Nitric oxide and endothelium-dependent effect of Tribulus terrestris feeding on aortic reactivity of streptozotocin-diabetic rats

1. Introduction

ardiovascular disorders continue to constitute major causes of morbidity and mortality in diabetic patients in spite of significant achievements in their diagnosis and treatment(1). Changes in vascular responsiveness to vasoconstrictors and vasodilators are mainly responsible for development of some vascular complications of diabetics (2). Most of these complications are due to increased serum glucose and augmented generation of reactive oxygen species (ROS) which finally lead to endothelium dysfunction (3).

Tribulus terrestris L. is a member of the Zygophyllaceae family. It is an annual herb about 30–70 cm high and has pinnate leaves (of unequal length), yellow flowers and  characteristic stellate shaped carpel fruits. It is widely distributed in

Africa, western Asia, China, Japan, Korea and Europe (4). Extracts from this plant have been used traditionally in treating a variety of diseases including hypertension and coronary heart disease, ocular inflammation and infertility in both sexes. The extracts have also been used as diuretics (4). Recent pharmacological studies tend to support these uses. For example, Al-Ali et al. (2003) have demonstrated diuretic activity in rats (5), while Adaikan et al. (2000) have shown that crude extract of Tribulus terrestris enhanced electrically- and nitroglycerine- induced relaxation of the rabbit corpus cavernosum consistent with a pro-erectile function (6). The mechanism responsible for the antihypertensive activity has not fully been understood (4). In a recent study, Sharifi et al. reported a significant antihypertensive effect of an aqueous extract of Tribulus terrestris in renin-dependent 2-kidney 1-clip (2K-1C) model of hypertension and suggested that this might be related to its inhibitory effect on angiotensin converting enzyme (ACE) activity (7). This was based on the observation that treatment with the aqueous extract Tribulus terrestris significantly reduced ACE activity in all tissues of the rat (7). This study was designed to assess for the first time the beneficial effect of chronic Tribulus terrestris feeding on improvement of aortic reactivity dysfunction of STZ-diabetic rats and to investigate some underlying mechanisms.

2. Materials and Methods

2.1. Animals

Male albino Wistar rats (n=28) (Pasteur’s institute, Tehran, Iran) weighing 225-310g were housed in an air-conditioned colony room at 21 2C 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 were rendered diabetic by a single intraperitoneal dose of 60 mg kg-1 STZ freshly dissolved in ice-cold 0.1 M citrate buffer (pH 4.5). Age-matched normal animals that received an injection of an equivalent volume of buffer comprised a non-diabetic control group. One week after STZ injection, overnight 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. Normal and hyperglycemic rats (a total of 48) were randomly allocated and similarly grouped into 4 groups (seven in each): normal vehicle-treated control, Tribulus terrestris (TT)-treated control, diabetic, and TT-treated diabetic. TT powder was mixed with standard food at a ratio of 6% and was freely available to rats throughout the experimental period for 7 weeks. Changes in body weight were regularly recorded during the study. The rats were finally 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) and SNP-(10-9-10-4 ) induced vasodilatation in rings with and without endothelium, they were preconstricted with a submaximal concentration of PE (10-6 M) which produced 70-80% of maximal response. To determine the participation of NO, rings were incubated 30 min before the experiment with L-NAME (100μM, a non-selective NOS inhibitor).

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.

2.3. Drugs

Phenylephrine, streptozotocin, ACh and L-NAME were purchased from Sigma Chemical (St. Louis, Mo., USA). All other chemicals were purchased from Merck (Germany) and Darupakhsh Co. (Iran).

2.4. 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

After 8 weeks, the weight of the vehicle-treated diabetic rats was found to be significantly decreased as compared to controls (p

Cumulative addition of KCl (10-50 mM) and PE (10-10-10-5 M) resulted in concentration dependent contractions in aortas of all groups (Figures 2-3). The maximum contractile responses to KCl and PE in the aortas from vehicle-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). As was expected, endothelium-dependent relaxation responses induced by ACh was significantly lower in vehicle-treated diabetic rats in relation to vehicle-treated controls (p

Pre-incubation of aortic rings with L-NAME non-significantly increased contractile response of aortic rings from all groups to PE. However, this increase was non-significantly lower in TT-treated diabetic group as compared to vehicle-treated diabetics. Regarding relaxation response to ACh, pre-incubation of aortic rings with L-NAME almost completely abolished the vasodilator response to ACh in segments from TT-treated diabetic rats, indicating the important role of endothelium-derived NO in the vascular effect of TT (Fig. 5).

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

Fig. 2: Cumulative concentration-response curves for KCl in aortic preparations 8 weeks after experiment in the presence (A) and absence (B) of endothelium (means ± S.E.M).

Fig. 3: Cumulative concentration-response curves for PE in aortic preparations 8 weeks after experiment in the presence (A) and absence (B) of endothelium (means ± S.E.M). * 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 (means ± SEM).
* p

Fig. 5: Cumulative concentration-response curves for ACh in endothelium-intact aortic rings precontracted with phenylephrine in the presence and absence of L-NAME 8 weeks after the experiment in control and diabetic rats. Relaxation responses are expressed as a percentage of the submaximal contraction induced by phenylephrine which produced 70-80% of maximal response (means ± SEM). * p