Low-dose dextromethorphan, a NADPH oxidase inhibitor, reduces blood pressure and enhances vascular protection in experimental hypertension

Abstract

Background: Vascular oxidative stress may be increased with age and aggravate endothelial dysfunction and vascular injury in hypertension. This study aimed to investigate the effects of dextromethorphan (DM), a NADPH oxidase inhibitor, either alone or in combination treatment, on blood pressure (BP) and vascular protection in aged spontaneous hypertensive rats (SHRs).

Methodology/principal findings: Eighteen-week-old WKY rats and SHRs were housed for 2 weeks. SHRs were randomly assigned to one of the 12 groups: untreated; DM monotherapy with 1, 5 or 25 mg/kg/day; amlodipine (AM, a calcium channel blocker) monotherapy with 1 or 5 mg/kg/day; and combination therapy of DM 1, 5 or 25 mg/kg/day with AM 1 or 5 mg/kg/day individually for 4 weeks. The in vitro effects of DM were also examined. In SHRs, AM monotherapy dose-dependently reduced arterial systolic BP. DM in various doses significantly and similarly reduced arterial systolic BP. Combination of DM with AM gave additive effects on BP reduction. DM, either alone or in combination with AM, improved aortic endothelial function indicated by ex vivo acetylcholine-induced relaxation. The combination of low-dose DM with AM gave most significant inhibition on aortic wall thickness in SHRs. Plasma total antioxidant status was significantly increased by all the therapies except for the combination of high-dose DM with high-dose AM. Serum nitrite and nitrate level was significantly reduced by AM but not by DM or the combination of DM with AM. Furthermore, in vitro treatment with DM reduced angiotensin II-induced reactive oxygen species and NADPH oxidase activation in human aortic endothelial cells.

Conclusions/significance: Treatment of DM reduced BP and enhanced vascular protection probably by inhibiting vascular NADPH oxidase in aged hypertensive animals with or without AM treatment. It provides the potential rationale to a novel combination treatment with low-dose DM and AM in clinical hypertension.

Figure 1. Effect of dextromethorphan (DM) monotherapy on blood pressure of spontaneous hypertension rats.

(A) systolic blood pressure (SBP) before and after treatment; (B) diastolic blood pressure (DBP) before and after treatment; (C) mean blood pressure (MBP) before and after treatment; (D) percentage changes of SBP by treatment; (E) percentage changes of DBP by treatment; (F) percentage changes of MBP by treatment, * means P<0.05; ** means P<0.01 ; *** means P<0.001.

Figure 2. Effect of amlodipine (AM) monotherapy on blood pressure of spontaneous hypertension rats.

(A) systolic blood pressure (SBP) before and after treatment; (B) diastolic blood pressure (DBP) before and after treatment; (C) mean blood pressure (MBP) before and after treatment; (D) percentage changes of SBP by treatment; (E) percentage changes of DBP by treatment; (F) percentage changes of MBP by treatment, *** means P<0.001.

Figure 3. Effects of dextromethorphan (DM) monotherapy or the combination of DM with amlodipine (AM) on blood pressure of spontaneous hypertension rats. SBP: systolic blood pressure; DBP: diastolic blood pressure; MBP: mean blood pressure.

* means P<0.05; ** means P<0.01; *** means P<0.001, compared to blood pressure before treatment.

Figure 4. Effect of dextromethorphan (DM) monotherapy or the combination of DM with amlodipine (AM) on acethylcholine-induced vasorelaxation of aortic segments of spontaneous hypertension rats (SHRs). +, P<0.05 compared to SHRs.

(A) WKY rats versus SHRs; (B) SHRs with different dose of M versus SHRs without treatment; (C) SHRs with low dose of AM combined with different dose of DM treatment; (D)SHRs with high dose of AM combined with different dose of DM treatment.

Figure 5. Effect of dextromethorphan (DM) monotherapy or the combination of DM with amlodipine (AM) on SNP-induced vasorelaxation of aortic segments of spontaneous hypertension rats (SHRs).

*, P<0.05 compared to SHRs. (A) WKY rats versus SHRs; (B) SHRs with different dose of DM versus SHRs without treatment; (C) SHRs with low dose of AM combined with different dose of DM treatment; (D) SHRs with high dose of AM combined with different dose of DM treatment.

Figure 6. Effect of dextromethorphan (DM) monotherapy or the combination of DM with amlodipine (AM) on vascular media area of aortas of spontaneous hypertension rats (SHRs).

(A) Effect of AM on the aortic media layer of SHRs. (B) Effect of DM on the aortic media layer of SHRs. (C) Effect of combination with DM and AM on the aortic media layer of SHRs, + means P<0.05, compared to SHRs. (D,E,F) Effect of DM, AM or combination therapy on the percentage change of reduction of media layer of SHRs. + means compared to SHR; * means P<0.05 ; ** means P<0.01.

Figure 7. Effect of dextromethorphan (DM) monotherapy or the combination of DM with amlodipine (AM) on the plasma NOx levels.

(A) and total antioxidant status (B) in spontaneous hypertension rats. The serum levels of renin (C) , angiotensin II (D) and aldosterone (E) were also measured. + means P<0.05; ++ means, P<0.01; +++ means P<0.001, compared to plasma NOx levels, total antioxidant capacity and renin-angiotensin system of SHRs.

Figure 8. In vitro effects of dextromethorphan (Dex) on human aortic endothelial cells (HAECs).

(A) Effects of various concentrations of Dex on cell viability in MTT assay. Data are expressed as mean±SD of three independent experiments. (B) Effect of Dex treatment on angiotensin II-induced superoxide production. Production of superoxide in HAECs was measured by lucigenin-enhanced chemiluminescence. (C) Effect of Dex treatment on angiotensin II-induced NADPH oxidase activity in HAECs. Production of NADPH oxidase activity was measured by adding lucigenin and NADPH. (D) Effect of Dex treatment on angiotensin II-induced membrane p47phox protein expression in HAECs. Membrane fraction was isolated for western blot analysis of membrane p47phox protein expression. Caveolin was used as an internal control. Bar graphs showed the ratio in percentage of expression intensity of each treatment to that of controls. Data are expressed as mean±SD of three independent experiments. *P<0.05 vs. Control; ^#P<0.05 compared with the angiotensin II-treated cells.