Cardiovascular disease (CVD) is the largest cause of premature death in most developed and developing countries and it is also an increasingly important source of disability and contributes in large part to the escalating costs of health care. Atherosclerosis is the most common pathologic process underlying CVD. It is clear that several risk factors such as oxidant stress [¹], lipid abnormalities [²] as well as chronic inflammation [³] have been correlated to both the initiation and the progression of atherosclerosis and subsequent CVD.

Rapeseed oil is one of the major and cheapest vegetable oils for human diet in china and many other countries. This kind of plant oil has the lowest concentration of saturated fatty acids in all commonly consumed oils and high level of monounsaturated fatty acids [⁴]. In addition, it is also the major source of linoleic acid and α-linolenic acid, and it is, so far, the closest to the optimum to meet the basic requirements of essential fatty acids in the body [⁵]. A lot of studies have shown that rapeseed oil can reduce serum total cholesterol (TC) and/or low-density lipoprotein cholesterol (LDL-C) when fed in place of saturated fatty acids [^6-8], which means that rapeseed oil possesses significant health benefits in reducing atherosclerosis risk. In addition to triacylglycerols, rapeseed oil also contains many micronutrients such as tocopherols, phytosterols and phenolic compounds which have been reported to impart health benefits or desirable physiological effects. For example, these micronutrients are the most important natural antioxidants which act as free radical scavengers or complexers of prooxidant metals, and the various bioavailable antioxidants present in rapeseed oil work in concert to upgrade the complex antioxidant network which increase antioxidant capacity higher than that provided by each separate compound [^9-11]. Phytosterols have been reported to exert hypocholesterolemic effect by inhibiting cholesterol absorption [¹²]. Studies also point out an independent effect of phenolics improving plasma lipid profiles [¹³,¹⁴]. In addition, all these compounds are also believed to have antiinflammatory effects [^14-16]. All these beneficial effects of these micronutrients might participate in preventing the initiation and development of atherosclerosis. However, although most unwanted components are removed, the traditional processing technology currently used in the world (extraction and refining) markedly decreases the content of these micronutrients, which will have an adverse effect on the protection of cardiovascular system.

In order to increase the levels of these micronutrients in rapeseed oil, some improving processing technologies [¹⁷] which can preserve the content of these micronutrients in rapeseed oil have been researched, and artificially adding micronutrients to refined oil may be another simple and expedient method. The present study tried to investigate that whether rapeseed oil fortified with micronutrients (polyphenols, tocopherols and phytosterols) can reduce atherosclerosis risk factors in rats fed a high-fat diet.

As one of the commonly consumed vegetable oils in the world, rapeseed oil has the potential to meet consumers' dietary needs because it has the lowest concentration of saturated fatty acids, a very favorable ratio of linoleic acid to α-linolenic acid and the predominantly of monounsaturated fatty acids. The optimum fatty acid composition of this oil can exert a hypo chotesterolsemic effect [^6-8,²³,²⁴] when fed in place of higher saturated fatty acids-containing fats and thus benefits the protection of CVD.

In recent years, an increasing interesting has been taken in the study of many micronutrients contained in rapeseed because of their excellent bioactivity. These micronutrients include phytosterols and various antioxidants such as polyphenols, coenzyme Q and tocopherols, and the joint actions of all these bioactive molecules may contribute to prevent the risk of atherosclerosis. Unfortunately, most of these micronutrients are lost during the oilseed oil refining. The present study examined the effect of reducing atherosclerosis risk factors by artificially adding micronutrients (polyphenols, tocopherols and phytosterols) to refined rapeseed oil.

The imbalance between the cellular free radical formation and the antioxidant defense leads to oxidative stress. The relative excessive production of free radicals can attack many different cellular components, including lipids, proteins and DNA, which initiates the processes of atherogenesis through cell dysfunction [²⁵]. In fact, oxidative stress is the unifying mechanism for many CVD risk factors [²⁶]. For example, free radicals mediate many signaling pathways which underlie vascular inflammation in atherogenesis [²⁷]. However, the deleterious effects of oxidative stress can be prevented by enzymatic and non-enzymatic antioxidant defense mechanism. In mammals, the most important antioxidant enzymes include SOD which converts superoxide to hydrogen peroxide, GPx and CAT which are responsible for converting hydrogen peroxide to water [²⁸]. As a very important non-enzymatic antioxidant, GSH can react directly with free radical or act an electron donor in the reduction of peroxides catalyzed by GPx [²⁹]. In addition to scavenge free radicals directly, supplement of these micronutrients in the present study also significantly elevated plasma activities of SOD, CAT and GPx as well as level of GSH, which led to the remarkable increase of T-AOC and were thus favorable to attenuate oxidative stress. As a result, plasma lipid peroxidation levels were significantly decreased with the supplement of these micronutrients. In accord with these findings, these micronutrients naturally abundant in rapeseed oil have been reported to increase antioxidant status and lipid peroxidation in plasma [¹⁷] as well as in brain [³⁰].

Hyperlipemia is also well known to be closely linked to arteriosclerosis and other cardiovascular disease. It has been reported that rapeseed oil possessed hypolipidemic properties [⁷,³¹] and which could be due to its optimum fatty acid composition. In line with expectations, the further reduction in plasma TG, TC and LDL-C were observed with the fortification of rapeseed oil with micronutrients in this study. The micronutrients phytosterols and polyphenols were both responsible for these beneficial changes. Many studies have shown that intake of phytosterols is effective at lowering plasma TC and LDL-C. Phytosterols have a similar chemical structure with cholesterol but themselves are absorbed only in trace amounts [³²], thus they inhibit cholesterol absorption including recirculating endogenous biliary cholesterol which is a key step in cholesterol elimination [³²]. These meant that although rodent diet contained little cholesterol in this study, inhibition of intestinal cholesterol absorption was still the main mechanism responsible for the cholesterol-lowering effect of phytosterols. Besides, the hypolipidaemic effect of the phytosterols were also associated with the down-regulation of hepatic acyl CoA:cholesterol acytransferase activity [²⁴] and the increasing LDL receptor expression [³³]. Polyphenols have been shown to reduce plasma TG, TC and LDL-C by altering hepatic triglyceride assembly and secretion, cholesterol absorption and the processing of lipoproteins in plasma [¹⁴]. Although these micronutrients did not affect plasma HDL-C level in the present study, the increase in the ratio of HDL to LDL cholesterol still meant that FRRO had a significant protective effect with regard to atherosclerosis. It should be pointed out that although rat was used as a model for lipid metabolism in our investigation and others [¹⁷,³⁴], the plasma lipid and lipoprotein profile in this species are very different from human, and therefore further experimentation is needed to verify the beneficial effects on lipid metabolism of human subjects.

Recent advances in both the basic and clinical science have recognized the critical role of inflammation in all stages of atherosclerosis [^35-37]. Various proinflammatory risk factors (oxidized LDL, infectious agents, etc) can trigger the production of proinflammatory cytokines which contribute to development and progression of atherosclerosis. IL-6 and CRP are two of the most important proinflammatory cytokines, and both of which have been served as inflammatory markers for evaluation of atherosclerotic risk [^38-40]. In parallel with the enhancement of these micronutrients in the present study, plasma levels of both IL-6 and CRP declined remarkably, which meant that FRRO was able to improve inflammation status. Each ingredient of these micronutrients has been suggested to play a role in inhibition of inflammatory cytokine production [^14-16]. For example, both polyphenols and tocopherols have been shown to retard LDL oxidation through their antioxidant property [¹⁵,⁴¹] and inhibit the synthesis of leukotriene B₄ (LTB₄) [⁴²,⁴³]. Polyphenols can also exert the anti-inflammatory effect by inactivating STAT1 and NF-κB [⁴⁴].

In conclusion, rapeseed oil fortified with tocopherols, polyphenols and phytosterols has the ability to ameliorate oxidative stress, lipid profile and inflammation of plasma. These results suggested that the rapeseed oil fortified with these micronutrients might contribute to prevent atherogenesis and then reduce the incidence of CVD.

CVD: cardiovascular disease; TG: total triglyceride; TC: total cholesterol; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; RRO: refined rapeseed oil; FRRO: fortified refined rapeseed oil; GPx: glutathione peroxidase; CAT: catalase; SOD: superoxide dismutase; GSH: glutathione; T-AOC: total antioxidant capability; TBARS: thiobarbituric acid reactive substances; IL-6: interleukin 6; CRP: C-reactive protein

The authors declare that they have no competing interests.

JX designed and wrote a first draft of the paper. XZ,QD and JY carried out all the experiments. QH performed the data analysis and created the figures. FH contributed to the design of the study, reviewed the manuscript and contributed to the final version. All authors contributed to and have approved the final manuscript.

This work was supported by the earmarked fund for Modern Agro-industry Technology Research System, china.