Effects of Black Soybean on Atherogenic Prevention in Hypercholesterolemic Rabbits and on Adhesion Molecular Expression in Cultured HAECs

Pi-Yu Chao^1*, Yuh-Lien Chen², Yi-Chu Lin³, Ju-Ing Hsu^1,4, Kuan-Hung Lin⁵, Yi-Fa Lu⁴, Tien-Joung Yiu⁶, Meng-Yuan Huang⁷, Chi-Ming Yang⁷

¹Department of Food, Health and Nutrition Science, Chinese Culture University, Taipei City.
²Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei City.
³Institute of Applied Life Science, Chinese Culture University, Taipei City.
⁴Department of Nutritional Science, Fu Jen Catholic University, Hsin-Chuang, Taipei City.
⁵Graduate Institute of Biotechnology, Chinese Culture University, Taipei City.
⁶Tainan District Agricultural Research and Extension Station-Potzu Branch Station, Council of Agriculture, Executive Yuan, Chiayi County.
⁷Research Center for Biodiversity, Academia Sinica, Taipei City.

DOI: 10.4236/fns.2013.48A002                

Abstract

The aim of the study was to investigate the effect of black soybean (BS) on the susceptibility of low-density lipoprotein (LDL) in hypercholesterolemic New Zealand white rabbits. Effects of the BS extract (BSE) and its components on monocyte adhesion of human aortic endothelial cells (HAECs), and adhesion molecule were investigated. Rabbits were divided into four groups, including control, 0.5% cholesterol with 20% casein (either with or without 0.5% vitamin E), and BS groups, all fed for 8 weeks. LDL was treated with 10 μM Cu²⁺ in vitro to determine the LDL lag time, and the vitamin E content of LDL was determined. The thickness of the tunica intima was measured on paraffin sections of thoracic aortas and aortic arches stained with Movat’s pentachrome. HAECs were pretreated with 100 μg/ml of BSE, and 10 μM of genistein, daidzein, cyanidin, and aspirin for 18 h, followed by tumor necrosis factor (TNF)-α (2 ng/ml) for 6 h, after which U937 cell adhesion was determined. Adhesion molecule expression was examined using ELISAs. The LDL lag time in the BS group was similar to that in the vitamin E group, while its lag time was significantly longer than those in the control and casein groups. The ratio of the intimal area/medial area of the aortic arch of the casein group was significantly higher than those in the control, BS, and vitamin E groups. The vitamin E group had the lowest value, and was closest to the control group. The BS group exhibited a significantly decreased atheroma region in the aortic arch compared to the casein group. Pre-incubation with BSE, genistein, daidzein, cyanidin, and aspirin significantly decreased adhesion by U937 monocytic cells to TNF-α stimulated HAECs. Genistein, daidzein, cyanidin, and aspirin significantly suppressed the expression of vascular cell adhesion molecule (VCAM)-1. Only genistein and aspirin significantly decreased intracellular adhesion molecule (ICAM)-1 expression compared to TNF-α treatment, while no treatments had any effect on E-selectin expression. BS significantly prolonged the LDL lag time and decreased the atheroma region of the aortic arch in hypercholesterolemic rabbits, thereby exerting an antiatherosclerotic effect. Presumably, the BSE downregulate intracellular redox-dependent signaling pathways in HAECs upon TNF-α stimulation through regulating NF-κB, thereby attenuating the inflammatory response in atherosclerosis. The antiatherogenic and anti-inflammatory effects of BS can be used as a nutraceutical for atherogenesis prevention.

Keywords

Black Soybean; Hypercholesterolemia; Human Aortic Endothelial Cells; Adhesion

TABLE I.    1. Introduction

TABLE II.                

The development of atherosclerosis is positively correlated with increasing levels of cholesterol and low-density lipoprotein (LDL) in blood, and oxidation of LDL in vessel walls is highly related to the development of atherosclerosis [1,2]. Human atherosclerotic plaque contains both oxidized lipids and relatively large amounts of α-tocopherol and ascorbate [3]. Dietary supplementation with

α-tocopherol increases the resistance of LDL subsequently isolated from the blood to oxidation in vitro [4,5]. High intake and elevated plasma levels of vitamin E are associated with low rates of ischemic heart disease in controlled intervention studies [6] and large-scale prospective studies [7,8]. Previous studies showed that the consumption of soy products may lower rates of cardiovascular disease, which was associated with the cholesterol-lowering action of soy and the antioxidant activity of soybean isoflavonoids [9-12]. Genistein and daidzein are the main isoflavone phytoestrogens found in soy products and a number of plants including soybeans, and have been shown to exert estrogen-like [13] and antioxidative actions [14]. High isoflavone aglycone attenuates atherosclerosis development in cholesterol-fed rabbits [15]. Esterified isoflavones can be incorporated into LDL particles, and some of them increase the in vitro oxidation resistance of LDL [16]. Esterified isoflavones contain several antioxidants, such as α-tocopherol, ubiquinol-10, lycopene, β-carotene, and lutein [17], and may be lost during the lag phase period. In addition to the antioxidant potential, genistein prevents endothelial cells from the cytotoxic effects of oxidized (ox)-LDL [11].

Atherosclerosis is a chronic inflammatory process characterized by increased oxidative stress [18]. The resulting adhesion of monocytes to the vascular endothelium and subsequent migration into the vessel wall are pivotal early events in atherogenesis [19,20]. The interaction between monocytes and vascular endothelial cells may be mediated by adhesion molecules, including vascular cell adhesion molecule (CAM; VCAM)-1, intercellular adhesion molecule (ICAM)-1, and E-selectin on the surface of the vascular endothelium [21]. The inflammatory cytokine, tumor necrosis factor (TNF)-α, activates nuclear factor (NF)-κB, and activator protein (AP)-1, which are the two major redox-sensitive eukaryotic transcription factors that regulate expressions of adhesion molecules [22- 24]. Because activation of NF-κB and AP-1 can be inhibited to various degrees by different antioxidants, endogenous reactive oxygen species (ROS) may play an important role in these redox-sensitive transcription pathways in atherogenesis [18,24]. For example, quercetin, the most abundant flavonoid in the human diet and an excellent free radical-scavenging antioxidant, attenuates expressions of ICAM-1 and E-selectin in human aortic endothelial cells (HAECs) [25]. Several in vitro and in vivo studies showed that the cardioprotective effects of soy isoflavones include improved serum lipid profiles [26,27] and vascular reactivity [28], protection against LDL oxidation [29], modulation of cytokines, and inhibition of platelet aggregation [28]. Soy isoflavones attenuate human monocyte adhesion to endothelial cell-specific protein ICAM-1 (or CD54) by inhibiting the monocyte, CD11a [30].

Black soybean (BS; Glycine max L. Merrilx), like the soybean, is a species in the genus of Glycine, but has a black testa (seed coat). BSs are abundant in natural antioxidants, such as isoflavones, saponins, anthocyanins, and vitamin E [31]. Delphinidin-3-0-β-D-glucoside from the seed coat of BS has strong antioxidant activity in an acidic environment [31]. The seed coats of 60 Chinese BS varieties contain high levels of cyanidin-3-glucoside and antioxidant activity of the oxygen radical absorbance capacity (ORAC) [32,33]. In addition to anthocyanins, the BS seed coat (BSSC) is also a good source of other phenolics, such as condensed tannins and phenolic acids [32, 34]. BSs abound in natural antioxidants, and their extracts prolong lag times of Cu-induced LDL oxidation, leading to reduced atheroma formation [35,36]. Furthermore, Kim et al. [37] demonstrated that anthocyanins from the BSSC inhibited TNF-α-induced ICAM-1 and cyclooxygenase (COX)-2 levels through an NF-κB-dependent pathway, and had anti-inflammatory effects on an immortalized epidermal keratinocyte cell line (HaCaT).

The majority of studies showed that soybeans inhibit LDL oxidation both in vitro and ex vivo, and reduce atherosclerosis in cholesterol-fed rabbits [12,15,38]. The effects of BS on atherosclerosis progression of hypercholesterolemic rabbits and monocyte-endothelial cell interactions have not yet been elucidated. The objectives of our study were to determinate the effects of BS on atherosclerosis progression and ox-LDL formation in rabbits fed a cholesterol-rich diet to elucidate the mechanism by which BS alleviates atherosclerosis, and provide evidence supporting the use of antioxidants of BS in preventing atherosclerosis. The antiinflammatory effects of BSE and its components, genistein, daidzein, and cyanidin, on TNF-α-induced cell adhesion, and adhesion molecule in an HAEC model were also investigated. The antiatherogenic and antiinflammatory effects of BS can be used as a nutraceutical for atherogenic prevention.

2. Materials and Methods

2.1. Chemicals

Medium 200, low-serum growth supplement (LSGS), fetal bovine serum (FBS), and RPMI-1640 were purchased from Gibco Invitrogen (Carlsbad, CA, USA). RayBio enzyme-linked immunosorbent assay (ELISA) kits were purchased from RayBiotech (Norcross, GA, USA). The 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluo-rescein acetoxymethyl ester (BCECF-AM) was obtained from Molecular Probes (Eugene, OR, USA). Other chemical reagents were purchased from Sigma (St. Louis, MO, USA).

2.2. Experimental Animal Treatment

Male New Zealand white rabbits were used in the study. The investigation conformed to the Guide for the Care of Laboratory Animals, published by the US National Institute of Health (NIH publication no. 85-23, revised 1996). All institutional and national guidelines for the care and use of laboratory animals were followed. The antiatherogenic effects of BS were evaluated using the following four rabbit groups: control, 0.5% cholesterol with 20% casein, 0.5% cholesterol with 20% casein and 0.5% vitamin E, and BS groups. Each rabbit was housed in a single cage, and fed 100 g daily for 8 weeks. In the week before the study began, animals were acclimatized and received a 100% standard rabbit diet. In the first and second weeks of the study period, animals received a 50% standard rabbit diet and a 50% semi-purified diet. In the third and fourth weeks, animals received a 25% standard rabbit diet and a 75% semi-purified diet. From the fifth study week to the end of the study period, animals received 100% semi-purified diets containing 0.5% cholesterol and individual proteins. Eight weeks after cholesterol feeding, rabbits were processed for further experiments.

2.3. Blood Samples and Serum Cholesterol, Triglyceride, and Anthocyanin Assays Serum cholesterol and triglyceride levels were monitored every 2 weeks throughout the study. Thirty milliliters of blood was drawn from the ear vein of each rabbit, and was collected in sterile microcapillary glass tubes containing 1.5 mg/ml EDTA. Twenty milliliters of blood was also drawn from the heart of each rabbit. Plasma was isolated for the cholesterol, triglyceride, and anthocyanin assays. Serum was isolated for LDL determination. The cholesterol and triglyceride assays were based on the respective procedures of Allain et al. [39] and Fossati and Prencipe [40]. Two milliliters of 100% methanol containing 1% HCl was used to extract anthocyanin from 0.5 ml of plasma by centrifugation (1500 g for 15 min at 4˚C) for 2 h. The supernatant was measured using absorbances at 657 (A₆₅₇) and 530 nm (A₅₃₀). Anthocyanin was calculated according to the formula: anthocyanin (units/ml of plasma) = (A₅₃₀ − A₆₅₇ × 0.33) × 2 ml × ml of plasma [41].

2.4. Vessel Samples

An aortic segment was removed from the ascending arch to the diaphragm. The abdominal aorta was then taken from the diaphragm to the iliac trifurcation. Tissue samples (aortic arch and thoracic aorta) were fixed by immersion in 10% formalin fixative overnight, followed by dehydration through a graded ethanol series. Samples were then embedded in paraffin, cut into 5-μm sections, and stained with Movat’s pentachrome stain [42]. The severity of atherosclerosis in the arch and thoracic aorta was determined morphometrically using an LV-2 computerized image analysis system.

2.5. LDL Isolation and Oxidative Susceptibility of the LDL Assay

Thirty milliliters of blood was centrifuged for 10 min at 3000 rpm and 4˚C. Fifteen milliliters of serum sample was collected for LDL isolation. Two steps of LDL fractionation (1.006 > d > 1.063 g/ml) were isolated by twostep sequential flotation ultracentrifugation [43]. A Hitachi CP85β ultracentrifuge (Tokyo, Japan) equipped with a P70AT₂-376 rotor at 127,980 g rpm was used for 16 h (d < 1.006 g/ml) to remove very LDL, and for 20 h (d < 1.063 g/ml) to collect LDL. The isolated LDL fraction from each study animal was separately dialyzed at 4˚C against saline buffer containing 0.15 M NaCl and phosphate (pH 7.4) for 22 h before determining the oxidative susceptibility of the LDL assay. Dialyzed LDL was then diluted with saline and incubated with 10 mM copper iron at a final concentration of 0.05 mg cholesterol/ml. The kinetics of LDL oxidation was determined by monitoring the change in absorbance at 234 nm and 25˚C with a spectrophotometer (Hitachi U-2000, Tokyo, Japan). The absorbance was recorded every 15 min for less than 5 h. Changes in absorbance at 234 nm against time were divided into three consecutive phases: lag, propagation, and decomposition [44].

2.6. Concentration of α-Tocopherol in LDL

The α-tocopherol content of LDL was determined by a high-performance liquid chromatographic (HPLC) system (Merck, Darmstadt, Germany) [45]. Briefly, 1 ml of α-tocopherol acetate (7.116 μg/ml EtOH with 1% BHT, as an internal standard) was added to 0.25 ml of LDL, followed by adding 50 μl of 12 N HCl to the samples, and extraction with 3 ml of n-hexane (0.25% BHT w/w) twice. The upper hexane layer was collected and dried with nitrogen. The residue was dissolved in 200 μl of methanol, followed by injection of 20 μl for HPLC. The extracts were analyzed by reversed-phase HPLC (Lichropher 100 RP-18, 5 μm, 125 × 4 mm I.D.) eluent with methanol-water (95:5, v/v) at a flow rate of 1.5 ml/min and a 292-nm wavelength.

2.7. BSE Preparation

BS seeds (Tainan no. 3 with a green cotyledon) were obtained from Dr. Tien-Joung Yiu, Tainan District Agricultural Research and Extension Station, Chiayi, Taiwan. Seeds were freeze-dried, ground, and stored at −80˚C until being analyzed. One gram of powdered seeds was sonicated for 20 min with 8 ml of 80% methanol containing 2 ml of 6 M HCl, followed by extraction for 24 h at 4˚C. After centrifugation (1500 g for 10 min at 4˚C), the supernatant was collected as crude extracts containing antioxidants, and the residue was extracted again. The collected supernatant was evaporated and dissolved in 20 ml of 80% methanol for the HPLC assay and HAEC treatment [12].

2.8. Concentrations of Isoflavones and Cyanidin in BSs Daidzein and genistein in the purified mixtures were individually determined by HPLC. One milliliter of the methanolic extract of the acid hydrolysate was filtered through a 0.45-μm filter prior to a 20-μl injection into a C18 reversed-phase column (Astec Silica-based, 110 Å, ODS, 25 cm × 4.6 mm, 5-µm particle size, Sigma-Aldrich, St. Louis, MO, USA), and eluted for 45 min using a ternary TSP Thermo Separation Products Pump (Agilent 1100 G1311A Quat Pump SpectraLab Scientific, Markham, Ontario, Canada). Solvents containing methanol-water (30%:70%; v/v) with 1% formic acid, 100% methanol, and aqueous 10% (v/v) acetic acid were used for the HPLC. The solvent gradient was allowed to equilibrate for 15 min (at a flow rate of 1 ml/min), and monitored at 528 nm using a Waters Photodiode Array Detector (Waters, Milford, MA, USA). Results are expressed as daidzein and genistein weight equivalents [46].

2.9. Cell Culture and Treatment

HAECs were grown in Medium 200 supplemented with 1% low-serum growth supplement and 10% FBS in an atmosphere of 95% air and 5% CO₂ at 37˚C in plastic flasks as described by Vielma et al. [47]. The U937 human monocytic cell line was grown in suspension culture in RPMI-1640 containing 10% FBS and 1% of an antibiotic-antimycotic mixture. After incubation with BSE, genistein, daidzein, cyanidin, aspirin, and TNF-α, cell viability was assessed using a 3-(4,5-dimethyl-thiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Mitochondrial dehydrogenase activity, by the reduction of MTT in active mitochondria to purple formazan, was used to determine cell survival in a colorimetric assay. Cell viability was calculated according to the formula: Cell viability = (absorbance sample tested/absorbance medium only) × 100%.

2.10. Cell Adhesion Assay

To explore the effects of BSE and its components, genistein, daidzein, cyanidin, and aspirin, on endothelial cell-monocyte interactions, the adherence of U937 cells to TNF-α-activated HAECs was examined under static conditions. HAECs were grown to confluence in 24-well plates. HAECs were then pretreated with 100 μg/ml of BSE, and 10 μM of genistein, daidzein, cyanidin, and aspirin for 18 h, followed by stimulation with TNF-α (2 ng/ml) for 6 h [48,49]. Adhesion assays of McCrohon et al. [50] were performed with minor modifications. Briefly, U937 cells were labeled with 10 μmol/l of the fluorescent dye, 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein acetoxymethyl ester at 37˚C for 1 h in RPMI-1640 medium, and subsequently washed by centrifugation. Confluent HAECs in 24-well plates were incubated with labeled U937 cells (10⁶ cells/ml) at 37˚C for 1 h. Nonadherent monocytes were removed, and plates were gently washed twice with PBS. Numbers of adherent monocytes were determined by counting four fields per 100- fold high-power-fields using fluorescence microscopy (Nikon, Tokyo, Japan) and photographed. Four randomly chosen high-power fields were counted per well. Experiments were performed in duplicate and repeated at least 3 times independently.

2.11. ELISA

The effects of BSE, genistein, daidzein, cyanidin, and aspirin on the HAEC surface expressions of VCAM-1, ICAM-1, and E-selectin were analyzed with an ELISA using RayBio ELISA kits. Briefly, HAECs cultured to 95% confluence in 24-well microplates were incubated for 18 h during a 6-h TNF-α activation period. Monolayers were washed 3 times with cool phosphate-buffered saline (PBS), and cells were lysed with 1 ml of Celytic reagent, vortexed, incubated on ice for 30 min, and centrifuged at 12,000 g for 30 min at 4˚C. Aliquots (100 μl) of the supernatant were frozen in liquid nitrogen, and stored at −70˚C until later use. The ICAM-1, VCAM-1, and E-selectin present in an aliquot were captured by an immobilized antibody after overnight incubation at 4˚C. Wells were washed 4 times with 0.1% Tween-20 in PBS, and 100 μl of 1-fold biotinylated primary antibody (specific for ICAM-1, VCAM-1, and E-selectin) was added for 1 h at room temperature. After washing, 100 μl of HRP-conjugated streptavidin was added to the wells for 45 min. After washing, 100 μl of a 3,3’,5,5’-tetramethylbenzidine substrate solution was added to the wells for 30 min in the dark. Finally, 50 μl of 2 M sulfuric acid was added to the cells, and the intensity of the color was measured at 450 nm using a spectrophotometer.

2.12. Statistical Analysis

Data were analyzed by a one-way analysis of variance (ANOVA), and the significance of differences between means was analyzed by the least significant difference (LSD) test.

3. Results

3.1. Levels of Plasma Cholesterol and Triglycerides

In the control group, the plasma cholesterol and triglyceride concentrations before the experiment were 0.72 ± 0.18 and 1.60 ± 0.33 g/l, respectively (n = 6) (unpublished data). Concentrations of plasma cholesterol (0.40 ± 0.06 g/l) and triglycerides (1.64 ± 0.10 g/l) in the control group did not change significantly during the 8-week feeding period (Table 1). In the treatment groups, plasma cholesterol and triglyceride concentrations before the experiment in the casein (n = 6), BS (n = 5), and vitamin E (n = 6) groups were 0.75 ± 0.42 and 1.72 ± 0.16, 0.77 ± 0.16 and 1.55 ± 1.11, and 0.54 ± 0.13 and 1.62 ± 0.88 g/l, respectively (unpublished data). In the treatment groups, in the 8 weeks after feeding cholesterol-containing chow to the casein, BS, and vitamin E groups, concentrations of plasma cholesterol significantly increased to 8.35 ± 6.26, 14.42 ± 4.30, and 10.60 ± 2.75 g/l, respectively (Table 1). After cholesterol feeding in the casein, BS, and vitamin E groups for 8 weeks, levels of plasma triglycerides in the treatment groups were 2.27 ± 0.84, 2.47 ± 1.00, and 3.32 ± 1.05 g/l, respectively (Table 1).

3.2. BS Attenuated the Atheroma Area and Oxidative Susceptibility of LDL in New Zealand White Rabbits

The ratio of the intimal to medial area in the aortic arch of the casein group (0.49% ± 0.29%) was significantly higher than those in the control (0.09% ± 0.02%), BS (0.25% ± 0.13%), and vitamin E groups (0.05% ± 0.03%) (Figure 1(a)). The atheroma region in the aortic arch of the BS group decreased 49% compared to the casein group.

Table 1. Plasma cholesterol and triglycerides of New Zealand white rabbits fed various 0.5% cholesterol-supplemented diets for 8 weeks.

All values in the experiments are presented as the mean ± S.D. of n = 6 (except for the black soybean group, n = 5). Values with different superscripts significantly differ at p < 0.05.

In addition, the ratio of the intimal to the medial area in the thoracic aorta of the casein group (0.56% ± 0.44%) was significantly higher than those in the control (0.10% ± 0.03%), vitamin E (0.11% ± 0.09%), and BS groups (0.23% ± 0.17%) (Figure 1(b)). The atheroma region of the thoracic aorta of the BS group decreased 58.9% compared to the casein group, while atheroma formation in the thoracic aorta in the vitamin E group was prevented and was close to the control level. Figure 2 demonstrates that the lag time of LDL in the BS group (319.6 ± 100.5 min) was similar to that in the vitamin E group (286.7 ± 97.0 min), which was significantly longer than those in the control (185.8 ± 61.6 min) and casein groups (187.2 ± 73.9 min).

3.3. Vitamin E Content in LDL and Anthocyanin in Plasma of New Zealand White Rabbits

The LDL vitamin E content in the vitamin E group (134.78 ± 87.67 nmole/mg LDL-cholesterol) was significantly higher than those in the control (58.77 ± 37.72 nmole/mg LDL-cholesterol), casein (30.23 ± 19.07 nmole/mg LDL-cholesterol), and BS groups (57.95 ± 68.94 nmole/mg LDL-cholesterol) (Table 2). The level of plasma anthocyanin in the BS group (2.10 ± 0.77 units/ml) was significantly higher than those in the control (0.22 ± 0.19 units/ml), casein (0.21 ± 0.10 units/ ml), and vitamin E groups (0.29 ± 0.08 units/ml) (Table 2).

3.4. Concentrations of Isoflavones and Cyanidin in the BSs

Table 3 illustrates contents of genistein, daidzein, and cyanidin in BSs were 198.7 ± 35.2, 342.0 ± 16.6, and 141.5 ± 1.8 μg/g of dry weight, respectively. Individual concentrations were equivalent to 0.074, 0.12, and 0.039 μM when treated with 100 μg/ml BSE in the HAECs model (unpublished data).

Conflicts of Interest

The authors declare no conflicts of interest.

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