Mustard Seeds Prevent Atherosclerosis In Hypercholesterolemic Zebrafish

Background and AimsAtherosclerosis is considered a form of chronic inflammation, resulting from interactions between modified lipoproteins, monocyte-derived macrophages, neutrophils and normal cellular elements of the arterial wall [1,2]. This inflammatory process may ultimately lead to the development of complex lesions, or plaques, that protrude into the arterial lumen [2]. Among the many genetic and environmental risk factors that have been identified, elevated levels of serum cholesterol is probably unique in being sufficient to drive the development of atherosclerosis in humans and experimental animals even in the absence of other known risk factors. As the second most important factor, many lines of evidence suggest oxidative modifications of LDL and its apo-lipoprotein B (apo B)[3-5].Although recruitment of monocytes to the arterial wall, and their subsequent differentiation into macrophages, may initially serve as a protective function by removing cytotoxic and pro-inflammatory oxLDL particles as well as apoptotic cells, progressive accumulation of macrophages and their uptake of oxLDL ultimately leads to development of foam cells, dystrophic calcification and established atherosclerotic lesions. Multiple steps in the atherogenic process might theoretically be available for intervention, including steps that control levels and oxidation of LDL, monocyte recruitment, and reverse cholesterol transport [2,3].Mustard seeds have been reported to contain components with powerful antioxidant capability[12]. Therefore, we hypothesized that MS may work as an anti-atherogenic food-product that might act synergistically with cholesterol-lowering drugs.In the Bible, a source of many good advices on what to eat or not, mustard seeds are described as a symbol of confidence. In Japan, the country with the world’s longest average life expectancy, citizens used to consume plenty of MS and in China it is considered a spice as well as a medicine and is now included in the Pharmacopoeia of the People’s Republic of China. According to recent research, MS is not only an excellent natural anti-oxidant but also provides unique anti-aging properties, supporting the idea that redox stress contributes to aging.To provide a basis for the use of MS in clinical applications, we here took advantage of hypercholesterolemic zebrafish as an experimental model to research the preventive function of MS on atherosclerosis [14].The zebrafish (Danio rerio) is a powerful model organism for the study of vertebrate biology, being well suited to both developmental and genetic analyses [16]. Large-scale genetic screenings have identified hundreds of mutant phenotypes, many of which develop human-like disorders [13,23]. Zebrafish are small (100+fish per20-liter tank), reproduce rapidly (100’s+of embryos per adult female and week) and develop quickly (most organs are formed within24h). An important property of zebrafish is their larvae being transparent until about the30th day of age, which enables temporal observations of fluorescent probes in live animals [21].Low-density lipoprotein (LDL) oxidation is an important factor in the development of atherosclerosis. In an earlier study on mice, we showed that mustard seeds (MS) suppress oxLDL-induced macrophage respiratory burst, suggesting that MS either prevents oxLDL uptake or diminishes its oxidizing effect within the lysosomal compartment. Therefore, MS may be expected to prevent atherosclerosis. Here we describe a novel hypercholesterolemic zebrafish model thathas been developed for the study of atherogenesis. It utilizes optically transparent zebrafish larvae, fed a high cholesterol diet (HCD), to monitor the early processes of atheromatosis in live animals. Initially, feeding Tg (flil:eGFP)larvae diets with varying(2to8%)concentrations of cholesterol, the most reproducible results were achieved using3%cholesterol that, consequently, was used in all further experiments. Subsequently, feeding Tg (flil.eGFP) larvae diets (all with a fluorescent cholesterylester added) with normal chow; or HCD; HCD+2.5%MS; HCD+5%MS; HCD+Ezetimibe (Eze-being an inhibitor of cholesterol uptake).We observed significantly reduced lipid accumulation following MS intervention, although the TC and LDL levels did not decline. SOD activity, however, increased significantly in the MS groups compared to the HCD-only group, while formation of MDA-like peroxidative degradation products were significantly decreased in Zebrafish homogenates, suggesting that MS acts by its antioxidant capacity rather than by reducing cholesterol uptake. Feeding HCD to Tg (lyz: DsRed) and Tg (coroninala: eGFP), we found that MS prevents accumulation of neutrophils and macrophages in the vascular wall. Collectively, the results suggest that MS protects blood vessels from atherosclerosis in zebrafish.MATERIALS AND METHODS ZebrafishAll animals were handled according to the rules of the Ethical Committee of Southern Medical University. The transgenic Tg (flkl:cherry), Tg (lyz: DsRed) and Tg (coroninala: eGFP) were kind gifts from The Hong Kong University of Science and Technology, while the other animals were raised in the Department of Cell Biology, Southern Medical University, P.R. China.AB zebrafish is a wild type fish, while Tg (flil.eGFP) is a transgenic animal with GFP-1labeled transcription factor ERGB, which expresses only in endothelial cells. Tg (flkl:cherry) is transfected with cherry-protein-labeled vascular endothelial growth factor receptor2(VEGFR-2),which also expresses only in endothelial cells. Tg (lyz: DsRed) fish, is transfected with DsRed-labeled lysozyme-3-protein, which expresses in granulocytes only. Tg (coroninala:eGFP) is transfected with coronin, being an actin-binding protein that interacts with microtubules and is labeled with GFP.High fat dietA high-cholesterol diet (HCD) was prepared by dissolving cholesterol (BioSharp) in diethyl ether to achieve3%(w/w) cholesterol in the food after ether evaporation [14].Fresh mustard seeds (MS) were crushed, using a mechanical blender (Ultra-Turrax model T25, Labotechnik, Staufen, Germany), and added to HCD with3%cholesterol at2.5or5%. In a positive control group, fish tank water was fortified with Ezetimibe (Eze),an inhibitor of intestinal cholesterol absorption, during the HCD feeding period [14]. Zebrafish maintenanceZebrafish embryos were obtained by in vitro fertilization and natural spawning of adults maintained in tanks with aerated and purified water (28.5℃) on a14-h light,10-h dark light cycle. Zebrafish larvae were fed three times a day, starting at the5th day after fertilization. The study was reviewed and approved by the University of Southern Medical University Ethics Committee [5,6]. Image analysis Fluorescent images were documented and analyzed by an Olympus Fluoview confocal microscope (FV10-ASW1.7, Tokyo, Japan) using Viewer software. Mean fluorescent intensity (MIF) was recorded and analyzed. To allow life observation, Zebrafish larvae were anesthetized with0.02%tricaine and mounted in0.5%low melting agarose.Zebrafish homogenateAt the end of the experiments, groups of50zebrafish larvae were euthanized by prolonged exposure to tricaine and non-digested food was removed from the animals’ abdomens [19]. The fish bodies were pooled, homogenized in200μl ice-cold PBS and filtered through0.45μm syringe filter to remove debris. Lipid analysisZebrafish homogenates were assayed for total cholesterol (TC), low-density lipoprotein (LDL), triglycerides (TG) and high-density lipoprotein (HDL) using colorimetric diagnostic reagent kits (Nanjing Jiancheng Bioenginering, Nanjing city, P.R.China) according to the manufacturer’s instructions. LPO and SODZebrafish homogenates were assayed for lipid peroxides (LPO) and superoxide dismutase (SOD) using colorimetric diagnostic reagent kits (Nanjing Jiancheng Bioenginering, Nanjing city, P.R.China) according to the manufacturer’s instructions. LPO was compared to a MDA standard and expressed as mmol/ml. StatisticsData in graphs are presented as means±SE. Differences between experimental groups were evaluated by one-way ANOVA.P<0.05was considered statistically significant.*:p<0.05,**:p<0.01;***:p<0.001.RESULTS3%cholesterol diet induces vessel lipid deposition Flil:eGFP larvae were for10days fed normal chow diet or a high cholesterol diet (HCD)containing2-8%cholesterol. The food was always supplemented with10μg/g of a red fluorescent cholesteryl ester analog (cholesteryl BODIPY576/589Cll; Invitrogen), obtained by soaking the food in a diethyl ether solution of the fluorochrome. Images of caudal veins were documented by an Olympus Fluoview confocal microscope, equipped with an x40objective, using488and559nm laser excitation. Different degrees of red fluorescent lipid deposits in caudal veins were observed and found correlated to the cholesterol concentrations. The cholesterol concentration in the HCD that consistently induced lipid accumulation (and therefore used in the main part of the study) was found to be3%(Figurel). MS reduces lipid deposit areasWe tested the effect of mustard seed at2.5,5,7.5and10%in HCD containing3%cholesterol and compared the results to standard chow and HCD only. It was found that MS at2.5to7%had similar good effects, while10%showed toxic effects and killed some larvae. Figure2shows MS and Eze to have significant preventive effect on HCD-induced lipid accumulation in zebrafish caudal veins. MS and Eze has different effects on lipid levelsSince MS decreased lipid deposit in the zebrafish caudal veins, we then checked if it reduced lipid concentrations as well. Figure3shows that MS did not reduce total cholesterol (TC), cholesterol in LDL (C-LDL) or cholesterol in HDL (C-HDL), while it significantly decreased triglycerides (TG). This was in opposition to the effect of Eze that did not only depress TG, but also TC and C-LDL. MS decreases LPO and enhances SOD activityCompared to normal chow food,3%cholesterol significantly increased lipid peroxides (LPO) and reduced superoxide dismutase (SOD) activity. Eze and MS, in both2.5%and5%concentrations, similarly counteracted the HCD-effect on LPO (Figure4A).MS prevents neutrophil recruitingTo investigate the recruitment of neutrophils to vessels, we used hybrids, namely Tg(flil: eGFP)*Tg(lyz:DsRed)[24,25].Neutrophil migration to lipid deposit areas was documented by measuring granulocyte red fluorescence. Three percent cholesterol was found to induce neutrophil accumulation, while both MS and Eze prevented it (Figure5).MS inhibits macrophage recruitingNeutrophil recruiting might be an early event in athero-pathogenesis, although most vessel foam cells are derived from macrophages [24]. We utilized another transgenic zebrafish-Tg (coroninala:eGFP), which expresses monocyte/macrophage GFP. Figure6shows that3%cholesterol induces substantial monocyte/macrophage accumulation in lipid deposit areas, while MS and Eze both inhibit such recruitment[8,22,25-27]. To further investigate the behavior of macrophages following high cholesterol feeding, we used a fourth kind of transgenic zebra fish, a hybrid of Tg (flk1:cherry) and Tg (coroninala: eGFP), namely Tg(flkl:cherry)*Tg(coroninala: eGFP). In this kind of hybrid zebrafish, monocytes/macrophages express GFP, while endothelial cells express proteins showing cherry fluorescence emission when excited. Figure7A shows that in monocytes/macrophages adhered to blood vessel in animals fed3%cholesterol, both MS and Eze block lipid infiltration. Statistic analysis of monocyte/macrophage adhesion is illustrated in Figure7B.CONCLUSION1. Continuous feeding zebrafish for a period of15days, we can duplicate the zebrafish model for the early stage of human atherosclerosis. We will take advantage of this model to research what has changed in the prophase of the AS, and explore the early pathogenesis of the disease, we can also do drug screening for AS.2. The mechanisms of classical anti-atherosclerosis drug EZE is different from mustard seeds. Mustard seeds can not reduce the level of lipid, but it can lessen the concentration of LPO and enhance the activity of SOD, sequentially inhibit oxidation of LDL. So, MS has the role of anti-atherosclerosis.3. MS can inhibit macrophage and neutrophil recruiting. It can alleviate the degree of inflammation of early AS. It can also decrease the number of foam cell source from macrophage. So MS can inhibit the progression of atherosclerosis lesions.