Dehydroepiandrosterone protects against oxidative stress-induced endothelial dysfunction in ovariectomized rats

Abstract

Cardiovascular disease is less frequent in premenopausal women than in age-matched men or postmenopausal women. Moreover, the marked age-related decline in serum dehydroepiandrosterone (DHEA) level has been associated to cardiovascular disease. The aim of this study was to evaluate the effects of DHEA treatment on vascular function in ovariectomized rats. At 8 weeks of age, female Wistar rats were ovariectomized (OVX) or sham (SHAM) operated and 8 weeks after surgery both groups were treated with vehicle or DHEA (10mg kg⁻¹ week⁻¹) for 3 weeks. Aortic rings were used to evaluate the vasoconstrictor response to phenylephrine (PHE) and the relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP). Tissue reactive oxygen species (ROS) production and SOD, NADPH oxidase and eNOS protein expression were analysed. PHE-induced contraction was increased in aortic rings from OVX compared to SHAM, associated with a reduction in NO bioavailability. Furthermore, the relaxation induced by ACh was reduced in arteries from OVX, while SNP relaxation did not change. The incubation of aortic rings with SOD or apocynin restored the enhanced PHE-contraction and the impaired ACh-relaxation only in OVX. DHEA treatment corrected the increased PHE contraction and the impaired ACh-induced relaxation observed in OVX by an increment in NO bioavailability and decrease in ROS production. Besides, DHEA treatment restores the reduced Cu/Zn-SOD protein expression and eNOS phosphorylation and the increased NADPH oxidase protein expression in the aorta of OVX rats. The present results suggest an important action of DHEA, improving endothelial function in OVX rats by acting as an antioxidant and enhancing the NO bioavailability.

Figure 1. Vascular reactivity to phenylephrine, acetylcholine and sodium nitroprusside

Concentration–response curve to phenylephrine (A), acetylcholine (B) and sodium nitroprusside (C) in endothelium-intact aortic rings from SHAM, OVX and DHEA-treated rats. Results are expressed as means ± SEM. Two-way ANOVA: *P < 0.01 in comparison to SHAM (n = 9–13).

Figure 2. Reactive oxygen species in aorta

Representative fluorescence photomicrographs of microscopic sections of thoracic aorta from SHAM, OVX and DHEA-treated rats. Vessels were labelled with the oxidative dye hydroethidine, which produces a red fluorescence when oxidized to ethidium bromide by superoxide anion. Quantitative analysis for fluorescence photomicrographs of microscopic sections of thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are expressed as means ± SEM. Two-way ANOVA: *P < 0.01 in comparison to SHAM and #P < 0.01 in comparison to OVX (n = 7).

Figure 3. eNOS protein expression and phosphorylation in aorta

Representative Western blots (top) and quantitative analysis (bottom) for total eNOS protein expression (A) and ser1177 eNOS phosphorylation (B) in thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are expressed as means ± SEM. Two-way ANOVA: *P < 0.01 in comparison to SHAM and #P < 0.01 in comparison to OVX (n = 9).

Figure 4. Cu/Zn-SOD and Mn-SOD protein expression in aorta

Representative Western blots (top) and quantitative analysis (bottom) for Cu/Zn-SOD (A) and Mn-SOD (B) protein expression in thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are expressed as means ± SEM. Two-way ANOVA: *P < 0.01 in comparison to SHAM and #P < 0.01 in comparison to OVX (n = 8).

Figure 5. NADPH oxidase subunits protein expression in aorta

Representative Western blots (top) and quantitative analysis (bottom) for gp91phox (A) and p22phox (B) protein expression in thoracic aorta from SHAM, OVX and DHEA-treated rats. Results are expressed as means ± SEM. Two-way ANOVA: *P < 0.01 in comparison to SHAM and #P < 0.01 in comparison to OVX (n = 8).