Biological effects of combined resveratrol and vitamin D3 on ovarian tissue

Abstract

Background: Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural antioxidant polyphenol able to exert a wide range of biological effect on several tissues. Despite its important beneficial properties, it has a low water solubility, which limits its therapeutic applications in humans. Resveratrol also acts as a phytoestrogen that modulates estrogen receptor (ER)-mediated transcription. In addition, it has been shown that ovarian tissues benefit greatly from vitamin D3, which exerts its beneficial effects through VDR receptors. The aim was to evaluate the cooperative effects of resveratrol combined with vitamin D3 on ovarian cells and tissues and some other organs as well. Moreover, the modulation of specific intracellular pathways involving ER and VDR receptors has been studied.

Methods: The experiments were performed both in vitro and in vivo, to analyze cell viability, radical oxygen species production, signal transductions through Western Blot, and resveratrol quantification by HPLC.

Results: Cell viability, radical oxygen species production, and intracellular pathways have been studied on CHO-K1 cells. Also, the relative mechanism activated following oral intake in female Wistar rats as animal model was investigated, evaluating bioavailability, biodistribution and signal transduction in heart, kidney, liver and ovarian tissues. Both in in vitro and in vivo experiments, resveratrol exerts more evident effects when administered in combination with vitD in ovarian cells, showing a common biphasic cooperative effect: The role of vitamin D3 in maintaining and supporting the biological activity of resveratrol has been clearly observed. Moreover, resveratrol plus vitamin D3 blood concentrations showed a biphasic absorption rate.

Conclusions: Such results could be used as a fundamental data for the development of new therapies for gynecological conditions, such as hot-flashes.

Keywords: Bioavailability; Hot flashes; Phytoalexins; Resveratrol; Vitamin D3.

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Figures

Fig. 1

Time-course and dose-response study of CHO-K1 viability measured by MTT test. a time-course of RES 10 μM; b time-course of RES 25 μM; c time-course of RES 50 μM; d time-course of RES 100 μM. Reported data are means ± SD of five independent experiments. * not significant vs control; point without symbol p < 0.05 vs control. RES = resveratrol. The effect of solvent alone is reported as well

Fig. 2

The cooperative effect of RES and vitD during time on cell viability and ROS production in CHO-K1 cells. a cell viability and b ROS production measured during time-course study in presence of RES and vitD alone and combined. Reported data are means ± SD in (a) and they are expressed as means ± SD(%) in (b) of five independent experiments. * not significant vs control; points without symbol, p < 0.05 vs control. RES = resveratrol 50 μM; vitD = vitamin D3 100 nM; RES + vitD = co-stimulation of RES 50 μM with vitD 100 nM

Fig. 3

Measure of intracellular concentration of RES alone and combined with vitD in CHO-K1 cells in a time-course study. Reported data are means ± SD of five independent experiments. * not significant vs control; points without symbol, p < 0.05 vs control. RES = resveratrol 50 μM; RES + vitD = co-stimulation of RES 50 μM with vitamin D 100 nM

Fig. 4

Western Blot, densitometric analysis and protein activation of CHO-K1 cells stimulated with RES and vitD alone and together. In a (ERK/MAPK), b (Akt), c (SOD) activations by ELISA are reported as means ± SD(%) of five independent experiments. In d (ERβ receptor) and e (VDR receptor) Western blot (on the left) and densitometric analysis (on the right) are reported. In the right column the specific densitometric analysis is reported and expressed as means ± SD(%) of five independent experiments. * p < 0.05 vs control; ** p < 0.05 vs RES alone; φp < 0.05 vs vitD; the bars, p < 0.05 between RES + vitD at different times. RES = resveratrol 50 μM; vitD = vitamin D3 100 nM; RES + vitD = co-stimulation of RES 50 μM with vitD 100 nM

Fig. 5

Bioavailability, vitamin D quantification and ROS production in in vivo experiments. Female rats (n = 48) were treated with RES 0.5 mg alone (n = 24), combined with vitD 0.4 μg (n = 24) and with vitD 0.4 μg alone (n = 18) by gavage. The animals were sacrificed at specific time-points (ranging 2–270 min) and plasma samples were collected. In a RES plasma concentration (μg/L), in b vitD plasma quantification and in c the ROS production are reported. The results are expressed as means ± SD in panel (a) and as means ± SD(%) in panel (b) of 4 independent experiments. All data p < 0.05 vs control. RES = resveratrol 0.5 mg; RES + vitD = preparation composed of RES 0.5 mg and vitD 0.4 μg

Fig. 6

Western blot, densitometric analysis and protein activity of ovarian tissue obtained from female rats (n = 32) treated with RES alone (n = 16) and combined with vitD (n = 16). In the upper (a) an example of Western Blot taken at different time (ranging 2–30 min) of Cyclin D1, ERβ receptor and VDR receptor is reported. In the downstream the specific densitometric analysis of Cyclin D1 (b), ERβ receptor (c), and VDR receptor (d) is reported and expressed as means ± SD(%) of 4 independent experiments. In e SOD activity by ELISA was reported as means ± SD(%) of 4 independent experiments. * p < 0.05 vs control; ** p < 0.05 vs RES alone; the bars, p < 0.05 between RES + vitD at different times. RES = resveratrol 0.5 mg; RES + vitD = preparation composed of RES 0.5 mg and vitD 0.4 μg

Fig. 7

Biodistribution of RES (μg/L) in in vivo experiments. Female rats (n = 40) were treated with RES 0.5 mg alone (n = 20) and combined with vitD 0.4 μg (n = 20) by gavage. The animals were sacrificed at specific time-points (ranging 30–270 min) and heart (a), kidney (b) and liver (c) were collected. The results are expressed as means ± SD (μg/L) of 4 independent experiments. * not significant vs control; point without symbol, p < 0.05 vs control. RES = resveratrol 0.5 mg; RES + vitD = preparation composed of RES 0.5 mg and vitD 0.4 μg