with the E group (P<0.001). In the HC group, levels of serum H2O2, platelet-neutrophil complex formation and neutrophil ROS status was significantly increased (P<0.001) (Table 2, Figures 1 and and2).2). On the other hand, in the HCR group, serum H2O2 levels, platelet-neutrophil complex formation and neutrophil ROS status was found to be decreased compared with the HC group (P<0.001).
Serum TC levels were positively correlated with serum H2O2 levels (r=0.709; P<0.001), platelet-neutrophil complex formation (r=0.856; P<0.001) and neutrophil ROS status (r=0.891; P<0.001). Serum LDL-C levels were positively correlated with serum H2O2 levels (r=0.629; P<0.001), platelet-neutrophil complex formation (r=0.825; P<0.001) and neutrophil ROS status (r=0.559; P<0.001). Serum HDL-C levels were negatively correlated with serum H2O2 levels (r=−0.357; P<0.001), platelet-neutrophil complex formation (r=−0.353; P<0.001) and neutrophil ROS status (r=−0.308; P<0.005). Serum H2O2 levels were positively correlated with platelet-neutrophil complex formation (r=0.827; P<0.001) and neutrophil ROS status (r=0.804; P<0.001). Platelet-neutrophil complex formation was positively correlated with neutrophil ROS status (r=0.970; P<0.001).É
Cholesterol feeding has often been used to elevate serum or tissue cholesterol levels to assess hypercholesterolemia-related metabolic disturbances in different animal models (10). We have previously shown a gradual increase in serum TC levels with cholesterol-enriched diet (5%) for 80 days in Wistar rats (9). In this study, we also used the same hypercholesterolemic rat model with or without resveratrol treatment to test the interaction between platelets and neutrophils. However, in the present study, compared with the control group, we used ethanol (50% v/v) to dissolve t-resveratrol, and this vehicle mimicked mild hypercholesterolemia in this study group. Ethanol administration significantly increased TC and LDL-C; however, there was no weight gain or altered food and water consumption compared with the control group. In rats receiving 20 mg/kg t-resveratrol, TC increase induced by ethanol administration was reversed, but LDL-C levels remained elevated. It has been demonstrated that chronic alcohol exposure for four weeks resulted in disturbed cholesterol homeostasis in rats (11). Our results are partly in agreement with these findings because the study does not mention the serum or plasma LDL-C levels, although they mention decreased LDL receptor levels in the liver. The discrepancy between these two studies may be attributed to different methodologies, exposure periods or doses. The possible underlying mechanism by which ethanol exerts total cholesterol-increasing effect may be elucidated in future studies.
Despite changes in lifestyle and the use of new pharmacological approaches to lower plasma cholesterol concentrations, cardiovascular disease continues to be the principal cause of death in the United States, Europe and much of Asia (12). In the current study, rats fed a high-cholesterol diet consumed considerably more food and water than the control rats throughout the experiment. Our results support the concept that hypercholesterolemia may lead to obesity because it facilitates the development of a positive energy balance leading to an increase in visceral fat deposition, which leads to abdominal obesity in particular (13). Interestingly, intraperitoneal administration of t-resveratrol to HC rats significantly reversed the hypercholesterolemia-induced weight gain and increases in TC and LDL-C levels without affecting the HDL-C level, appetite and thirst of study animals. Although this finding was not the main goal of our study, the effect of high dietary cholesterol consumption and resveratrol treatment may be of particular interest in obesity models. Ren et al (14) reported that the TC content in C57BL/6J mice can be decreased by t-resveratrol (22.5 mg/kg body weight) but we have not been able to find a diet-induced HC animal or human study that has assessed the effect of t-resveratrol on body weight and serum lipid content.
Thrombin-activated platelets adhere to monocytes and neutrophils through P-selectin (15). Elevated levels of platelet-leukocyte interactions, especially platelet-monocyte and platelet-neutrophil aggregation, have been demonstrated in patients with vascular diseases (16). In the present study, the platelet-neutrophil complex formation increased significantly in the HC group compared with the control group. Our study also demonstrated that platelet-neutrophil complex formation was positively correlated with neutrophil ROS status. This association may demonstrate that an increase in neutrophil-platelet aggregate formation could be an important mechanism by which hypercholesterolemia contributes to neutrophil burst or vice versa. In the literature, there are confounding data about the state of neutrophil burst in hypercholesterolemia. Kanashiro et al (17) have reported that polymorphonuclear leukocytes of Golden Syrian hamsters fed a high-fat diet showed no significant changes in ROS generation when compared with animals fed a normal diet. Li et al (18) reported an increase in neutrophil oxidative burst in hypercholesterolemia following CD40 ligand stimulation. We have previously reported increased platelet CD40 ligand expressions in hypercholesterolemia (9). The increased neutrophil ROS production in HC animals may, therefore, be attributed to increased platelet surface receptors as well as increased platelet-neutrophil complex formation.
The platelet-neutrophil complex formation decreases after t-resveratrol treatment in HC rats. Although we are not aware of research evaluating the degree of platelet-neutrophil interactions in HC patients and animals or the effect of t-resveratrol on platelet-neutrophil interactions in hypercholesterolemia, our results corroborate an in vitro study by Kaneider et al (19), who have investigated the possible role of resveratrol in neutrophil-platelet interactions and found that resveratrol-treated platelets decrease the oxidative burst of neutrophils. This effect was significantly diminished when platelets were exposed to t-resveratrol at concentrations as low as 100 pmol/L (19). The data presented here support the previously accumulated data that t-resveratrol decreases hypercholesterolemia-induced platelet activation (9) and the contribution of platelet-neutrophil complex formation to neutrophil burst. The beneficial role of t-resveratrol on platelet-neutrophil complex formation and neutrophil ROS status is not dependent on hypercholesterolemia, as suggested by the significant difference between R and E groups.
The data presented here strongly suggest that t-resveratrol reverses the increased serum H2O2 levels caused by ethanol administration or hypercholesterolemia. We attribute this effect to diminished neutrophil ROS status and platelet-neutrophil complex formation. Although we are not aware of in vivo studies to compare our results with others that assess the effect of t-resveratrol on H2O2 levels, in vitro results are confounding; in a study by Long et al (20), t-resveratrol did not generate significant H2O2 levels in a cell culture medium. In vivo results from our study may reveal that serum H2O2 concentrations are altered as a result of a protective role of t-resveratrol against oxidative stress at the cellular level.
There are several limitations to the present study. First, animals were three months of age at the beginning of the study and we cannot evaluate the effect of aging on t-resveratrol functions or hypercholesterolemia. Thus, there is uncertainty in assessing the effect of t-resveratrol on platelet-neutrophil interactions in different age groups. Second, the effect of different t-resveratrol doses was not studied in vivo or in vitro. Therefore, we cannot discuss the effect of different t-resveratrol doses on platelet-neutrophil interactions or concomitant neutrophil burst. Finally, the expression of platelet activation markers was not studied in the same animal groups, and we have only been able to cite our previous work in a similar rat model.
In conclusion, this is the first study that demonstrates the protective effect of t-resveratrol against hypercholesterolemia-induced platelet-neutrophil complex formation and neutrophil ROS burst. These findings may be pointing out another mechanism to explain the cardiovascular protective role of this natural product found in fresh grapes, grape juice and wine. Further investigations to understand the main mechanisms of t-resveratrol on cell-to-cell interactions between platelets and neutrophils as well as its plausible place in treatment are warranted.
This study was supported by Akdeniz University Research Projects Unit. None of the authors has any potential conflict of interest regarding this work. The first author developed the hypothesis and design of the study. The corresponding author provided the research laboratory facilities and permissions. All other authors contributed to the acquisition, analysis and interpretation of the data and to the drafting of the article. The final version of the manuscript was reviewed by all authors.