Oral Administration of a Soy Extract Improves Endothelial Dysfunction in Ovariectomized Rats

• Abstract
• Materials and Methods
• References

Abstract

Effects of oral administration for 4 weeks of a soy fraction with mainly isoflavones (Glycine max, Leguminosae) (SOYPH; 5 mg/kg) on vascular dysfunction induced by bilateral ovariectomy (OVX) in rats were studied. We evaluated vascular reactivity of aortic rings after acetylcholine (ACh 10 nM-10 μM), sodium nitroprussiate (SN 15 - 30 nM) and N ^G-L-arginine (L-NMA; 10 - 100 μM). Uterine weight and nitric oxide synthase (NOS) activity were also investigated. The same parameters were observed after 4 weeks treatment with 17β-estradiol. In OVX rats endothelial-dependent vascular responses were changed: reduction of induced contraction (L-NMA 100 mM: sham OVX 2.1 ± 0.2 g/mg tissue; OVX 1.7 ± 0.4 g/mg tissue). Ovariectomy produced a reduction of constitutive NOS activity. Uterine weight was increased in animals treated with 17β-estradiol but not with SOYPH. Either SOYPH or 17β-estradiol produced a similar improvement of endothelial dysfunction and increased NOS activity. Our data suggest that soy isoflavones produce an improvement of endothelial dysfunction induced by ovariectomy so as 17β-estradiol, but probably without changes in reproductive system.

In previous years many authors have shown a growing interest for the possible protective role of estrogens on cardiovascular system [1]. However, it has been observed that estrogens increase the risk of cancer in women [2]. An alternative to estrogens is represented by plant chemicals soy isoflavones [3].

The most important isoflavone genistein binds to estrogen receptors and inhibits atherosclerotic lesion development [4]. Isoflavones may exert their effects also on platelet aggregation [5] and serotonin uptake [6], thus contributing to reduce vasospasm and thrombosis in cardiovascular diseases [7]. Evidences also indicates that supplementation of isoflavones in diet inhibits low-density lipoprotein (LDL) oxidation [8].

It has been shown that subcutaneous administration of genistein produces protective effects on vascular dysfunction induced by bilateral ovariectomy (OVX) [9]. In the present work we investigated whether oral administration of an extract of soy (SOYPH; 5 mg/kg) for 4 weeks produces similar effects on the same experimental model. In the same experimental conditions we observed the effects of estrogenic therapy with 17β-estradiol.

The weight of uteri of OVX rats treated with vehicle or SOYPH were decreased (35 - 40 %) with respect to sham OVX rats. OVX rats treated with 17β-estradiol showed a mean uterine weight significantly greater than the weight of vehicle and SOYPH groups (Fig. [1]).

Contractile responses to PE were not different between the groups. In OVX rats we observed a significant reduction of the relaxant effect induced by ACh (Fig. [2] A, Fig. [2] B) and we did not observe changes of relaxant response to SN in animals treated with SOYPH or estradiol (data not shown). Treatment with estradiol (Fig. [2] B) or SOYPH (Fig. [2] A) did not modify the relaxant effects of ACh in sham OVX rats. Either estradiol (Fig. [2] A) or SOYPH (Fig. [2] B) modified in an overlapping way changes in endothelium dependent relaxation of OVX rats.

Vasoconstriction induced by L-NMA (10 - 100 μM) was significantly attenuated in OVX rats with respect to sham OVX animals (Fig. [3] A, Fig. [3] B). Both estradiol (Fig. [3] B) and SOYPH (Fig. [3] A) did not modify this response in sham OVX rats. Either estradiol (Fig. [3] B) or SOYPH (Fig. [3] A) caused an increase in this constrictory response.

OVX rats showed a significant decrease in NOS activity in lung homogenates with respect to sham OVX rats. Estradiol or SOYPH treatment produced a similar and significant increase in the activity of constitutive form of the enzyme. The addition of L-NMA in the incubation buffer inhibited NOS activity in lung homogenates, thus providing further evidence that NOS was being measured (Table [1]).

Isoflavones could have a role as nutritional agents against the risk of cardiovascular diseases related to menopause [12]. Soy foods produce a hypocholesterolemic effect, but probably various components of soy may modify cardiovascular diseases independently of effects on plasmatic lipoproteins [7]. The mechanisms of action of isoflavones include their roles as weak estrogens, inhibitors of tyrosine kinase-dependent signal transduction processes and as cellular antioxidants [13].

Reduction of the risk of cardiovascular diseases induced by estrogenic therapy in post-menopausal women could be produced by an improvement of vessel reactivity [14] and it has been hypothesized that protective effects on the cardiovascular system could be produced by increasing nitric oxide formation from vascular endothelium [15]. This point of view has been recently supported by studies showing that treatment with genistein improves endothelial dysfunction caused by ovariectomy in the rat [13]. In this experimental model there is a marked reduction of vasodilatation (nitric oxide dependent) to ACh response together with a reduced contractile response to inhibitors of NOS [9].

Our data indicate that supplementation of soy isoflavones (SOYPH) is capable, as well as estradiol, to repair normal endothelial function damaged by ovariectomy in the rat. We treated intact animals with estradiol or soy with the aim to evaluate possible soy estradiol-like effects. While estradiol causes an increase in uterine weight, treatment with isoflavones, did not cause changes in uterine weight, indicating that these compounds did not have estradiol-like effects influencing the reproductive system and they could be used alternatively to estrogens in disorders following menopause. Finally, whereas both estradiol therapy and isoflavones therapy enhanced NOS activity in OVX animals, the cardiovascular protection produced by isoflavones seems to be mediated by increased formation of nitric oxide.

In conclusion, orally administered soy isoflavones (SOYPH) seem to improve endothelial dysfunction following ovariectomy in the rat. This protective effect is probably due to increase of nitric oxide formation, one of the factors that could contribute to protection of phytoestogens on the cardiovascular system.

Materials and Methods

Female mature Sprague-Dawley rats (Charles River laboratories; 250 - 280 g) were subjected to a bilateral ovariectomy (OVX rats). Sham operated (Sham OVX) rats served as controls. Adaptation and experiments were carried out in accordance with the internationally accepted principles and the national laws concerning the care and the use of laboratory animals.

Three weeks after surgery, OVX and sham OVX rats were randomly assigned to one of the four groups treated daily for four weeks as follows: 1) subcutaneous (s. c.) injection of 17β-estradiol 20 μg/kg in 100 μl of cotton seed oil (vehicle); 2) 17β-estradiol vehicle s. c. 3) oral administration of soy isoflavones (SOYPH; 5 mg/kg in 1 ml of saline); 4) oral administration of the vehicle of SOYPH (1 ml of saline). SOYPH (Pharmalife, Research) is composed as follows: 42.3 % soy isoflavones (17 % genistein and genistin, 22.2 % daidzein and daidzin, 3.1 % glycitein and glycitin), 15 % proteins, 2 % mono- and oligosaccharides, 10 % saponins, 2 % lectin, 4 % minerals, 3.7 % water, 15 -17 % non detectable.

At the end of treatment heparinized rats were euthanized with an intraperitoneal overdose of sodium pentobarbital (75 mg/kg). The uteri were removed immediately after perfusion fixation and weights were subsequently measured. Thoracic aortas were removed, placed in a cold Krebs solution and cut into rings about 2 mm in length. Rings were then placed under 1 g of tension in an organ bath where developed tension was measured with an isometric force transducer and recorded on a polygraph (Ugo Basile, Varese, Italy). After an equilibration period (60 min), basal tension was readjusted to 1 g and tissue was exposed to phenylephrine (PE, 100 nM). When the contraction was stable, the functional integrity of endothelium was assessed by a relaxant response to acetylcholine (ACh, 100 nM). Endothelium response was evaluated with cumulative concentrations of ACh (10 nM-1 μM) in aortic rings precontracted with PE (100 nM). Endothelium independent response was investigated by analizing the relaxant effects of sodium nitroprusside (SN; 15 - 30 nM) in endothelium denuded aortic rings. Relaxation of the rings was calculated as percentage decrease of contractile force. Other rings were precontracted with PE and then incubated with N ^G-L-arginine (L-NMA 10 - 100 μM). Results were expressed as g of tension X mg of tissue.

To measure NOS activity the rings were removed at the end of the experiments and immediately frozen in liquid nitrogen and later assaied according to the method of Bredt and Snyder [10], [11].

Data analysis was performed using one-way analysis of variance (ANOVA) with the Scheffè posthoc test for multiple comparisons. The data are expressed as the means ± S.D. Statistical significance was set at P < 0.05.

References

• 1 Grady D, Rubin S M, Pettiti D B, Fox C S, Black D, Ettinger B, Ernster V L, Cummings S R. Hormone therapy to prevent diseases and prolong life in postmenopausal women.  Annals of Internal Medicine. 1992;  117 549-56
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  CrossrefPubMedSearch in Google Scholar
• 10 Bredt D S, Snyder S H. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme.  Proceedings of National Academy of Science of United States of America. 1990;  87 682-5
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• 12 Scheiber M D, Liu J H, Subbiah M T, Rebar R W, Setchell K D. Dietary inclusion of whole soy foods results in significant reductions in clinical risk factors for osteoporosis and cardiovascular desease in normal postmenopausal women.  Menopause. 2001;  8 384-92
  PubMedSearch in Google Scholar
• 13 Barnes S, Boersma B, Patel R, Kirk M, Darley-Usmar V M, Kim H, Xu J. Isoflavonoids and chronic disease: mechanisms of action.  Biofactors. 2000;  12 209-15
  PubMedSearch in Google Scholar
• 14 Lieberman E H, Gerhard M D, Uehata A, Walsh B W, Selwyn A P, Ganz P. Estrogen improves endothelium-dependent, flow mediated vasodilation in postmenopausal women.  Ann Intern Med. 1994;  121 936-41
  PubMedSearch in Google Scholar
• 15 Luscher T F, Dbey R K. Endothelium and platelet-derived vasoactive substances: role in the regulation of vascular tone and growth.  In: Hypertension: pathophysiology, diagnosis and management. 2^nd ed. Laragh J.H., Brenner B.M. editors New York; Raven Press 1994: 609-30
  Search in Google Scholar

Prof. Gioacchino Calapai MD

Department of Clinical and Experimental Medicine and Pharmacology

Section of Pharmacology

School of Medicine

University of Messina

Via Consolare Valeria

Policlinico Universitario Torre Biologica 5° piano

98125 Messina

Italy

Phone: +39 90 2213646

Fax: +39 90 2213300

Email: gcalapai@unime.it

References

• 1 Grady D, Rubin S M, Pettiti D B, Fox C S, Black D, Ettinger B, Ernster V L, Cummings S R. Hormone therapy to prevent diseases and prolong life in postmenopausal women.  Annals of Internal Medicine. 1992;  117 549-56
  PubMedSearch in Google Scholar
• 2 McNagny S E. Prescribing hormone replacement therapy for menopausal symptoms.  Annals of Internal Medicine. 1999;  131 605-16
  PubMedSearch in Google Scholar
• 3 Setchell K DR, Adlercreutz H. 1988 Mammalian lignans and phyto-oestrogens. Recent studies on their formation, metabolism and biological role in health and disease.  In: Role of the Gut Flora in Toxicity and Cancer. (Rowland IR ed.) pp. 315-345 Academic Press London, UK;
• 4 Anderson J W, Smith B M, Washnock C S. Cardiovascular and renal benefits of dry bean and soybean intake.  American Journal of Clinical Nutrition. 1999;  70 (Suppl.) 464S-74S
  PubMedSearch in Google Scholar
• 5 Williams J K, Clarkson T B. Dietary soy isoflavones inhibit in vitro constrictor responses of coronary arteries to collagen-induced platelet activation. Cor.  Artery Dis. 1998;  9 759-64
  PubMedSearch in Google Scholar
• 6 Helmeste D M, Tang S W. Tyrosine kinase inhibitors regulate serotonin uptake in platelets.  European Journal of Pharmacology. 1995;  280 R5-7
  CrossrefPubMedSearch in Google Scholar
• 7 Anthony M S. Soy and cardiovascular disease: Cholesterol lowering and beyond.  Journal of Nutrition. 2000;  130 662S-663S
  PubMedSearch in Google Scholar
• 8 Tikkanen M J, Wahala K, Ojala S, Vihma V, Adlercreutz H. Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistence.  Proceedings of National Academy of Science of United States of America. 1998;  95 3106-10
  CrossrefPubMedSearch in Google Scholar
• 9 Squadrito F, Altavilla D, Squadrito G, Saitta A, Cucinotta D, Minutoli L, Deodato B, Ferlito M, Campo G M, Bova A, Caputi A P. Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats.  Cardiovascular Research. 2000;  45 454-62
  CrossrefPubMedSearch in Google Scholar
• 10 Bredt D S, Snyder S H. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme.  Proceedings of National Academy of Science of United States of America. 1990;  87 682-5
  PubMedSearch in Google Scholar
• 11 Bradford M M. A rapid and sensitive method for the quantification of protein dye binding.  Journal of Biological Chemistry. 1976;  72 700-3
  PubMedSearch in Google Scholar
• 12 Scheiber M D, Liu J H, Subbiah M T, Rebar R W, Setchell K D. Dietary inclusion of whole soy foods results in significant reductions in clinical risk factors for osteoporosis and cardiovascular desease in normal postmenopausal women.  Menopause. 2001;  8 384-92
  PubMedSearch in Google Scholar
• 13 Barnes S, Boersma B, Patel R, Kirk M, Darley-Usmar V M, Kim H, Xu J. Isoflavonoids and chronic disease: mechanisms of action.  Biofactors. 2000;  12 209-15
  PubMedSearch in Google Scholar
• 14 Lieberman E H, Gerhard M D, Uehata A, Walsh B W, Selwyn A P, Ganz P. Estrogen improves endothelium-dependent, flow mediated vasodilation in postmenopausal women.  Ann Intern Med. 1994;  121 936-41
  PubMedSearch in Google Scholar
• 15 Luscher T F, Dbey R K. Endothelium and platelet-derived vasoactive substances: role in the regulation of vascular tone and growth.  In: Hypertension: pathophysiology, diagnosis and management. 2^nd ed. Laragh J.H., Brenner B.M. editors New York; Raven Press 1994: 609-30
  Search in Google Scholar

Prof. Gioacchino Calapai MD

Department of Clinical and Experimental Medicine and Pharmacology

Section of Pharmacology

School of Medicine

University of Messina

Via Consolare Valeria

Policlinico Universitario Torre Biologica 5° piano

98125 Messina

Italy

Phone: +39 90 2213646

Fax: +39 90 2213300

Email: gcalapai@unime.it