Soybean feeding improves vascular dysfunction and atte-nuates oxidative stress in streptozotocin-diabetic rats

1. Introduction

The incidence of diabetes mellitus (DM) as a prevalent metabolic disorder is increasing worldwide and it is regarded as a major concern in this century (1). In general, cardiovascular disorders are considered major causes of morbidity in diabetic individuals despite major developments in health systems (2). Changes in vascular reactivity to vasoconstrictors and vasorelaxants lead to development of some vascular complications in DM (3). Most of these complications are due to hyperglycemia and enhanced oxidative stress burden which finally lead to endothelium dysfunction (4).

Soybean has been regarded for long time

as one important source of dietary  supplement. In addition to protein, soybean also has various nutrients and is known as a rich source of bioactive components with many clinical advantages (5). Epidemiological studies indicate that soy product feeding in population reduces incidence of cardiovascular disorders and for this reason, soybean and its bioactive ingredients may be helpful for health of cardiovascular system due to its protective activity (6, 7, 8). Studies on experimental animals in which cardiovascular responses to soybean have been evaluated are indicative of its protective function. In this regard, soybean could improve vascular function, partly via nitric oxide (9, 10). Endothelium-dependent relaxation response of aorta and pulmonary artery by soybean has previously been reported (11). In addition, there is some evidence that soybean consumption could be beneficial to normalize blood glucose and lipids in diabetic population and soybean could also attenuate oxidative stress and this property is responsible for protection of diabetic patients (12). Meanwhile, soybean consumption corrects glucose imbalance and lowers complications in diabetics (13). Since protective effect of soybean extract on vascular reactivity in diabetics has not been reported, this study was undertaken to investigate the possible effect of soybean aqueous extract on aortic reactivity in STZ-diabetic rats

2. Materials and Methods

2.1. Animals

Male Wistar rats (Pasteur’s institute, Tehran) weighing 205-285 g were housed in an air-conditioned colony room at 21 ± 2 °C and supplied with standard pellet diet and tap water ad libitum. Procedures involving animals and their care were conducted in conformity with NIH guidelines for the care and use of laboratory animals.

2.2. Experimental protocol

The rats (n = 32) were rendered diabetic by a single intraperitoneal dose of 60 mg kg-1 of STZ. STZ was freshly dissolved in cold normal saline. Control animals received an injection of an equivalent volume of normal saline. One week after STZ injection, fasting blood samples were collected and serum glucose concentrations were measured using glucose oxidation method (Zistchimie, Tehran). Only those animals with a serum glucose level higher than 250 mg/dl were selected as diabetic. During the next weeks, diabetes was reconfirmed by the presence of polyphagia, polydipsia, polyuria, and weight loss. Control and diabetic rats were randomly allocated and similarly grouped into four groups (eight in each): normal control, soybean extract-treated control, diabetic, and soybean extract-treated diabetic. Aqueous extract of soybean was prepared as follows:

Soybean was purchased from the local market (Tehran, July 2007), 100 g of soybean powder was mixed with 2 l of boiling water, after 10 min, it was filtered several times until a waxy extract with a final concentration of 18% (w/w) was obtained. Then, it was frozen until being used. For final preparation, it was dissolved in normal saline. The extract was administered p.o. on alternate days at a dose of 100 mg/kg for 2 months. Changes in body weight were regularly recorded during the study.

Finally, the rats were anesthetized with diethyl ether, decapitated, and through opening the abdomen, descending thoracic aorta was carefully excised and placed in a petri dish filled with cold Krebs solution containing (in mM): NaCl 118.5, KCl 4.7, CaCl2 1.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 11. The aorta was cleaned of excess connective tissue and fat and cut into rings of approximately 4 mm in length. Aortic rings were suspended between the bases of two triangular-shaped wires. One wire was attached to a fixed tissue support in a 50 ml isolated tissue bath containing Krebs solution (pH 7.4) maintained at 37 C and continuously aerated with a mixture of 5% CO2 and 95% O2. The other end of each wire attached by a cotton thread to a F60 isometric force transducer (Narco Biosystems, USA) connected to a computer. In all experiments, special care was taken to avoid damaging the luminal surface of endothelium. Aortic rings were equilibrated at a resting tension of 1.5 g for at least 45 min. In some experiments, the endothelium was mechanically removed by gently rubbing the internal surface with a filter paper. Isometric contractions were induced by the addition of phenylephrine (PE, 1) and once the contraction stabilized, a single concentration of acetylcholine (1) was added to the bath in order to assess the endothelial integrity of the preparations. Endothelium was considered to be intact when this drug elicited a vasorelaxation ≥50% of the maximal contraction obtained in vascular rings precontracted with PE. The absence of acetylcholine relaxant action in the vessels indicated the total removal of endothelial cells. After assessing the integrity of the endothelium, vascular tissues were allowed to recuperate for at least 30 min.

At the end of the equilibration period, dose–response curves with KCl (10-50 mM) and PE (10-10-10-5 M) in the presence and absence of endothelium were obtained in aortic rings in a cumulative manner. To evaluate ACh (10-9-10-4 M)-induced vasodilatation in rings with endothelium, they were preconstricted with a submaximal concentration of PE (10-6 M) which produced 70-80% of maximal response. The sensitivity to the agonists was evaluated as pD2, which is the negative logarithm of the concentration of the drug required to produce 50% of the maximum response.

To determine the participation of NO, rings were incubated 30 min before the experiment with L-NAME (100 μM, a non-selective NOS inhibitor). To determine the participation of endothelial vasodilator factors in response to ACh, segments were incubated with INDO (10 μM, an inhibitor of COX-derived prostanoid synthesis) 30 min before the experiment with ACh.

After each vasoreactivity experiment, aortic rings were blotted, weighed, and the cross-sectional area (csa) was calculated using the following formula: Cross-sectional area (mm2) = weight (mg) [length (mm) density (mg mm3-1)]-1. The density of the preparations was assumed to be 1.05 mg/mm2 (14).

2.3. Determination of MDA concentration in aortic rings

After removing aortic segments and cleansing them of extra tissues, they were blotted dry and weighed, then made into 5% tissue homogenate in ice-cold 0.9% saline solution. A supernatant was obtained from tissue homogenate by centrifugalization (1000×g, 4 ºC, 5 min). The MDA concentration (thiobarbituric acid reactive substances, TBARS) in the supernatant was measured as described before (15). Briefly, trichloroacetic acid and TBARS reagent were added to supernatant, then mixed and incubated at 100 ºC for 80 min. After cooling on ice, samples were centrifuged at 1000×g for 20 min and the absorbance of the supernatant was read at 532 nm. TBARS results were expressed as MDA equivalents using tetraethoxypropane as standard.

2.4. Measurement of SOD activity in aortic rings

The supernatant of tissue homogenate were obtained as described earlier (16). Briefly, supernatant was incubated with xanthine and xanthine oxidase in potassium phosphate buffer (pH 7.8, 37 ºC) for 40 min and NBT was added. Blue formazan was then monitored spectrophoto-metrically at 550 nm. The amount of protein that inhibited NBT reduction to 50% maximum was defined as 1 nitrite unit (NU) of SOD activity.

2.5. Drugs

Phenylephrine, streptozotocin, ACh, INDO, and L-NAME were purchased from Sigma Chemical (St. Louis, Mo., USA). All other chemicals were purchased from Merck (Germany) and Darupakhsh Co. (Tehran, Iran). Indomethacin solution was prepared in ethanol in such a way that the maximal ethanol concentration of the medium was less than 0.001% (v/v).

2.6. Data and statistical analysis

All values were given as means SEM. Contractile response to PE was expressed as grams of tension per cross-sectional area of tissue. Relaxation response for ACh was expressed as a percentage decrease of the maximum contractile response induced by PE. Statistical analysis was carried out using repeated measure ANOVA and one-way ANOVA followed by Tukey post-hoc test. A statistical p value less than 0.05 considered significant.

3. Results

The obtained data showed that after 8 weeks, the weight of the diabetic rats was found to be significantly lower versus control rats (p

Fig. 1: Body weight and serum glucose concentration of animals in different weeks (means ± S.E.M). * p

Cumulative addition of KCl (10-50 mM) and PE (10^-10-10^-5M) produced concentration dependent contractions in aortas in all groups (Figures 2-3). The maximum contractile responses to KCl and PE in the aortas from extract-treated diabetic rats in the presence of endothelium were found to be significantly (p

Addition of ACh resulted in concentration-dependent relaxations in all aortic rings precontracted with PE (Fig. 4). It was found out that endothelium-dependent relaxation responses to ACh was significantly lower in treated diabetic rats in relation to controls at concentrations higher than 1 nanomolar (p-5M. Relaxation response of soybean-treated control rats was not significantly higher versus controls.

Pre-incubation of aortic rings with L-NAME (Fig. 5) almost completely abolished the vasodilator response to ACh in segments from soybean extract-treated diabetics, indicating the important role of endothelium-derived NO in the vascular effect of soybean extract. Pre-incubation of aortic segments from soybean extract-treated diabetic rats with INDO did not significantly change the vascular reactivity to ACh (Fig. 6).

Regarding aortic oxidative stress, as shown in  Table 1, STZ- diabetes resulted in an elevation of MDA and decreased SOD (p

Fig. 4: Cumulative concentration-response curves for ACh in endothelium-intact aortic rings precontracted with PE 8 weeks after experiment. Relaxation responses are expressed as a percentage of the submaximal contraction induced by phenylephrine which produced 70-80% of maximal response.
 * p

Table 1: Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in aortic tissue of studied groups

* p