シロスタゾール（２） ２０１３

Probucol and cilostazol exert a combinatorial anti-atherogenic effect in cholesterol-fed rabbits

 

 

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Abstract

Introduction

Probucol (PB) and cilostazol (CZ) both exhibit anti-atherogenic effects. However, their combinatorial effects are unclear. This study was designed to investigate their combinatorial anti-atherogenic effect in cholesterol-fed rabbits.

Materials and Methods

Rabbits were fed a cholesterol diet with PB or CZ alone or both PB and CZ for 16 weeks. The plasma levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, C-reactive protein, superoxide dismutase, malondialdehyde, and nitric oxide (NO) were measured. The aortic atherosclerotic lesions were grossly and microscopically evaluated. Additionally, in vitro experiments were conducted using human umbilical vein endothelial cells.

Results and Conclusion

We found that the PB group and the PB + CZ group exhibited a reduction in the lesion areas (70% in the PB + CZ group, 56% in the PB group) compared with the vehicle group. However, although PB alone and PB + CZ led to a reduction in the lesion size, the histological analysis revealed that only PB + CZ significantly decreased the macrophage accumulation and smooth muscle cell proliferation in the lesions compared with the vehicle group. The plasma levels of total cholesterol in the PB + CZ group were decreased compared with the vehicle group, Moreover, PB + CZ exerted obvious anti-oxidant and anti-inflammatory effects. Interestingly, the PB + CZ treatment led to a marked increase in the levels of plasma NO. The in vitro experiments showed that the combinatorial treatment up-regulated the levels of NO and protein S-nitrosylation in endothelial cells treated with oxidized LDL. In summary, these results suggest that PB and CZ exert combinatorial anti-atherogenic effects.

Abbreviations

• CRP, C-reactive protein;
• CZ, cilostazol;
• eNOS, endothelial nitric oxide synthase;
• HCD, high cholesterol diet;
• HDL, high-density lipoproteins;
• HDL-C, high-density lipoprotein cholesterol;
• HUVECs, human umbilical vein endothelial cells;
• LDL-C, low-density lipoprotein cholesterol;
• MDA, malondialdehyde;
• MФ, macrophages;
• NO, nitric oxide;
• PB, probucol;
• ROS, reactive oxygen species;
• SMC, smooth muscle cell;
• SOD, superoxide dismutase;
• TC, total cholesterol

Keywords

• Probucol;
• Cilostazol;
• Atherosclerosis;
• S-nitrosylation;
• Rabbits

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Introduction

Atherosclerosis is a major cause of mortality in both developed and developing countries [1], and hypercholesterolemia is an important risk factor for the development of atherosclerosis. The extensive use of lipid-lowering agents, such as statins, has led to a marked reduction in the number of cardiovascular events in recent years. Despite this achievement, many patients with cardiovascular disease cannot be effectively treated with the use of statins alone [2]. Therefore, it is necessary to develop new therapeutics for these patients.

 

 

Probucol (PB) not only is a lipid-lowering agent but also possesses strong antioxidant properties against low-density lipoproteins (LDL). To date, many studies have shown that PB exhibits anti-atherogenic effects [3]. It has been shown that PB exerts its anti-atherogenic effects through diverse molecular mechanisms, including anti-inflammatory effects, the inhibition of smooth muscle cell (SMC) proliferation, the enhancement of the expression of scavenger receptor class B type I, and the improvement of the functions of high-density lipoproteins (HDL) to enhance reverse cholesterol transport [4], [5], [6] and [7].

 

 

Cilostazol (CZ) is a specific phosphodiesterase type III inhibitor that is currently used for the treatment of thrombotic vascular disease due to its anti-platelet aggregation functions [8]. As an anti-thrombotic drug, CZ is a preferred alternative to aspirin because CZ has fewer side effects, such as increased bleeding time [9]. Previous studies have shown that CZ also exhibits anti-atherogenic effects [10]. In vitro studies have revealed that CZ suppresses the production of intracellular reactive oxygen species (ROS) [11], and increases the production of nitric oxide (NO) [12]. Furthermore, CZ promotes reverse cholesterol transport and inhibits the inflammation and proliferation of SMCs [13], [14], [15] and [16].

 

 

Because both PB and CZ have anti-atherogenic effects, we hypothesized that the combination of minimal doses of PB and CZ may exert additional beneficial effects and may result in a greater athero-protective response. If so, this combinatorial therapy may provide a novel strategy for the treatment and prevention of atherosclerosis for those patients who cannot be effectively treated by statins.

 

 

In the current study, we compared the anti-atherogenic effects of the combination of PB and CZ in cholesterol-fed rabbits with those obtained with either PB or CZ alone. Our results showed that the combination of PB and CZ clearly reduced the aortic atherosclerotic lesion area, and markedly inhibited macrophage (MФ) accumulation and SMC proliferation in the lesions compared with the vehicle group.

 

 

The anti-atherogenic effects of PB and CZ may be collectively mediated by multiple mechanisms, including anti-oxidant effects, anti-inflammatory effects, a decrease in the plasma lipid levels, an increase in the levels of NO, and the maintenance of the endothelial protein S-nitrosylation.