Supplementation with the Symbiotic Formulation Prodefen® Increases Neuronal Nitric Oxide Synthase and Decreases Oxidative Stress in Superior Mesenteric Artery from Spontaneously Hypertensive Rats

Abstract

In recent years, gut dysbiosis has been related to some peripheral vascular alterations linked to hypertension. In this work, we explore whether gut dysbiosis is related to vascular innervation dysfunction and altered nitric oxide (NO) production in the superior mesenteric artery, one of the main vascular beds involved in peripheral vascular resistance. For this purpose, we used spontaneously hypertensive rats, either treated or not with the commercial synbiotic formulation Prodefen^® (10⁸ colony forming units/day, 4 weeks). Prodefen^® diminished systolic blood pressure and serum endotoxin, as well as the vasoconstriction elicited by electrical field stimulation (EFS), and enhanced acetic and butyric acid in fecal samples, and the vasodilation induced by the exogenous NO donor DEA-NO. Unspecific nitric oxide synthase (NOS) inhibitor L-NAME increased EFS-induced vasoconstriction more markedly in rats supplemented with Prodefen^®. Both neuronal NO release and neuronal NOS activity were enhanced by Prodefen^®, through a hyperactivation of protein kinase (PK)A, PKC and phosphatidylinositol 3 kinase-AKT signaling pathways. The superoxide anion scavenger tempol increased both NO release and DEA-NO vasodilation only in control animals. Prodefen^® caused an increase in both nuclear erythroid related factor 2 and superoxide dismutase activities, consequently reducing both superoxide anion and peroxynitrite releases. In summary, Prodefen^® could be an interesting non-pharmacological approach to ameliorate hypertension.

Keywords: Nrf2; PI3K-AKT; hypertension; neuronal nitric oxide; oxidative stress; perivascular mesenteric innervation; protein kinase A; protein kinase C; synbiotic.

Figure 1

(a) Effect of supplementation with Prodefen^® on systolic blood pressure. Results (Mean ± S.E.M.) are expressed in mm Hg. n = 12–14 animals each group. * p < 0.05 (Student’s t-test). (b) Serum endotoxin levels in SHR-Ctrl and SHR-SYNB. Results (Mean ± S.E.M.) are expressed in Endotoxin units (E.U.)/mL. n = 8–10 animals each group. * p < 0.05 (Student’s t-test).

Figure 2

Levels of acetic (a), butyric (b), lactic (c), formic (d) and propionic (e) acids in fecal samples from SHR-Ctrl and SHR-SYNB animals. Results (Mean ± S.E.M.) are expressed in mmol/L. n = 10–12 animals each group. * p < 0.05 (Student’s t-test).

Figure 3

(a) Vasoconstriction induced by electrical field stimulation (EFS) in endothelium-denuded mesenteric segments from SHR-Ctrl and SHR-SYNB animals (n = 6–8 segments from different rats in each experimental group). Results (mean ± S.E.M.) are expressed as a percentage of the previous tone elicited by KCl. Analysis of the functional role of neuronal NO on EFS-induced vasoconstriction by preincubation with the unspecific nitric oxide synthase (NOS) inhibitor L-NAME, in mesenteric arteries from SHR-Ctrl (b) and SHR-SYNB (c). Results (mean ± S.E.M.) are expressed as a percentage of previous tone induced by KCl. n = 5–6 segments from different animals in each experimental group. Insert panel: Differences in the area under the curve (dAUC) in presence/absence of L-NAME. * p < 0.05 SHR-Ctrl vs. SHR-SYNB (Student’s t-test).

Figure 4

(a) Vasodilator response to NO donor DEA-NO in noradrenaline-precontracted mesenteric segments from SHR-Ctrl and SHR-SYNB rats (n = 6 segments from different animals in each experimental group). Results are expressed as mean ± S.E.M. (b) EFS-induced NO release in mesenteric arteries from SHR-Ctrl and SHR-SYNB. Data (Mean ± S.E.M.) are expressed as arbitrary fluorescence units/mg tissue. n = 7–8 segments in each group. * p < 0.05 (Student’s t-test). (c) Analysis of nNOS expression and phosphorylation (Ser 1417) in mesenteric rings from SHR-Ctrl and SHR-SYNB. The figure is representative of 9 isolated segments from each group. Lower panel: Densitometry analysis for the expression of each protein. Results (mean + S.E.M.) are expressed as the relation between the signal obtained for the analyzed protein and the signal obtained for β-actin. * p < 0.05 (Student’s t-test).

Figure 5

(a) Inhibitory effect of H89 (PKA inhibitor, 1 µmol/L), calphostin C (PKC inhibitor, 0.1 µmol/L) or LY 294002 (PI3K inhibitor, 10 µmol/L) on EFS-induced NO release in endothelium-denuded mesenteric rings from SHR-Ctrl and SHR-SYNB (n = 4–5 segments in each experimental group). Data (arbitrary fluorescence units/mg tissue) are expressed as mean ± S.E.M. * p < 0.05 SHR-Ctrl vs. SHR-SYNB (Student’s t-test). # p < 0.05 conditions without inhibitor vs. conditions with inhibitor in each group (Student’s t-test). (b) PKA activity, and (c) PKC activity in mesenteric arteries from SHR-Ctrl and SHR-SYNB (n = 5 segments from different animals in each group). Results (optical density (OD) units/µg protein) are represented as (mean ± S.E.M). * p < 0.05 (Student’s t-test). (d) Analysis for AKT and PI3K (P85 subunit) expression, and AKT phosphorylation at the T308 residue (P-AKT) in mesenteric arteries from SHR-Ctrl and SHR-SYNB (8–9 isolated arterial segments from different animals in each group). Lower panel: Densitometry analyses of the protein expression. Results (mean ± S.E.M) are expressed as protein expression relative to β-actin expression. * p < 0.05 (Student’s t-test).

Figure 6

Effect of preincubation with 0.1 mmol/L tempol (a superoxide anion tempol) on the vasodilator response to NO donor DEA-NO in mesenteric segments from SHR-Ctrl (a) and SHR-SYNB (b). Results (mean ± S.E.M.) are expressed as a percentage of the previous tone elicited by noradrenaline. n = 6 segments from different animals in each experimental group. (c) Effect of preincubation with tempol on EFS-induced NO release in mesenteric arteries from SHR-Ctrl and SHR-SYNB. Data (Mean ± S.E.M.) are expressed as arbitrary fluorescence units/mg tissue. * p < 0.05 SHR-Ctrl vs. SHR-SYNB (Student’s t-test). # p < 0.05 conditions without tempol vs. conditions with tempol in each group (Student’s t-test). n = 5 segments in each experimental group. (d) Superoxide anion formation in mesenteric segments from SHR-Ctrl and SHR-SYNB. Results (mean ± S.E.M.) are expressed as chemiluminiscence units (U)/min mg tissue. n = 6 segments in each group. p < 0.05 (Student’s t-test). (e) EFS-induced peroxynitrite release in mesenteric arteries from SHR-Ctrl and SHR-SYNB. Data (Mean ± S.E.M.) are expressed as arbitrary fluorescence units/mg tissue. n = 6 segments in each group * p < 0.05 (Student’s t-test).

Figure 7

Western blot analysis for (a) total Nrf2, phosphorylated Nrf2 in Ser40 residue (P-Nrf2), and (b) SOD-1 and SOD-2 in mesenteric segments from SHR-Ctrl and SHR-SYNB. (7–9 isolated arterial segments from different animals in each group). Lower graphs show densitometry analyses of the protein expression. Results (mean ± S.E.M) are expressed as protein expression relative to β-actin expression. * p < 0.05 (Student’s t-test). (c) Superoxide dismutase activity in mesenteric segments from SHR-Ctrl and SHR-SYNB. Results (mean ± S.E.M.) are expressed as a percentage of inhibition (% inhibition). n = 6-8 segments in each group. * p < 0.05 (Student’s t-test).