Ellagic Acid Prevents L-NAME-Induced Hypertension via Restoration of eNOS and p47phox Expression in Rats

The effect of ellagic acid on oxidative stress and hypertension induced by Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME) was investigated. Male Sprague-Dawley rats were administrated with L-NAME (40 mg/kg/day) for five weeks. L-NAME induced high systolic blood pressure (SBP) and increased heart rate (HR), hindlimb vascular resistance (HVR) and oxidative stress. Concurrent treatment with ellagic acid (7.5 or 15 mg/kg) prevented these alterations. Co-treatment with ellagic acid was associated with up-regulation of endothelial nitric oxide synthase (eNOS) protein production and alleviation of oxidative stress as indicated by decreased superoxide production in the vascular tissue, reduced plasma malondialdehyde levels, reduced NADPH oxidase subunit p47phox expression and increased plasma nitrate/nitrite levels. Our results indicate that ellagic acid attenuates hypertension by reducing NADPH oxidase subunit p47phox expression, which prevents oxidative stress and restores NO bioavailability.


Blood Pressure Measurement
Systolic blood pressure (SBP) was measured once a week using non-invasive tail-cuff plethysmography (IITC/Life Science Instrument model 229 and model 179 amplifiers, Woodland Hills, CA, USA). Briefly, conscious rats were placed in a restrainer and allowed to rest for 10-15 min prior to blood pressure measurement. The tail was placed inside the cuff, which automatically inflated and released and SBP values were obtained from the mean of three measurements.

Hemodynamic Assessments
On the last day of the experiment, rats were anaesthetized with an intraperitoneal injection of Pentobarbital (60 mg/kg) and placed on heating pad to maintain body temperature at 37 • C. A tracheotomy was performed for measurement of spontaneous breathing, and a polyethylene catheter was inserted into the lower abdominal aorta via the left femoral artery for continuous monitoring of blood pressure using a pressure transducer and the Acknowledge data acquisition and analysis software (BIOPAC Systems Inc., Santa Barbara, CA, USA). The catheters were filled with heparinized saline to prevent clotting. SBP, diastolic blood pressure (DBP), mean arterial blood pressure (MAP) and heart rate (HR) were continuously monitored.
Hindlimb blood flow (HBF) was continuously measured by an electromagnetic flow meter (Carolina Medical Electronics, Carolina, NC, USA) connected to an electromagnetic flow probe placed around the abdominal aorta below the kidneys. Hindlimb vascular resistance (HVR) was calculated from baseline MAP and mean HBF.
After collection of the hemodynamic measurements, rats were sacrificed with an over dosage of the anesthetic drug. Blood samples were drawn from the bifurcation of the abdominal aorta into EDTA tubes for assay of the oxidative stress markers plasma MDA and NO metabolites. The carotid arteries were rapidly excised for analysis of vascular O 2 •− production and the thoracic aorta was isolated to evaluate eNOS and NADPH oxidase subunit (p47 phox ) protein expression.

Assay of Vascular O 2 •− Production
Vascular O 2 •− production was measured using a lucigenin-enhanced chemiluminescence method [36]. The carotid arteries (about 3-5 mm in length) were placed in ice-cold saline and adipose and connective tissues were removed. The concentration of plasma MDA was measured by a spectrophotometric method as previously described [37]. Briefly, plasma samples (150 µL) were reacted with 10% TCA, 125 µL of 5 mM EDTA, 125 µL of 8% SDS, and 10 µL of 0.5 µg/mL BHT for 10 minutes at room temperature. Then, an equal volume of 0.6% TBA was added and the mixture was boiled in a water bath for 30 min. After cooling to room temperature, the mixture was centrifuged at 10,000 g for 5 min at 25 • C. The absorbance of the supernatant was measured at 532 nm by spectrophotometer (Amersham Bioscience, Arlington, MA, USA) and compared to a standard curve generated with 0.3 to 10 µmol/L 1,1,3,3-tetraethoxypropane.

Assay of Plasma Nitrate and Nitrite
The concentrations of plasma nitrate and nitrite, the end products of NO metabolism, were measured using an enzymatic conversion method [38] with some modifications [39]. Plasma samples were first deproteinized by ultrafiltration using centrifugal concentrators (Pall Corp., Ann Arbor, MI, USA). The supernatants were mixed with 1.2 µmol/L NADPH, 4 mmol/L glucose-6-phosphate disodium (G-6-P), 1.28 U/mL glucose-6-phosphate dehydrogenase (G-6-PD) and 0.2 U/mL nitrate reductase, and then incubated at 30 • C for 30 min. The mixture was reacted with a Griess solution (4% sulfanilamide in 0.3% naphthalenediamine dichloride, NED) for 15 min. The absorbance of samples was measured on a microplate reader with a filter wavelength of 540 nm (Tecan GmbH., Groding, Austria).

Western Blot Analysis of p47 phox NADPH Oxidase Subunit and eNOS Protein
Expressions of the p47 phox NADPH oxidase subunit and eNOS proteins in aortic homogenates were determined following a previously described method with some modifications [17,40]. The aortic homogenates (15 µg) were electrophoresed on an SDS polyacrylamide gel and electrotransferred onto a polyvinylidenedifluoride membrane. Membranes were blocked with 5% skimmed milk in phosphate buffered saline (PBS) with 0.1% Tween-20 at room temperature for 2 h and incubated overnight at 4 • C with primary antibody of mouse monoclonal anti-eNOS (1:2500, BD Biosciences, CA, USA) or mouse monoclonal anti-p47 phox (1:2500, Santa Cruz Biotechnology, CA, USA). The membranes were washed with PBS 3 times for 7 min before being incubated with the secondary antibody, horseradish peroxidase goat anti-mouse IgG (1:2500, Santa Cruz Biotechnology, CA, USA), for 2 h at room temperature. The blots were developed in Amersham™ ECL™ Prime solution (Amersham Biosciences Corp., Piscataway, NJ, USA), and densitometric analysis was performed using an ImageQuant™ 400 imager (GE Healthcare Life Sciences, Piscataway, NJ, USA). β-actin (1:5000, Santa Cruz Biotechnology, CA, USA) was used as a loading control. The intensity of the specific eNOS and p47 phox protein bands were normalized to that of β-actin, and data were expressed as a percentage of the values determined in control aorta from the same gel.

Statistical Analysis
Data were expressed as mean ± SEM. The significance of differences between means were analyzed by one-way analysis of variance (ANOVA) and followed by Student Newman-Keul's test to show specific group differences. Statistical significance was assigned at a p value of less than 0.05.

Effect of Ellagic Acid on Systolic Blood Pressure
At the beginning of the experiment, average baseline SBP was similar among groups ( Figure 1). L-NAME administration induced a rapid progressive increase in SBP. Rats receiving L-NAME with concurrent administration of ellagic acid showed increased SBP compared to controls but reduced SBP compared to L-NAME treatment alone. The L-NAME+EA 15 mg/kg/day group showed a significantly lower SBP than the L-NAME+EA 7.5 mg/kg/day group (157.8 ± 4.8 mmHg vs. 165.7 ± 4.3, p < 0.05).

Statistical Analysis
Data were expressed as mean ± SEM. The significance of differences between means were analyzed by one-way analysis of variance (ANOVA) and followed by Student Newman-Keul's test to show specific group differences. Statistical significance was assigned at a p value of less than 0.05.

Effect of Ellagic Acid on Systolic Blood Pressure
At the beginning of the experiment, average baseline SBP was similar among groups ( Figure 1). L-NAME administration induced a rapid progressive increase in SBP. Rats receiving L-NAME with concurrent administration of ellagic acid showed increased SBP compared to controls but reduced SBP compared to L-NAME treatment alone. The L-NAME+EA 15 mg/kg/day group showed a significantly lower SBP than the L-NAME+EA 7.5 mg/kg/day group (157.8 ± 4.8 mmHg vs. 165.7 ± 4.3, p < 0.05).

Figure 1.
Effect of ellagic acid on systolic blood pressure during N ω -Nitro-L-arginine methyl ester hydrochloride (L-NAME) administration for five weeks. Values are expressed as mean ± SEM, n = 8. * p < 0.05 when compared to control group, # p < 0.05 when compared to L-NAME group and † p < 0.05 when compared to L-NAME+EA 7.5 mg/kg group.

Effect of Ellagic Acid on Hemodynamic Status
Animals that received only L-NAME were hypertensive as evidenced by significant increases in SBP, DBP, MAP and HR compared to control rats (Table 1). Concurrent treatment with ellagic acid significantly attenuated the elevation of the BP parameters and HR in L-NAME hypertensive rats (Table 1). L-NAME administered rats also showed significantly lower HBF levels and significantly higher HVR levels compared with control rats (4.2 ± 0.3 vs. 8.3 ± 0.8, p < 0.05 and 39.5 ± 3.5 vs. 12.1 ± 1.4, p < 0.001, respectively). Both the 7.5 mg/kg and 15 mg/kg doses of ellagic acid ameliorated the L-NAME induced changes in HBF and HVR in hypertensive rats (Table 1). Figure 1. Effect of ellagic acid on systolic blood pressure during N ω -Nitro-L-arginine methyl ester hydrochloride (L-NAME) administration for five weeks. Values are expressed as mean ± SEM, n = 8. * p < 0.05 when compared to control group, # p < 0.05 when compared to L-NAME group and † p < 0.05 when compared to L-NAME+EA 7.5 mg/kg group.

Effect of Ellagic Acid on Hemodynamic Status
Animals that received only L-NAME were hypertensive as evidenced by significant increases in SBP, DBP, MAP and HR compared to control rats (Table 1). Concurrent treatment with ellagic acid significantly attenuated the elevation of the BP parameters and HR in L-NAME hypertensive rats (Table 1). L-NAME administered rats also showed significantly lower HBF levels and significantly higher HVR levels compared with control rats (4.2 ± 0.3 vs. 8.3 ± 0.8, p < 0.05 and 39.5 ± 3.5 vs. 12.1 ± 1.4, p < 0.001, respectively). Both the 7.5 mg/kg and 15 mg/kg doses of ellagic acid ameliorated the L-NAME induced changes in HBF and HVR in hypertensive rats (Table 1). Values are expressed as mean ± SEM, n = 10. * p < 0.05 when compared to control group and # p < 0.05 when compared to vehicle L-NAME group.

Effects of Ellagic on Nitrate/Nitrite Production and eNOS Protein Expression
The concentration of nitrate/nitrite NO metabolites in plasma is shown in Figure 2A and the expression of eNOS protein in isolated rat aortas is shown in Figure 2B. The levels of NO metabolites in plasma were significantly reduced in L-NAME rats when compared with normal control rats (2.9 ± 0.2 µmol/L vs.11.2 ± 0.6 µmol/L, p < 0.05) and administration of L-NAME was associated with down-regulation of eNOS protein expression compared to control rats (p < 0.05). There was no change in plasma nitrate/nitrite levels or eNOS protein expression in control rats treated with ellagic acid alone (Figure 2A,B). Co-treatment of L-NAME administered rats with either 7.5 mg/kg or 15 mg/kg ellagic acid for five weeks significantly restored nitrate/nitrite production (6.4 ± 0.5 µmol/L vs. 7.1 ± 0.4 µmol/L, p < 0.05, respectively) and eNOS protein expression, p < 0.05, respectively).
Nutrients 2015, 7 7 Values are expressed as mean ± SEM, n = 10. * p < 0.05 when compared to control group and # p < 0.05 when compared to vehicle L-NAME group.

Effects of Ellagic on Nitrate/Nitrite Production and eNOS Protein Expression
The concentration of nitrate/nitrite NO metabolites in plasma is shown in Figure 2A and the expression of eNOS protein in isolated rat aortas is shown in Figure 2B. The levels of NO metabolites in plasma were significantly reduced in L-NAME rats when compared with normal control rats (2.9 ± 0.2 µmol/L vs.11.2 ± 0.6 µmol/L, p < 0.05) and administration of L-NAME was associated with down-regulation of eNOS protein expression compared to control rats (p < 0.05). There was no change in plasma nitrate/nitrite levels or eNOS protein expression in control rats treated with ellagic acid alone (Figure 2A,B). Co-treatment of L-NAME administered rats with either 7.5 mg/kg or 15 mg/kg ellagic acid for five weeks significantly restored nitrate/nitrite production (6.4 ± 0.5 µmol/L vs. 7.1 ± 0.4 µmol/L, p < 0.05, respectively) and eNOS protein expression, p < 0.05, respectively). Values are expressed as mean ± SEM, * p < 0.05 when compared to control group, # p < 0.05 when compared to L-NAME group and † p < 0.05 when compared to L-NAME+EA 7.5 mg/kg group. Values are expressed as mean ± SEM, * p < 0.05 when compared to control group, # p < 0.05 when compared to L-NAME group and † p < 0.05 when compared to L-NAME+EA 7.5 mg/kg group.

Effect of Ellagic Acid on Oxidative Stress
Oxidative stress was determined by measuring the concentration of plasma MDA and by assaying vascular superoxide production. The level of plasma MDA in the L-NAME group was three-fold higher than in the control group (10.9 ± 0.9 µmol/L vs. 3.3 ± 0.1 µmol/L, p < 0.05). Administration of both 7.5 and 15 mg/kg ellagic acid significantly reduced the level of plasma MDA in a dose dependent manner (6.9 ± 0.1 µmol/L and 5.0 ± 0.5 µmol/L, p < 0.05, respectively, Figure 3). L-NAME also induced a three-fold elevation in vascular superoxide production compared to the vehicle control (175.3 ± 11.2 count/min/mg dry weight vs. 59.9 ± 8.9 count/min/mg dry weight, p < 0.05) ( Figure 4A). Again, ellagic acid at both the 7.5 and 15 mg/kg doses significantly inhibited this elevation (137.1 ± 11.8 count/min/mg dry weight and 90.4 ± 4.6 count/min/mg dry weight, p < 0.05, respectively). In addition, there was a strong correlation between vascular superoxide production and SBP (R = 0.856; Figure 4B).

Effect of Ellagic Acid on Oxidative Stress
Oxidative stress was determined by measuring the concentration of plasma MDA and by assaying vascular superoxide production. The level of plasma MDA in the L-NAME group was three-fold higher than in the control group (10.9 ± 0.9 µmol/L vs. 3.3 ± 0.1 µmol/L, p < 0.05). Administration of both 7.5 and 15 mg/kg ellagic acid significantly reduced the level of plasma MDA in a dose dependent manner (6.9 ± 0.1 µmol/L and 5.0 ± 0.5 µmol/L, p < 0.05, respectively, Figure 3). L-NAME also induced a three-fold elevation in vascular superoxide production compared to the vehicle control (175.3 ± 11.2 count/min/mg dry weight vs. 59.9 ± 8.9 count/min/mg dry weight, p < 0.05) ( Figure 4A). Again, ellagic acid at both the 7.5 and 15 mg/kg doses significantly inhibited this elevation (137.1 ± 11.8 count/min/mg dry weight and 90.4 ± 4.6 count/min/mg dry weight, p < 0.05, respectively). In addition, there was a strong correlation between vascular superoxide production and SBP (R = 0.856; Figure 4B). Values are expressed as mean ± SEM, n = 7. * p < 0.05 when compared to control group, # p < 0.05 when compared to L-NAME group and † p < 0.05 when compared to L-NAME+EA 7.5 mg/kg group.  Values are expressed as mean ± SEM lucigenin-enhanced chemilumiescence counts/min/mg dry weight, n = 7. * p < 0.05 when compared to control group, # p < 0.05 when compared to L-NAME group and † p < 0.05 when compared to L-NAME+EA 7.5 mg/kg group. (B) Correlation between SBP and vascular superoxide production.

Effect of Ellagic Acid on p47 phox Protein Expression in Aortic Tissues
The expression of p47 phox protein in L-NAME treated rats was significantly up-regulated compared to control rats (p < 0.05, Figure 5A). Positive correlation of p47 phox protein expression and the values of the vascular O2 •− production was observed ( Figure 5B). Treatment of the L-NAME group with 7.5 mg/kg ellagic acid reduced the over-expression of p47 phox protein (p < 0.05) and treatment with 15 mg/kg ellagic acid restored p47 phox protein levels to normal ( Figure 5A).  Values are expressed as mean ± SEM lucigenin-enhanced chemilumiescence counts/min/mg dry weight, n = 7. * p < 0.05 when compared to control group, # p < 0.05 when compared to L-NAME group and † p < 0.05 when compared to L-NAME+EA 7.5 mg/kg group. (B) Correlation between SBP and vascular superoxide production.

Effect of Ellagic Acid on p47 phox Protein Expression in Aortic Tissues
The expression of p47 phox protein in L-NAME treated rats was significantly up-regulated compared to control rats (p < 0.05, Figure 5A). Positive correlation of p47 phox protein expression and the values of the vascular O 2 •− production was observed ( Figure 5B). Treatment of the L-NAME group with 7.5 mg/kg ellagic acid reduced the over-expression of p47 phox protein (p < 0.05) and treatment with 15 mg/kg ellagic acid restored p47 phox protein levels to normal ( Figure 5A).
Nutrients 2015, 7 9 Figure 4. (A) Effect of ellagic acid on vascular superoxide production. Values are expressed as mean ± SEM lucigenin-enhanced chemilumiescence counts/min/mg dry weight, n = 7. * p < 0.05 when compared to control group, # p < 0.05 when compared to L-NAME group and † p < 0.05 when compared to L-NAME+EA 7.5 mg/kg group. (B) Correlation between SBP and vascular superoxide production.

Effect of Ellagic Acid on p47 phox Protein Expression in Aortic Tissues
The expression of p47 phox protein in L-NAME treated rats was significantly up-regulated compared to control rats (p < 0.05, Figure 5A). Positive correlation of p47 phox protein expression and the values of the vascular O2 •− production was observed ( Figure 5B). Treatment of the L-NAME group with 7.5 mg/kg ellagic acid reduced the over-expression of p47 phox protein (p < 0.05) and treatment with 15 mg/kg ellagic acid restored p47 phox protein levels to normal ( Figure 5A).

Discussion
The present study examined the antihypertensive effects of ellagic acid and possible mechanisms involved in L-NAME-induced hypertensive rats. The findings demonstrate that L-NAME-induced hypertension exhibited hemodynamic alterations, including high blood pressure, high HVR, and low HBF. There was a reduction of plasma NOx that was consistent with down-regulation of eNOS expression in hypertensive rats. Oxidative stress markers were significantly increased in this animal model of experimental hypertension. The protein expression of p47 phox NADPH oxidase subunit was markedly increased in hypertensive rats. Administration of ellagic acid reduced blood pressure, HR, HVR as well as increased HBF in L-NAME hypertensive rats. Decreased plasma NOx and eNOS expression in hypertensive rats were normalized by daily treatment with ellagic acid. Furthermore, ellagic acid also reduced plasma MDA, vascular O 2 •− production and alleviated the expression of the NADPH oxidase subunit in these rats. It is well established that long-term administration of the L-arginine analogue L-NAME to normotensive rats can induce NO-deficient hypertension. The precise mechanism is based on the fact that NO is synthesized and released from endothelial cells to mediate vasorelaxation [6] and L-NAME reduces NO production resulting in increased total peripheral resistance and high blood pressure [41,42]. This mechanism is supported by findings that disruption of plasma NOx level and reduced protein eNOS expression have been reported in long-term L-NAME treatment [15,43,44].
Our results demonstrate that L-NAME treatment for five weeks caused a progressive increase in blood pressure resulting from increased HR and HVR. These observations were also associated with a reduction in plasma NOx levels and down-regulation of protein eNOS expression. The doses of ellagic acid (7.5 and 15 mg/kg/day) chosen for this study represent pharmacological doses. The higher dose of 15 mg/kg/day would equate to human consumption of approximately 850 mL/day of pomegranate juice [45,46]. However, this dose did not lead to any evident toxicity as indicated by the fact that the body weight of these rats was not different to that of the vehicle-treated rats. Moreover, our results showed that ellagic acid markedly improved the symptoms of hypertension in L-NAME treated rats. The protective effect of ellagic acid was characterized by reductions in HR and HVR and augmentation of HBF. While this report is the first to directly demonstrate the protective effect of ellagic acid on L-NAME-induced hypertension, several previous studies have shown beneficial effects of consumption of pomegranate juice, which contains a high concentration of ellagic acid [31,47,48]. For example, Aviram and coworkers found that consumption of pomegranate juice (50 mL/day) for a year was able to reduce SBP in atherosclerotic patients and they suggested that the pomegranate juice had a potent antioxidative capacity as one possible mechanism [47]. In another study, NOS activity was improved in diet-induced atherosclerosis in mice after ellagic acid supplementation [48]. Finally, in diabetic Wistar rats, chronic administration of pomegranate juice extract (100 and 300 mg/kg/day) for four weeks caused a reduction in MAP presumably related to its antioxidant capacity [31].
Oxidative stress is an imbalance between the production of reactive oxygen species and the antioxidant capacity, and has been found to link with the pathogenesis of hypertension [19][20][21]. The association between oxidative stress and hypertension has been extensively demonstrated ever since the plasma level of MDA, a biomarker of lipid peroxidation, was found to be increased in patients with hypertension [49][50][51]. In L-NAME hypertensive rats, we found increases in the level of plasma MDA and vascular O 2 •− . In addition, a positive correlation between SBP and vascular O 2 •− production was shown in this study, suggesting that O 2 •− rapidly reacts with NO to produce peroxynitrite (ONOO − ) resulting in impaired NO bioavailability [52]. These findings are supported by several studies in L-NAME hypertensive rats [44,53,54]. The increase in O 2 •− production in L-NAME hypertensive rats is probably due to the augmentation of NADPH oxidase activity, a major source of vascular O 2 •− production. It has been well documented that O 2 •− production is primarily produced from NADPH oxidase, which has several subunits [23]. In this study, we found the up-regulation of p47 phox protein expression, an NADPH oxidase subunit, in the vascular tissues. This observation has been reported that in nitric oxide deficient hypertensive rats showed an over expression of NADPH oxidase subunit p47 phox with lipid peroxidation and high levels of vascular O 2 •− production [24,55]. Thus, our findings suggested that increased vascular O 2 •− level observed in L-NAME hypertensive rats play an important role for decreasing NO bioavailability as it was confirmed by the low level of plasma NOx.
Here we report that ellagic acid reduced the development of L-NAME-induced hypertension in rats by significantly reducing oxidative stress markers, and NADPH oxidase subunit p47 phox expression. There is substantial evidence to show that ellagic acid has potent antioxidative effects. Goswami and coworkers found that dietary supplementation with reduced oxidative stress and improved sexual dysfunction in type 1 diabetic rats [56]. In addition, ellagic acid was reported to prevent diet-induced atherosclerosis in WT mice and this was related to improvements in NO availability, reduced oxidative stress and activation of the Nrf2 antioxidant transcription factor [48]. It has also been reported that oxidation can alter the extracellular matrix of the arterial wall [57] and changes to the arterial wall have been implicated as a key factor in the pathogenesis of hypertension [58]. Therefore, any effects of ellagic acid on the extracellular matrix of the vascular wall of the L-NAME induced-hypertensive rat should be investigated.

Conclusions
This study demonstrates that ellagic acid exhibits protective effects on the development of hypertension induced by a NOS inhibitor. These protective effects are associated with improved NO bioavailability, higher eNOS protein expression and reduced oxidative stress status, possibly due to a reduction in NADPH oxidase subunit p47 phox expression.