Hyperlipidemia is recognized as one of the most critical risk factors in the development of atherosclerosis. High fat diets have been used to induce hypercholesterolemia and atherosclerosis in ApoE−/− mice. A major finding in this study is that the combination therapy with simvastatin and pinocembrin effectively reduces serum lipid, protects vascular endothelial cells from dietary cholesterol– induced dysfunction, and reduces atherosclerotic plaque progression in the ApoE−/− mice. The benefits of simvastatin alone or in combination with pinocembrin on plaque size are associated with amelioration of vascular endothelial cells function. The potency and effectiveness of the combination were not less than that of simvastatin alone, although the dose of simvastatin was small in the combination group. This study demonstrated for the first time that pinocembrin was able to cooperate with simvastatin in inhibiting the progression of atherosclerosis in the ApoE−/− mice and the combined effects are synergistic.
In recent years, propolis has been used extensively in food and beverages because it is thought to improve human health and to prevent diseases such as heart disease, diabetes and even cancer. Because of its broad spectrum of biological activities and uses in health food, there is a renewed interest in the active constituents and pharmacological mechanisms of propolis. Pinocembrin is one of the most abundant flavonoids in propolis and has been reported to have multiple actions such as anti-inflammatory, anti-oxidative and anti-apoptotic activities [18, 19]. Zhu et al.  reported that pinocembrin induced relaxation of rat aortic rings through an endothelium-dependent pathway, while our results suggested that the combination of simvastatin and pinocembrin inhibited atherosclerotic lesion, and the effect may be partially dependent on the protecting vascular endothelium and anti-oxidative properties of pinocembrin.
Clinical trials have demonstrated that statins treatment inhibit the progression of atherosclerosis and reduce the frequency of acute coronary events and stroke, attributed to the lowering of circulating lipid concentrations. Interestingly, statins treatment can not affect lipid levels in rodents, even with a high dose statin [14, 15]. The ApoE−/− mouse is a well-established genetic mouse model of atherogenic hypercholesterolemia, even with normal diet which spontaneously develops atherosclerosis with similar features to those observed in human hyperlipoproteinemia, and high fat diet can accelerate atherosclerosis lesion formation . Unlike other studies that employed a much higher dose of simvastatin (100 mg/kg/day, or 300 mg/kg/day) [14, 15], the simvastatin dose (10 mg/kg/day) in the present study was substantially lower than many reported in the literature. Consistent with previous reports [13–15] that this dose did not significantly decrease lipid levels since the animals were fed a high fat diet, which greatly increased plasma cholesterol levels. However, the smaller dose of simvastatin (5 mg/kg/day) combinating with pinocembrin lowered blood lipid levels in apoE−/− mice. There was no significant difference in lipid-lowering effect between combination therapy and the single pinocembrin, we thus speculated that this effect would be mainly mediated by pinocembrin in the combination therapy.
Dyslipidemia plays an important role in the vascular endothelial cells dysfunction. Endothelial function is impaired in experimental model of atherosclerosis, including apoE−/− mice . Impaired endothelium-dependent vasodilation is considered as the hallmark of endothelial dysfunction, which precedes the development of atherosclerosis. When the balance between vasoconstriction and vasodilation is upset, endothelial dysfunction occurs and subsequently initiates a number of processes that promote or exacerbate atherosclerosis. The maintenance of vascular homeostasis is accomplished by the release of numerous dilator and constrictor substances. NO mediates endothelium-dependent vasodilation by opposing the effects of endothelium-derived vasoconstrictors such as ET-1. A defect in NO production or activity has been proposed as a major mechanism of endothelial dysfunction and a contributor factor in the progression of atherosclerotic lesions. Previous studies  reported that simvastatin preserves endothelial function in experimental hypercholesterolemia in association with an increase in eNOS levels independent of cholesterol-lowering activity. Statins also prevent the enhanced vasoconstrictor response to ET-1 of aortas in ApoE−/− mice . Our results showed that the combination of simvastatin and pinocembrin increased the level of serum NO, reduced the levels of serum ET, and reversed unfavorable imbalance between NO and ET.
Hypercholesterolemia also results in oxidative stress and generates excessive reactive oxygen species (ROS) such as superoxide anions (·O2-). Under physiological conditions, ROS can modulate cell proliferation, apoptosis and gene expression through the activation of transcription factors, and excessive ROS can be rapidly scavenged by endogenous antioxidant defenses such as SOD . But under pathological conditions, ·O2- reacts with NO to form ·ONOO-, resulting in decreasing NO bioavailability [16, 23]. In this study, we found that the combination of simvastatin and pinocembrin stimulated endothelial NO production compared to vehicle-treated group. We supposed that the restoring endothelium function mainly be dependent on antioxidant property of the combination, therefore MDA content and SOD activity in serum were determined. Notably, MDA content decreased and SOD activity increased in the combination therapy. Of course, the mechanisms by which the combination therapy exerts these effects on the vascular endothelial cells remain to be further evaluated.
Low NO bioavailability induces VEGF expression, which not only promotes normal and pathological angiogenesis but also plays an important role in atherosclerotic lesions. Increased VEGF levels have been reported in plasma from hypercholesterolemic individuals , in coronary arteries from hypercholesterolemic pigs  and in the vascular wall of ApoE−/−mice . Compared to normal vascular endothelium, in the area of high VEGF expression, endothelial cell adhesion molecule expression is increased, vascular permeability is enhanced, and mononuclear cell and lipid infiltration to the subendothelial area is elevated . In this study, we showed that increases in plaque area were accompanied by increased VEGF expression in ApoE−/− mice. Simvastatin could reduce the expression of VEGF which was not dependent on the hypolipidemic effect . Simvastatin combinating with pinocembrin not only reduced the plaque areas but also restrained the expression of VEGF in the ApoE−/− mice. The striking benefit achieved with the combination treatment in the ApoE−/− mice cannot be attributed to simvastatin effect alone, while the smaller dose of simvastatin was administered.