Combined use of probucol and cilostazol with atorvastatin attenuates atherosclerosis in moderately hypercholesterolemic rabbits
© Wang et al. 2015
Received: 18 March 2015
Accepted: 21 July 2015
Published: 29 July 2015
Atherosclerotic cardiovascular disease is one of the major diseases that seriously impacts human health. Combined drug therapy may be efficacious in delaying the occurrence of cardiovascular events.
The current study was designed to investigate whether combined use of probucol (an anti-oxidant agent) with cilostazol (a platelet aggregation inhibitor) would increase the inhibitory effect of statins (a lipid-lowering agent) on atherosclerosis in moderately hypercholesterolemic rabbits.
Methods and Results
Thirty Japanese white rabbits were fed with a high cholesterol diet for 12 weeks, which was supplemented with either 0.005 % atorvastatin alone or 0.005 % atorvastatin plus 0.3 % probucol and 0.3 % cilostazol. Except for high-density lipoprotein cholesterol, no difference was found in plasma lipids among vehicle, statin, and the combined treatment group. However, atherosclerotic lesions were significantly reduced by statin treatment compared with vehicle. Moreover, we found that the anti-atherogenic effect of statin was further enhanced by the combined treatment, which was due to increased anti-inflammatory and anti-oxidant properties.
These data demonstrated that combined drug treatment exhibits potent athero-protective effects via pleiotropic functions, such as anti-inflammatory and anti-oxidative stress, which is independent of the lipid-lowering effect.
Atherosclerotic cardiovascular disease (ASCVD) is the foremost cause of disability and mortality in both developed and developing countries [1–3]. Atherosclerosis is a multifactorial disease and progresses slowly throughout the human life; therefore treatment of atherosclerosis requires many therapeutic strategies. Statins, hydroxylmethyl glutaryl coenzyme A reductase inhibitors, are widely used for treating hyperlipidemia [4–7]. Although statins are the first choice for treatment of atherosclerosis [8–11], there are still many patients who are not responsive to statins. Actually, statins intolerance is frequently encountered in clinical practice [12, 13]. Therefore, it may be practical to consider the combined use of statins with other drugs for those unresponsive patients. Previous studies have shown that atorvastatin combined with probucol exhibited a stronger anti-atherogenic effect than single drug treatment . Probucol, a diphenolic compound with anti-inflammatory and anti-oxidant properties, can reduce atherosclerosis and restenosis in coronary arteries [15, 16]. Cilostazol, an inhibitor of type 3 phosphodiesterase, is widely used for treating thrombotic vascular disease and exerts antiplatelet activity via suppression of cyclic adenosine monophosphate degradation [17, 18]. Our previous studies found that the combined use of probucol with cilostazol has a greater anti-atherogenic effect than single probucol treatment . Rabbits fed with a cholesterol diet are readily to develop atherosclerotic lesions, which mimics the lesions observed in ASCVD patients . However, whether combination of statins with probucol and cilostazol has an add-on effect on atherosclerosis in moderately hypercholesterolemic rabbits is still unknown.
In the current study, a rabbit model with moderately hypercholesterolemia was established by feeding a cholesterol diet. The effect of statins combined with probucol and cilostazol on atherosclerosis was investigated. We found that combined drug treatment significantly attenuated atherosclerosis through inhibiting anti-inflammatory and antioxidant properties independent of the lipid-lowering function.
Materials and methods
Animals and diets
Thirty Japanese white rabbits (male, 4-mon) were supplied by Vital River Laboratories, Beijing, China. The rabbits were randomly divided into three groups: vehicle group (n = 10); atorvastatin group (0.005 % atorvastatin, n = 10); and APC group (0.005 % atorvastatin + 0.3 % probucol + 0.3 % cilostazol, n = 10). All rabbits were fed a cholesterol diet containing 0.3 % cholesterol and 3 % soybean oil for 12 weeks.
The probucol and cilostazol were provided by Otsuka Pharmaceutical Co., Ltd. Tokushima, Japan. Atorvastatin calcium was purchased from Sequoia Research Products Ltd, Pangbourne, UK. The drugs were mixed with cholesterol diets and prepared by Ke’ao Xieli Diet Co., Ltd., Beijing, China. The drug concentrations in diet were measured using gas chromatography or high-performance liquid chromatographic methods as previously described [21, 22]. All rabbits were given a restricted diet (100 g/rabbit per day) and free access to water. The animal experiments were approved by the Laboratory Animal Administration Committee of Xi’an Jiaotong University and carried out according to the Guidelines for Animal Experimentation of Xi’an Jiaotong University and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85–23, revised 2011).
Determination of plasma lipid levels and other biochemical parameters
Blood samples were collected from the ear artery using an EDTA anticoagulant tube after 16 h fasting. Plasma was obtained after centrifuging at 3000 rpm for 20 min. The plasma triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were determined by commercial assay kits (Biosino Bio-technology and Science Inc., Beijing, China). Plasma TC and TG levels were measured biweekly, while plasma HDL-C and LDL-C levels were measured every 4 weeks. In order to compare whole plasma lipid levels in 12 weeks among three groups, the incremental area under the curve (AUC) was calculated according to the trapezium rule .
The C-reactive protein (CRP) levels were quantified using an ELISA kit (Immunology Consultants Laboratory, Inc., Newberg, OR, USA). The plasma levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by xanthine oxidase assay and thiobarbituric acid assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively, and oxidized LDL (ox-LDL) was measured by an ELISA kit (R&D Systems, Minneapolis, MN,USA).
Quantification of gross atherosclerotic lesions
At the end of the experiment, all rabbits were euthanized by intravenous injection of an overdose of sodium pentobarbital. Rabbit aortas were subsequently collected for analysis of the aortic lesions. Aortic en face atherosclerosis was evaluated after the aortic trees were stained with Sudan IV as previously described . Sudanophilic area was quantified using image analysis software (WinROOF Ver.6.5, Mitani Co., Ltd., Fukui, Japan) and expressed as a percentage of the aorta.
Histology and immunohistochemistry
For the microscopic quantification of lesions, the aortic arch of each rabbit was cut into 8 to 10 sections (4 μm) as previously described . To evaluate the microscopic lesion area of each aorta, all sections were stained with hematoxylin and eosin (HE), and measured by the image analysis system described above. For microscopic evaluation of cellular components in the lesions, serial paraffin sections of the aorta were immunohistochemically stained with the following antibodies (Abs) against macrophage (MФ) (RAM11, Dako, Carpinteria, CA, USA) and smooth muscle cells (SMC) (α-smooth muscle actin, Thermo Fisher Scientific Pierce, Rockford, IL, USA). Secondary Abs included anti-murine IgG (Beijing Zhongshan Biotechnology, Beijing, China) for MФ and SMC staining .
Lesion type and quantitation
We analyzed whether the combined drug treatment had any effect on the progression of atherosclerosis according to American Heart Association guidelines in which atherosclerotic lesions are divided into I-VI morphologically characteristic types [26, 27]. To quantify lesion types, the total length of each lesion in the aortic arch was calculated in three groups using a method as reported in our previous study .
The statistical analyses were carried out by one-way ANOVA followed by LSD test using the SPSS 13.0 software. In all cases, data were expressed as the mean ± SEM. P values less than 0.05 were considered statistically significant.
Plasma lipid levels
Drug concentrations in diet from each group
Gross lesion of aortic atherosclerosis
Lesion type analysis
Inflammatory and oxidation markers
Risk factors for atherosclerosis, such as dyslipidemia, inflammation, oxidative stress, abnormal levels of coagulant and central obesity often co-exist . The present study provides evidence that the combined drug therapy consisting of probucol, cilostazol and atorvastatin markedly enhances the statin anti-atherogenic effect independent of a lipid-lowering manner in moderately hypercholesterolemic rabbit model.
In this experiment, rabbit model had moderate hypercholesterolemia (plasma TC = 350–500 mg/dl) compared to our previous studies (rabbit plasma TC = 800–1200 mg/dl) [19, 24, 25, 30, 31]. We used this model to investigate the effect of combined drug treatment in the early stages of atherosclerosis and found that, except for HDL-C, plasma lipids were not affected after 12 weeks of drug treatment. This was possibly due to the “relatively low” hypercholesterolemia baseline in these rabbits. However, APC treated group had lower HDL-C levels, which was caused by the presence of probucol. In spite of this, HDL functions were not impaired but were actually more efficient for reverse cholesterol transport(RCT) [32–35]. Previous studies have revealed that cholesterol ester transfer protein (CETP) plays a crucial role in mediating HDL functions and probucol may enhance reverse cholesterol transport by increasing CETP expression. HDLs mediated by enhanced CETP activity showed potentially anti-atherogenic functions . In our previous study, we measured hepatic LDL receptor, SR-B1, ABCA1, CETP mRNA and protein expression levels and found that all drug treatment groups significantly increased hepatic LDL receptor mRNA expression by which hepatic uptake of LDLs would be enhanced (data not shown). Interestingly, we found combination treatment synergistically increased CETP mRNA and protein expression level in liver (data not shown). CETP as a plasma glycoprotein transfers CE from HDL to apoB-containing lipoproteins in exchange for triglycerides. Thus, directly through hepatic SR-B1 receptor uptake or indirectly through transfer of HDL-CE to ApoB-containing lipoproteins with subsequent receptor-mediated hepatic uptake, CETP could contribute significantly to the RCT pathway [36, 37].
Statins dramatically reduced cardiovascular events in patients with normal lipid levels or without established ASCVD, independently from lipid-lowering properties . Moreover, plasma cholesterol lowering does not necessarily lead to protection against cardiovascular disease. In the present study, the attenuation of atherosclerotic lesions in APC treated group was independent of lipid-lowering function. These findings may be consistent with the “Mevalonate hypothesis” proposed recently  and further studies are needed for verifying. The anti-atherosclerosis in APC treatment may be due to their multiple pharmacological properties, such as enhanced anti-inflammatory and anti-oxidant effects in APC treatment group. Elevations in inflammatory markers, such as CRP, prospectively define the risk of atherosclerotic complications [31, 40, 41]. In the present study, plasma levels of CRP and ox-LDL were notably reduced in APC treated group compared to vehicle. Furthermore, SOD levels were significantly increased, while MDA levels simultaneously decreased in the APC treated group compared to vehicle. Statins as well as cilostazol, also known as platelet-activating factor inhibitors, play an important role in the crosstalk of dyslipidemia, inflammation and atherogenesis [42–46]. In the moderate hypercholesterolemia rabbit model, we found that combination treatment (statins, cilostazol and probucol) decreases atherogenesis via pleiotropic effects, such as anti-inflammation, anti-oxidation and inhibition of platelet-activating factor.
Although it remains to be verified clinically whether combined APC treatment exhibits a “potent anti-atherogenic function”, it seems that APC more strongly attenuates the progression of atherosclerosis than statin alone in cholesterol-fed rabbits. These insights may provide us with a new concept with which to effectively delay the occurrence of cardiovascular events by APC combined drug treatment in the early stages for those patients with or without established ASCVD.
This work was partly supported by the National Natural Science Foundation of China (81200207, 81270348), by a Public Service Platform Grant of Shaanxi Province (2014FWPT-07), and by Otsuka Pharmaceutical Co., Ltd.
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