Resveratrol Inhibition of Rac1-Derived Reactive Oxygen Species by AMPK Decreases Blood Pressure in a Fructose-Induced Rat Model of Hypertension

Abstract

Recent studies have reported that the activation of AMP-activated protein kinase (AMPK) suppressed oxidative stress. The aim of this study was to examine whether the activation of AMPK in the brain decreased Rac1-induced ROS generation, thereby reducing blood pressure (BP) in rats with fructose-induced hypertension. The inhibition of ROS by treatment with an AMPK activator (oral resveratrol, 10 mg/kg/day) for 1 week decreased the BP and increased the NO production in the rostral ventrolateral medulla (RVLM) of fructose-fed rats but not in control Wistar-Kyoto (WKY) rats. In addition, resveratrol treatment abolished the Rac1-induced increases in the activity of the NADPH oxidase subunits p22-phox and reduced the activity of SOD2, while treatment with an AMPK inhibitor (compound C, 40 μM/day) had the opposite effect, in the fructose-fed rats. Interestingly, the activation of AMPK abolished Rac1 activation and decreased BP by inducing the activities of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and ribosomal protein S6 kinase (RSK) and nNOS phosphorylation in the fructose-fed rats. We conclude that the activation of AMPK decreased BP, abolished ROS generation, and enhanced ERK1/2-RSK-nNOS pathway activity by negatively regulating Racl-induced NADPH oxidase levels in the RVLM during oxidative stress-associated hypertension.

Figure 1. Resveratrol reversed the superoxide-dependent NO production and elevated SBP in the RVLM of rats with fructose-induced hypertension.

(A) The graph reveals the effects of resveratrol on SBP in the fructose groups after one week. The SBP significantly recovered after the resveratrol treatment compared with the fructose group. (B) Quantitative analysis of intracellular NO, measured by flow cytometry of DAF-2 DA florescence intensity, in control, fructose and fructose + resveratrol (n = 10,000 cells per group; One-way ANOVA). Treatment with resveratrol significantly increased the NO levels in the RVLM in the fructose group compared with the control. (C) Confocal microscopy analysis of green fluorescence was used to estimate p-AMPKT172 levels in the RVLM after treatment with resveratrol. The representative images shown demonstrate that the elevation of the AMPK phosphorylation level was significantly increased in the RVLM after treatment with resveratrol. (D) Representative images of DHE-treated brain sections. Sections of the RVLM from the fructose group showed significant increases in DHE fluorescence compared with the control group sections. Furthermore, the DHE fluorescence in the RVLM were significantly attenuated by the resveratrol treatment. (E) Confocal microscopy analysis of green fluorescence was used to estimate nitrotyrosine levels in the RVLM after treatment with resveratrol. The representative images shown demonstrate that the elevation of the nitrotyrosine level was significantly decreased in the RVLM after treatment with resveratrol. The images were photographed at ×400 magnification. We examined 3 groups (control; 10% fructose-treated; and 10% fructose+resveratrol; n = 6 for each). The values shown are the means ± SEM, n = 6. *P < 0.05, **P < 0.01. Scale bar: 20 μm.

Figure 2. Resveratrol abolished the Rac1-induced increases in NADPH oxidase subunits (p67 and p22-phox) in the RVLM, respectively, and reduced the levels of SOD2 in the RVLM of rats with fructose-induced hypertension.

(A) Bar graph showing the Rac1 activation ratio after treatment with fructose and/or resveratrol. Note the significant decrease in fructose-induced Rac1 activation after the administration of resveratrol. (B) Quantitative immunoblot analysis demonstrating that the NADPH oxidase subunit p22-phox ratio in the fructose group was reduced in the RVLM after treatment with resveratrol. (p22, p47, p67-phox, rac1 and actin the position of the 22, 47, 67, 21 and 43-kDa molecular weight marker is indicated, respectively). (C) Quantitative immunoblot analysis demonstrating that the level of SOD2 protein in the RVLM was significantly increased by the administration of resveratrol. (SOD1, SOD2 and actin the position of the 23, 25 and 43-kDa molecular weight marker is indicated, respectively). (D) Bar graph showing the NADPH oxidase activity ratio after treatment with fructose and/or resveratrol. Note the significant decrease in fructose-induced NADPH oxidase activity after the administration of resveratrol. (E) Bar graph showing the Total SOD activity ratio after treatment with fructose and/or resveratrol. Note the significant increase in fructose-reduced total SOD activity after the administration of resveratrol. The data shown represent the mean ± SEM of six independent experiments. *P < 0.05, **P < 0.01.

Figure 3. Activation of AMPK abolished the Rac1-induced increases in Rac1 and NADPH oxidase activities and reduced the activities of SOD2 in the RVLM of rats with fructose-induced hypertension.

(A) Graph showing the effects of resveratrol on SBP with or without the administration of compound C. The SBP after treatment with resveratrol was significantly increased by compound C. (B) Levels of NO in the RVLM after the administration of compound C. The bar graph shows that the concentration of NO after treatment with resveratrol was significantly reduced by compound C. (C) Confocal microscopy analysis of green fluorescence was used to estimate p-AMPK^T172 levels in the RVLM after treatment with resveratrol and compound (C). The representative images shown demonstrate that the elevation of the AMPK phosphorylation level in the RVLM after treatment with resveratrol was reduced by compound C. (D) Confocal microscopy analysis of DHE-treated brain sections in the RVLM after treatment with resveratrol and compound C. The representative images shown demonstrate that the elevation of the DHE fluorescence level in the RVLM after treatment with resveratrol was reduced by compound C. (E) Bar graph showing the activation ratio of Rac1 after treatment with resveratrol and compound C. The Rac1 activation in the RVLM after treatment with resveratrol was significantly inhibited by treatment with compound C. (F) Immunoblot showing P-ACC^S79 and P-AMPK^T172 protein levels after treatment with resveratrol and the AMPK inhibitor, compound C. The elevated ACC and AMPK phosphorylation levels in the RVLM after resveratrol treatment were reduced by treatment with compound C. (P-ACC^S79, P-AMPK^T172 and actin the position of the 280, 62 and 43-kDa molecular weight marker is indicated, respectively) (G) Immunoblot showing p22-phox protein levels after treatment with resveratrol and compound C. The resveratrol-reduced increase in the p22-phox activity ratios in the RVLM were further enhanced by compound C. However, the resveratrol-induced increase in the SOD2 activity in the RVLM was inhibited by compound C. (p22-phox, SOD2 and actin the position of the 22, 25 and 43-kDa molecular weight marker is indicated, respectively) The values are shown as the means ± SEM, n = 6. *P < 0.05, **P < 0.01. Scale bar: 20 μm.

Figure 4. Activation of AMPK enhanced the activity of the ERK1/2-RSK-nNOS pathway in the RVLM of rats with fructose-induced hypertension.

(A,B) Immunoblot showing the levels of P-ERK1/2^T202/Y204, P-RSK^T359/S363, P-eNOS^S1177, P-nNOS^S1416, and iNOS after treatment with resveratrol and compound C. (P-ERK1/2^T202/Y204, P-RSK^T359/S363, P-nNOS^S1416, P-eNOS^S1177, iNOS and actin the position of the 44/42, 90, 165, 140, 130 and 43-kDa molecular weight marker is indicated, respectively) The elevated ERK1/2, RSK, and nNOS phosphorylation ratios after treatment with resveratrol were reduced by compound C in the RVLM. The values shown are the means ± SEM, n = 6. *P < 0.05, **P < 0.01.

Figure 5. The proposed mechanism by which the AMPK signaling pathway regulates blood pressure (BP) in the RVLM of rats with fructose-induced hypertension.

Treatments with an AMPK activator (resveratrol) and inhibitor (compound C) demonstrated that AMPK acts as an important regulator of BP through Rac1-NADPH oxidase activity. AMPK decreased blood pressure, abolished the generation of ROS and enhanced the activity of the ERK1/2-RSK-nNOS pathway by negatively regulating Racl-induced NADPH oxidase levels in the RVLM of rats with oxidative stress–induced hypertension.