Diosmetin Ameliorates Vascular Dysfunction and Remodeling by Modulation of Nrf2/HO-1 and p-JNK/p-NF-κB Expression in Hypertensive Rats

Abstract

Diosmetin is a citrus flavonoid that has antioxidant and anti-inflammatory effects. This study examined the effect of diosmetin on blood pressure and vascular alterations and its underlying mechanisms in experimentally hypertensive rats. Male Sprague rats were administered Nω-nitro-l-arginine methyl ester L-NAME for five weeks and were given diosmetin at doses of 20 or 40 mg/kg or captopril (5 mg/kg) for two weeks. Diosmetin alleviated hypertension, improved endothelial dysfunction, and suppressed the overactivity of sympathetic nerve-mediated vasoconstriction in aorta and mesentery hypertensive rats (p < 0.05). Increases in plasma and aortic tissue malondialdehyde (MDA) and carotid superoxide generations and reductions of plasma superoxide dismutase, catalase, and nitric oxide in hypertensive rats were ameliorated by diosmetin (p < 0.05). Diosmetin increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in hypertensive rats. Furthermore, diosmetin mitigated hypertrophy and collagen accumulation of the aortic wall in L-NAME rats. It exhibited an anti-inflammatory effect by reducing interleukin-6 (IL-6) accumulation and by overexpressing the phospho-c-Jun N-terminal kinases (p-JNK) and the phospho-nuclear factor-kappaB (p-NF-κB) proteins in the aorta (p < 0.05). Captopril was a positive control substance and had similar effects to diosmetin. In summary, diosmetin reduced blood pressure and alleviated vascular abnormalities in L-NAME-treated rats. These effects might be related to antioxidant and anti-inflammatory effects as well as to the modulation of the expression of the Nrf2/HO1 and p-JNK/NF-κB proteins.

Keywords: diosmetin; inflammation; oxidative stress; vascular function; vascular remodeling.

Figure 1

Changes in systolic blood pressure in conscious rats throughout the experimental period. ^a p < 0.05 vs. control group, ^b p < 0.05 vs. LN + vehicle group, ^c p < 0.05 vs. LN + diosmetin20 group, ^d p < 0.05 vs. LN + diosmetin40 group. LN, L-NAME; LN + diosmetin20, L-NAME treated with 20 mg/kg of diosmetin; LN + diosmetin40, L-NAME treated with 40 mg/kg of diosmetin; LN + captopril, L-NAME treated with captopril (n = 8/group). Statistical analysis between the five groups was performed using the one-way ANOVA followed by Turkey’s post hoc test.

Figure 2

Effect of diosmetin and captopril on the contractile response to EFS (A) and exogenous NE (B) in the mesenteric vascular bed. ^a p < 0.05 vs. control group, ^b p < 0.05 vs. LN + vehicle group, ^c p < 0.05 vs. LN + diosmetin20 group. LN, L-NAME; LN + diosmetin20, L-NAME treated with 20 mg/kg of diosmetin; LN + diosmetin40, L-NAME treated with 40 mg/kg of diosmetin; LN + captopril, L-NAME treated with captopril (n = 8/group). Statistical analysis between the five groups was performed using the one-way ANOVA followed by Turkey’s post hoc test.

Figure 3

Effect of diosmetin and captopril on the vascular response to exogenous acetylcholine (A) and sodium nitroprusside (B) in the mesenteric vascular bed and exogenous acetylcholine (C) and sodium nitroprusside (D) aortic rings. ^a p < 0.05 vs. control group, ^b p < 0.05 vs. LN + vehicle group, ^c p < 0.05 vs. LN + diosmetin20 group. LN, L-NAME; LN + diosmetin20, L-NAME treated with 20 mg/kg of diosmetin; LN + diosmetin40, L-NAME treated with 40 mg/kg of diosmetin; LN + captopril, L-NAME treated with captopril (n = 8/group). Statistical analysis between the five groups was performed using the one-way ANOVA followed by Turkey’s post hoc test.

Figure 4

Effect of diosmetin and captopril on vascular O₂^•− production (A), aortic MDA levels (B), plasma MDA levels (C), plasma SOD activity (D), and plasma CAT activity (E) in LN-induced hypertensive rats. ^a p < 0.05 vs. control group, ^b p < 0.05 vs. LN + vehicle group, ^c p < 0.05 vs. LN + diosmetin20 group. LN, L-NAME; LN + diosmetin20, L-NAME treated with 20 mg/kg of diosmetin; LN + diosmetin40, L-NAME treated with 40 mg/kg diosmetin; LN + captopril, L-NAME treated with captopril (n = 7–8/group). Statistical analysis between the five groups was performed using the one-way ANOVA followed by Turkey’s post hoc test.

Figure 5

Effect of diosmetin and captopril on the plasma NOx concentration in LN-induced hypertensive rats. ^a p < 0.05 vs. control group, ^b p < 0.05 vs. LN + vehicle group. LN, L-NAME; LN + diosmetin20, L-NAME treated with 20 mg/kg of diosmetin; LN + diosmetin40, L-NAME treated with 40 mg/kg of diosmetin; LN + captopril, L-NAME treated with captopril (n = 7–8/group). Statistical analysis between the five groups was performed using the one-way ANOVA followed by Turkey’s post hoc test.

Figure 6

Effect of diosmetin and captopril on Nrf2 (A) and HO-1 (B) protein expression in vascular tissue. ^a p < 0.05 vs. control group, ^b p < 0.05 vs. LN + vehicle group, ^c p < 0.05 vs. LN + diosmetin20 group. LN, L-NAME; LN + diosmetin20, L-NAME treated with 20 mg/kg of diosmetin; LN + diosmetin40, L-NAME treated with 40 mg/kg of diosmetin; LN + captopril, L-NAME treated with captopril (n = 4/group). Statistical analysis between the five groups was performed using the one-way ANOVA followed by Turkey’s post hoc test. scale bar = 100 μm.

Figure 7

Effect of diosmetin and captopril on the vascular morphology in LN-induced hypertensive rats. Representative figures of a vascular section stained with H&E (A) and picrosirius red staining (B). Results of the quantitative analyses of vascular remodeling are represented by cross-sectional areas (C), thoracic wall thickness (D), luminal diameter (E), ratios of the wall to lumen (F), vascular smooth muscle cell numbers (G), and the percentage of collagen deposition (H). ^a p < 0.05 vs. control group, ^b p < 0.05 vs. LN + vehicle group, ^c p < 0.05 vs. LN + diosmetin20 group. LN, L-NAME; LN + diosmetin20, L-NAME treated with 20 mg/kg of diosmetin; LN + diosmetin40, L-NAME treated with 40 mg/kg of diosmetin; LN + captopril, L-NAME treated with captopril (n = 7–8/group). Statistical analysis between the five groups was performed using the one-way ANOVA followed by Turkey’s post hoc test.

Figure 8

Effect of diosmetin and captopril on interleukin-6 (IL-6) immunohistochemical staining for vascular tissue (A) and IL-6 level (B) in LN-induced hypertensive rats. ^a p < 0.05 vs. control group, ^b p < 0.05 vs. LN + vehicle group. LN, L-NAME; LN + diosmetin20, L-NAME treated with 20 mg/kg of diosmetin; LN + diosmetin40, L-NAME treated with 40 mg/kg of diosmetin; LN + captopril, L-NAME treated with captopril (n = 7–8/group). Statistical analysis between the five groups was performed using the one-way ANOVA followed by Turkey’s post hoc test.

Figure 9

Effect of diosmetin and captopril on the p-JNK/p-NF-κB protein expression in vascular tissue (A,B) in LN-induced hypertensive rats. ^a p < 0.05 vs. control group, ^b p < 0.05 vs. LN + vehicle group. LN, L-NAME; LN + diosmetin20, L-NAME treated with 20 mg/kg of diosmetin; LN + diosmetin40, L-NAME treated with 40 mg/kg of diosmetin; LN + captopril, L-NAME treated with captopril (n = 4/group). Statistical analysis between the five groups was performed using the one-way ANOVA followed by Turkey’s post hoc test.

Figure 10

The possible mechanism action of diosmetin on vascular dysfunction and remodeling in L-NAME hypertensive rats.