Soy Isoflavone Protects Myocardial Ischemia/Reperfusion Injury through Increasing Endothelial Nitric Oxide Synthase and Decreasing Oxidative Stress in Ovariectomized Rats

Abstract

There is a special role for estrogens in preventing and curing cardiovascular disease in women. Soy isoflavone (SI), a soy-derived phytoestrogen, has similar chemical structure to endogenous estrogen-estradiol. We investigate to elucidate the protective mechanism of SI on myocardial ischemia/reperfusion (MI/R) injury. Female SD rats underwent bilateral ovariectomy. One week later, rats were randomly divided into several groups, sham ovariectomy (control group), ovariectomy with MI/R, or ovariectomy with sham MI/R. Other ovariectomy rats were given different doses of SI or 17β-estradiol (E2). Four weeks later, they were exposed to 30 minutes of left coronary artery occlusion followed by 6 or 24 hours of reperfusion. SI administration significantly reduced myocardial infarct size and improved left ventricle function and restored endothelium-dependent relaxation function of thoracic aortas after MI/R in ovariectomized rats. SI also decreased serum creatine kinase and lactate dehydrogenase activity, reduced plasma malonaldehyde, and attenuated oxidative stress in the myocardium. Meanwhile, SI increased phosphatidylinositol 3 kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) signal pathway. SI failed to decrease infarct size of hearts with I/R in ovariectomized rats if PI3K was inhibited. Overall, these results indicated that SI protects myocardial ischemia/reperfusion injury in ovariectomized rats through increasing PI3K/Akt/eNOS signal pathway and decreasing oxidative stress.

Figure 1

Experimental procedure. One week after ovariectomy, rats were maintained with soy free chow and given different treatments. Four weeks later, rats were subjected to 30 min myocardial ischemia and 6 h or 24 h reperfusion.

Figure 2

SI improved cardiac function after I/R in ovariectomized rats. (a) Representative echocardiography in each group after 6 h perfusion. (b) Ejection fraction after 6 h perfusion. (c) Fractional shortening after 6 h perfusion. Plots represent the mean ± SEM, n = 4–6. Statistical significance: ^∗∗ P < 0.01 compared with OVS; ^# P < 0.05, ^## P < 0.01 compared with CMC.

Figure 3

SI decreased MI size after I/R in ovariectomized rats. (a) Representative staining of heart by Evans blue/triphenyltetrazolium chloride (TTC) after 24 h perfusion. Evans blue-stained areas (blue) indicate nonischemic/reperfused area; TTC stained areas (red staining) indicate ischemic but viable tissue; Evans blue/TTC staining negative areas (white staining) indicate infarcted myocardium; red staining plus white staining indicates area at risk (AR). (b) Ratio of area at risk (AR)/left ventricle (LV) in each group after 24 h perfusion. (c) Ratio of area of infarction size (IS)/AR after 24 h perfusion. Plots represent the mean ± SEM, n = 3–5. Statistical significance: ^∗∗ P < 0.01, compared with OVS; ^## P < 0.01 compared with CMC.

Figure 4

SI decreased serum CK and LDH activity after I/R in ovariectomized rats. (a) Level of serum CK activity. (b) Level of serum LDH activity after 24 h perfusion. Plots represent the mean ± SEM, n = 4–6. Statistical significance: ^∗∗ P < 0.01 compared with OVS; ^# P < 0.05, ^## P < 0.01 compared with CMC.

Figure 5

SI increased myocardial PI3K/Akt/eNOS pathway during I/R in ovariectomized rats. Expression of p85α (a), phosphorylation of Akt (Ser473) (b), and eNOS (Ser1177) (c) with western blotting after 24 h perfusion. Plots represent the mean ± SEM, n = 5-6. Statistical significance: ^∗∗ P < 0.01, compared with OVS; ^# P < 0.05, ^## P < 0.01 compared with CMC.

Figure 6

SI decreased myocardial oxidative stress and iNOS expression but increased IκBα expression after I/R injury in ovariectomized rats. (a) Dihydroethidium (DHE) fluorescence staining of myocardium. (b) Lever of plasma MDA. ((c) and (d)) Expression of iNOS (c) and IκBα (d) in myocardium with western blotting after 24 h perfusion. Plots represent the mean ± SEM, n = 6. Statistical significance: ^∗ P < 0.05, ^∗∗ P < 0.01 compared with OVS; ^# P < 0.05 compared with CMC.

Figure 7

SI improved endothelium-dependent vasorelaxation, decreased oxidative stress and iNOS expression, and increased IκBα expression and PI3K/Akt/eNOS phosphyration in thoracic aorta in ovariectomized rats. (a and b) Endothelium-dependent vasorelaxation to acetylcholine of precontracted aortic sections was assessed. (c) Dihydroethidium (DHE) fluorescence staining of thoracic aorta. ((d)–(h)) Expression of iNOS (d), IκBα (e), and p85α (f), phosphorylation of Akt (Ser473) (g) and eNOS (Ser1177) (h) in thoracic aorta with western blotting after 24 h perfusion. Plots represent the mean ± SEM, n = 5–7. Statistical significance: ^∗ P < 0.05, ^∗∗ P < 0.01 compared with OVS; ^# P < 0.05, ^## P < 0.01 compared with CMC.

Figure 8

SI failed to decrease the infarct size of hearts with I/R in ovariectomized rats if PI3K was inhibited. One week after ovariectomy, the female SD rats were given 0.5% CMC (CMC group) or SI of 270 mg/kg·d by gavage (SI group). LY294002 (a specific PI3K inhibitor) at dose of 0.3 mg/kg was intraperitoneally injected immediately after SI was given (SI + LY294002 group). (a) Representative staining of heart by Evans blue/TTC after 24 h perfusion. (b) Ratio of area at risk (AR)/left ventricle (LV) in each group after 24 h perfusion. (c) Ratio of area of infarction size (IS)/AR after 24 h perfusion. Plots represent the mean ± SEM, n = 4. Statistical significance: ^## P < 0.01, compared with CMC; ^$$ P < 0.01 compared with SI.