Chronic administration of the probiotic kefir improves the endothelial function in spontaneously hypertensive rats

Abstract

Background: The beverage obtained by fermentation of milk with kefir grains, a complex matrix containing acid bacteria and yeasts, has been shown to have beneficial effects in various diseases. However, its effects on hypertension and endothelial dysfunction are not yet clear. In this study, we evaluated the effects of kefir on endothelial cells and vascular responsiveness in spontaneously hypertensive rats (SHR).

Methods: SHR were treated with kefir (0.3 mL/100 g body weight) for 7, 15, 30 and 60 days and compared with non-treated SHR and with normotensive Wistar-Kyoto rats. Vascular endothelial function was evaluated in aortic rings through the relaxation response to acetylcholine (ACh). The balance between reactive oxygen species (ROS) and nitric oxide (NO) synthase was evaluated through specific blockers in the ACh-induced responses and through flow cytometry in vascular tissue.

Results: Significant effects of kefir were observed only after treatment for 60 days. The high blood pressure and tachycardia exhibited by the SHR were attenuated by approximately 15 % in the SHR-kefir group. The impaired ACh-induced relaxation of the aortic rings observed in the SHR (37 ± 4 %, compared to the Wistar rats: 74 ± 5 %), was significantly attenuated in the SHR group chronically treated with kefir (52 ± 4 %). The difference in the area under the curve between before and after the NADPH oxidase blockade or NO synthase blockade of aortic rings from SHR were of approximately +90 and -60 %, respectively, when compared with Wistar rats. In the aortic rings from the SHR-kefir group, these values were reduced to +50 and -40 %, respectively. Flow cytometric analysis of aortic endothelial cells revealed increased ROS production and decreased NO bioavailability in the SHR, which were significantly attenuated by the treatment with kefir. Scanning electronic microscopy showed vascular endothelial surface injury in SHR, which was partially protected following administration of kefir for 60 days. In addition, the recruitment of endothelial progenitor cells was decreased in the non-treated SHR and partially restored by kefir treatment.

Conclusions: Kefir treatment for 60 days was able to improve the endothelial function in SHR by partially restoring the ROS/NO imbalance and the endothelial architecture due to endothelial progenitor cells recruitment.

Figures

Fig. 1

Photomicrographs of kefir grains obtained at the unmagnified level (a) and scanning electronic micrographs of the exterior (b–d) and interior (e, f) surface of a kefir grain. The external surface (c, d) shows the prevalence of the bacilli in tight association with a polysaccharide matrix (kefiran). The inside surface shows rod-shaped bacilli growing in association with yeasts (e, f)

Fig. 2

Time-course of the effects of kefir on hemodynamic parameters in SHR. The bar graphs show average values (mean ± SEM) of the mean arterial pressure (a) and heart rate (b) of SHR treated with kefir compared to non-treated SHR and Wistar rats. *p < 0.05 vs. Wistar group; #p < 0.05 vs. SHR (two-way ANOVA)

Fig. 3

Time-course of the effects of kefir administration on the endothelial dysfunction of SHR. Dose–response curves to acetylcholine-induced relaxations of aortic rings from SHR-kefir compared to the non-treated SHR and to the normotensive Wistar rats (a). The bar graphs show the maximum relaxation (b), the area under the curve (c) and the sensitivity (pEC50, d) to acetylcholine. The values shown are the mean ± SEM. *p < 0.05 vs. Wistar group; #p < 0.05 vs. SHR (two-way ANOVA)

Fig. 4

Effects of kefir administration on ROS contribution to the endothelial dysfunction in SHR. Dose–response curves to acetylcholine-induced relaxations of aortic rings from SHR-kefir compared to the non-treated SHR and to the normotensive Wistar rats after the pre-blockade with apocynin (a). The bar graphs show the difference in the area under the curve (b) and the maximum relaxation (c) in response to acetylcholine. The values are the mean ± SEM (n = 7–8 animals per group)

Fig. 5

Time-course of changes in the ROS content and the number of endothelial cells in the aorta from the hypertensive rats administered kefir. The graphs show production of superoxide anion (a), hydrogen peroxide (b), peroxynitrite/hydroxyl radical (c), nitric oxide (d), and the number of endothelial cells (e) measured through flow cytometry and comparing SHR-kefir group with non-treated SHR and Wistar rats. The values are the mean ± SEM. *p < 0.05 vs. Wistar group; #p < 0.05 vs. SHR (two-way ANOVA)

Fig. 6

Contribution of the nitric oxide bioavailability to the endothelial dysfunction in SHR administered kefir for 60 days. The line graph (a) shows the changes in the dose–response to acetylcholine following the endothelial NO synthase blockade with N(G)-nitro-l-arginine methyl ester (L-NAME). The bar graphs show the average values of the maximum response (b) and the difference in the area under the curve (ΔAUC, c) calculated from the dose–response curve obtained during the blockade of the basal NO/cGMP molecular pathway with L-NAME. The bar graphd shows the basal NO/cGMP molecular pathway activation, indicated by the phenylephrine-induced contraction of the aortic rings during the setting of endothelial NO synthase blockade via L-NAME, comparing the 3 groups of animals. The values are the mean ± SEM (n = 7–8 per group). *p < 0.05 vs. Wistar group; #p < 0.05 vs. SHR [two-way (a) and one-way (b–d) ANOVA]

Fig. 7

Recovery of vascular endothelial surface architecture in SHR administered with kefir. Scanning electron microscopy showing representative endothelial structure of aortas from a normotensive Wistar rat, a non-treated SHR and a SHR treated with kefir for 60 days. Scale bar 10 µm. White arrow endothelial cell; white arrow head endothelial surface denudation; black arrow gaps

Fig. 8

Time-course effect of the kefir administration on the endothelium-independent relation in SHR. The dose–response curves to the NO donor, sodium nitroprusside, of aortic rings from SHR administered kefir compared to the non-treated SHR and to the normotensive Wistar rats (a). The values are the mean ± SEM. *p < 0.05 vs. Wistar group (two-way ANOVA)

Fig. 9

Beneficial effects of kefir in arterial hypertension. Simplified scheme of main effects of chronic administration of kefir on the endothelial dysfunction in SHR