Resveratrol decreases fructose-induced oxidative stress, mediated by NADPH oxidase via an AMPK-dependent mechanism

Abstract

Background and purpose: Oxidative stress is an important pathogenic factor in the development of hypertension. Resveratrol, the main antioxidant in red wine, improves NO bioavailability and prevents cardiovascular disease. The aim of this study was to examine whether resveratrol decreases the generation of reactive oxygen species (ROS), thereby reducing BP in rats with fructose-induced hypertension.

Experimental approach: Rats were fed 10% fructose with or without resveratrol (10 mg·kg(-1) ·day(-1) ) for 1 week or for 4 weeks with resveratrol treatment beginning at week 2; systolic BP (SBP) was measured by tail-cuff method. Endogenous in vivo O2 (-) production in the nucleus tractus solitarii (NTS) was determined with dihydroethidium. Real-time PCR and immunoblotting analyses were used to quantify RNA and protein expression levels.

Key results: In fructose-fed rats, ROS levels in the NTS were higher, whereas the NO level was significantly decreased. Also, RNA and protein levels of NADPH oxidase subunits (p67, p22-phox) were elevated, superoxide dismutase 2 (SOD2) reduced and AMP-activated PK (AMPK) T172 phosphorylation levels in the NTS were lower in fructose-fed rats. Treatment with the AMPK activator resveratrol decreased levels of NADPH oxidase subunits and ROS, and increased NO and SOD2 levels in the NTS of fructose-fed rats. Administration of resveratrol, in combination with fructose at week 0 and later at week 2, significantly reduced the SBP of fructose-fed rats.

Conclusions and implications: Collectively, resveratrol decreased BP through the phosphorylation of AMPK, Akt and neuronal NOS in fructose-fed rats. These novel findings suggest that resveratrol may be a potential pharmacological candidate for the treatment of hypertension.

Keywords: AMPK; hypertension; nitrogen oxides; nucleus tractus solitarii; resveratrol.

Figure 1

Superoxide-dependent NO production elevates SBP in the NTS of fructose-induced hypertensive rats. (A) The graph shows the SBP in the fructose groups after 1 week. The SBP was significantly increased in the fructose group compared with the control group. (B) Quantification of NOx concentrations in the NTS and cerebellum of fructose-fed rats. The bar graph shows the NOx concentration (as µM nitrate µg^-1 of total protein). NOx levels significantly decreased in the NTS of the fructose-fed rats compared with the control rats. However, NOx levels in the cerebellum sections did not change in the fructose-fed group compared with in the control groups. (C) Representative images of DHE-treated brain sections. The images were photographed at ×280 magnification. Bar graph representation of ROS index in the NTS and cerebellum of the fructose groups as compared with WKY controls. The ROS index is the relative mean intensity of fluorescence of DHE. Sections including the NTS of fructose-fed rats displayed a significant increase in DHE fluorescence compared with the control group sections. However, DHE stain levels in the cerebellum sections did not change in the fructose-fed group compared with the control groups. Values are shown as the means ± SEM, n = 6. *P < 0.05, **P < 0.01.

Figure 2

Fructose increases RAGE expression, which increases NADPH oxidases and decreases SOD1/2 in the NTS of fructose-induced hypertensive rats. (A) In the bar graph showing the real-time PCR analysis of relative NADPH oxidase subunits p22-phox p47-phox and p67-phox mRNA expression levels in the NTS of fructose groups after 1 week. Note the significant increase in the mRNA expression of the NADPH oxidase subunits p22-phox and p67-phox in fructose-fed rats compared with control rats. (B) Quantitative immunoblotting analysis demonstrates that the ratio of NADPH oxidase subunits p22-phox and p67-phox was significantly increased in the NTS of the fructose-fed group after 1 week. RAGE, was significantly increased in the NTS of fructose-fed rats compared with control rats. (C) Bar graph showing the effect of after fructose-fed 1 week on SOD1, SOD2 and SOD3 mRNA expression. Note the significant decrease in SOD2 mRNA expression in fructose-fed rats compared with control rats. (D) Quantitative immunoblotting analysis demonstrates that the SOD2 ratio was significantly decreased in the NTS of fructose-fed rats after 1 week. The data shown are the means ± SEM of six independent experiments. *P < 0.05, **P < 0.01.

Figure 3

AMPK is significantly decreased in the NTS of fructose-induced hypertensive rats. (A) Confocal analysis of green fluorescence was used to estimate p-AMPK^T172 levels in the NTS of fructose-fed rats after 1 week. The representative images demonstrate that the AMPK phosphorylation ratio was reduced in the NTS by fructose. (B) DHE fluorescence was use to estimate the ROS levels in the NTS of animals fed fructose or resveratrol for 1 week. We examined three groups (control; 10% fructose-treated; and 10% fructose + resveratrol; n = 6 for each). The images were photographed at ×100 and ×200 magnifications. Bar graph representation of ROS index in the NTS of the fructose groups as compared with WKY controls or 10% fructose + resveratrol group compared with fructose groups. The ROS index is the relative mean intensity of fluorescence of DHE. Sections of the NTS from the fructose group showed significant increases in DHE fluorescence compared with the control group sections. Furthermore, the DHE fluorescence in the NTS was significantly attenuated by the resveratrol treatment. Data are expressed as the means ± SEM (n = 6). **P < 0.01.

Figure 4

Resveratrol elevates SBP and reverses superoxide-dependent NO production in the NTS of rats with fructose-induced hypertension. (A) The graph shows the effects of resveratrol on SBP in the study groups during weeks 1–4 of treatment. We examined five groups (control group, fructose-fed group, resveratrol group, fructose + resveratrol group, fructose-fed week 2 + resveratrol group; n = 6 for each). The SBP significantly recovered after resveratrol treatment compared with the fructose group. (B) Quantification of the NOx concentration in the NTS of rats. The rats were randomly divided into five groups (control; fed with 10% fructose; fed with resveratrol 10 mg·kg⁻¹·day⁻¹; fed with 10% fructose and resveratrol 10 mg·kg⁻¹·day⁻¹; fructose-fed for 2 weeks then fructose + resveratrol for 2 weeks) of six rats each. The bar graph shows the NOx concentration (as µM nitrate µg^-1 of total protein). Fructose significantly decreased the NOx levels in the NTS of the fructose group compared with the control group. However, treatment with resveratrol significantly increased the NOx levels in the NTS of the fructose + resveratrol group or fructose-fed week 2 + resveratrol group compared with the frucotse group. Data are expressed as the means ± SEM (n = 6). *P < 0.05.

Figure 5

Resveratrol increases the activity of the AMPK-Akt-nNOS pathway in the NTS of rats with fructose-induced hypertension. (A–D) Immunoblot showing the levels of P-AMPK^T172, P-Akt^S473, P-nNOS^S1416 and P-eNOS^S1177 after treatment with resveratrol. The AMPK, Akt and nNOS phosphorylation ratios were significantly higher in the NTS after treatment with resveratrol. The values shown are the means ± SEM, n = 6. *P < 0.05, **P < 0.01.

Figure 6

The proposed mechanism by which the AMPK signalling pathway regulates BP in the NTS of rats with fructose-induced hypertension. Treatment with an AMPK activator (resveratrol) demonstrated that AMPK acts as an important regulator of BP through NADPH oxidase activity. AMPK decreased BP, decreased the generation of ROS, and enhanced the activity of the Akt-nNOS pathway by down-regulating NADPH oxidase levels and up-regulating SOD2 levels in the NTS of rats with oxidative stress-induced hypertension.