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Sesamin improves endothelial dysfunction in renovascular hypertensive rats fed with a high-fat, high-sucrose diet
European Journal of Pharmacology 620 (2009) 84–89
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Sesamin improves endothelial dysfunction in renovascular hypertensive rats fed with a high-fat, high-sucrose diet Xiang Kong, Jie-ren Yang ⁎, Li-qun Guo, Ying Xiong, Xiang-qi Wu, Kai Huang, Yong Zhou Department of Pharmacology, Third-Grade Pharmacology Laboratory of State Administration of Traditional Chinese Medicine, Wannan Medical College, 10 Weiliu Road, Wuhu 241001, Anhui Province, China
a r t i c l e
i n f o
Article history: Received 10 May 2009 Received in revised form 3 July 2009 Accepted 6 August 2009 Available online 20 August 2009 Keywords: Endothelial dysfunction Endothelial nitric oxide synthase NADPH oxidase Sesamin
a b s t r a c t The present study was designed to evaluate the possible in vivo protective effects of sesamin on hypertension and endothelial function in two-kidney, one-clip renovascular hypertensive rats fed with a high-fat, high-sucrose diet (2K1C rats on HFS diet). Sesamin was orally administered for 8 weeks in 2K1C rats on HFS diet. Then, the serum malondialdehyde level was determined. The protein expression of endothelial nitric oxide synthase (eNOS), nitrotyrosine and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47phox in aortas was detected by Western blotting. Vasorelaxation response to acetylcholine and nitroprusside, and functional assessment of nitric oxide (NO) bioactivity were also determined in aortic rings. Sesamin treatment reduced systolic blood pressure, improved vasodilatation induced by acetylcholine and enhanced NO bioactivity in the thoracic aortas. These changes were associated with increased eNOS, decreased malondialdehyde content, and reduced nitrotyrosine and p47phox protein expression. All these results suggest that chronic treatment with sesamin reduces hypertension and improves endothelial dysfunction through upregulation of eNOS expression and reduction of NO oxidative inactivation in 2K1C rats on HFS diet. © 2009 Elsevier B.V. All rights reserved.
1. Introduction It is well known that diminished nitric oxide (NO) production and/ or increased reactive oxygen species, in particular superoxideinduced oxidative inactivation may lead to decrease NO availability, contributing to endothelial dysfunction and maintenance of hypertension. Several studies have shown that endothelial nitric oxide synthase (eNOS) expression and/or NO concentration are significantly increased although endothelium-dependent vasorelaxation is decreased in spontaneously hypertensive (SHR), stroke-prone spontaneously hypertensive (SHRSP), and two-kidney, one-clip renovascular hypertensive rats (Hong et al., 2000; Ülker et al., 2003a,b; McIntyre et al., 1999; García-Saura et al., 2005). These results suggest that only inactivation of NO is responsible for endothelial dysfunction in these models. However, Ma et al. (2001) reported that severe endothelial dysfunction occurred and caused not only an increase in NO inactivation, but also a decrease in its production from eNOS in SHRSP supplemented with a high-salt, high-fat diet. In fact, a link between high-fat or high-fat, high-sucrose (HFS) diet mediated oxidative stress and a decrease in NO concentration has been recently demonstrated (Rodríguez et al., 2002; Yamamoto and Oue, 2006). Chronic consumption of HFS diet induces dyslipidemia and hyperten-
⁎ Corresponding author. Tel.: +86 553 3932464. E-mail address: [email protected] (J. Yang). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.08.023
sion in genetically normal rats. Endothelial dysfunction in this model is associated with NO inactivation and downregulation of eNOS expression (Roberts et al., 2005, 2006; Bourgoin et al., 2008). Thus, we hypothesized that NO inactivation in combination with decreased eNOS expression could contribute to endothelial dysfunction in twokidney, one-clip renovascular hypertensive rats fed with a HFS diet (2K1C rats on HFS diet). Sesamin, one of the major lignan in sesame seeds has received a great deal of interest. The literatures reported that sesamin has hypolipidemic (Gu et al., 1998) and antihypertensive effect (Matsumura et al., 1995, 1998; Kita et al., 1995). Recent works demonstrated that sesamin metabolites induced a vasorelaxation in vitro (Nakano et al., 2006) and sesamin feeding enhanced endothelium-dependent relaxation in deoxycorticosterone acetate (DOCA)-salt hypertensive rats (Nakano et al., 2003). Konan et al. (2008) reported that the aqueous extract of leaves from sesame induced dose-dependent vasorelaxation in guinea-pig aortas. Nevertheless, the underlying mechanisms of in vivo protective effects of sesamin on blood pressure and endothelial function are not completely understood. In a series of recent studies, Nakano et al. (2003, 2008) suggested that sesamin decreased superoxide production and suppressed mRNA expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in aortas from DOCA-salt hypertensive rats. Lee et al. (2004) reported that sesamin increased NO concentration and induced eNOS mRNA and protein expression in the medium of human umbilical vein endothelial cells. Moreover, sesamin metabolite-induced relaxations
X. Kong et al. / European Journal of Pharmacology 620 (2009) 84–89
in isolated rat aortic rings were attenuated by pretreatment with NGnitro-L-arginine (L-NOARG, a NOS inhibitor) (Nakano et al., 2006). The aqueous extract of leaves from sesame caused relaxation was attenuated in the presence of the L-NOARG (Konan et al., 2008). These results suggested that the effects of sesamin on biosynthesis and/or reduced superoxide-mediated inactivation of NO may be involved in its protective action. Therefore, the purpose of this study was to evaluate the effects of sesamin in the development of hypertension and endothelial dysfunction in 2K1C rats on HFS diet. In addition, we further examined the roles of eNOS, NADPH oxidase subunit p47phox, and nitrotyrosine (the footprint of NO interaction with reactive oxygen species) expression in sesamin-mediated protection. 2. Materials and methods 2.1. Drugs and reagents Sesamin (>94% purity) was provided by Tianyi Lvbao Technology Co., Ltd. (Wuhu, China) with an invention patent number ZL 03113181.6 of China to extract sesamin. Its structure is shown in Fig. 1. Norepinephrine, phenylephrine, acetylcholine (an endothelium-dependent vasodilator), nitroprusside (an endothelium-independent vasodilator), and NG-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NOS) were purchased from Sigma (St. Louis, MO, USA). Total cholesterol and triglyceride assay kits were purchased from Rongsheng Biotechnology Co. (Shanghai, China). Malondialdehyde analysis kit was obtained from Jiancheng Institute of Biotechnology (Nanjing, China). The composition of Krebs solution was as follows (in mM): NaCl 118.3, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 11.1. 2.2. Animals, diet and surgery Thirty-six 6–7 week old (weighing 200–240 g) male Sprague– Dawley rats [Certificate No: SCXK(jing) 2007-0001] were purchased from Weitong Lihua Experimental Animal Co., Ltd. (Beijing, China). The rats were housed in individual cages at 24–26 °C with a 12h light–dark cycle, and acclimatized to these conditions for 1 week. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Rat chow [Certificate No: SCXK(jun) 2002-018] was purchased from the Experimental Animal Center at the Academy of Military Medical Sciences of PLA in Beijing. High-fat, high-sucrose (HFS) diet was prepared as described previously (Xu et al., 2006) with slight modification, and composed of 18% protein, 22% fat (containing 15% lard oil and 2% cholesterin), 46% carbohydrate (containing 25% sucrose), and standard vitamins and mineral mix. Specifically, the percent distribution of calories and caloric density for the two diets were as follows: 28% protein, 13% fat, 59% carbohydrate and 3.1 kcal/g for the standard diet, and 16% protein, 44% fat, 40% carbohydrate and 4.5 kcal/g for the HFS diet. Rats were anesthetized with 3% sodium pentobarbital (30 mg/kg, i. p.) after the acclimatization period. 2K1C renovascular hypertension was induced as described previously (Ono et al., 1989). Briefly, a Ushaped silver clip (0.25 mm internal diameter) was placed around the
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left renal artery in 28 rats through a midline laparotomy. Shamoperated rats underwent the same procedure, except that the clip was not applied. The wound was closed in layers, and penicillin G (100,000 U/kg) was administered intramuscularly for 3 days. 2.3. Experimental design Seven days after surgery, the 2K1C and Sham-operated rats received the HFS and standard diet, respectively. Systolic blood pressure (SBP) was measured in conscious rats using the tail-cuff method (ALC-NIBP, Alcott Biotech, Shanghai, China) after the animals were maintained in a warm chamber for about 10 min. Before the first measurement of SBP, rats were trained for 3 days to adapt to the procedure. SBP values were derived from an average of 5 measurements per animal. Five weeks after placement of the clip, 22 hypertensive rats (SBP greater than 160 mm Hg) were randomly assigned to three groups, namely a model group (2K1C on HFS, n = 8), and two treatment groups (Ses 120 and Ses 60 groups, received the same HFS diet, given sesamin by gavage at the daily dose of 120 or 60 mg/kg, n = 7 each). In addition, the sham-operated group (with standard diet, n = 8) was set up. Sesamin was suspended in 0.5% carboxymethylcellulose sodium and orally administrated at 9:00– 10:00 AM everyday for 8 weeks in two treatment groups. Untreated groups received an equal volume of carboxymethylcellulose sodium as control. At the end of the experiment, the rats were fasted overnight and anesthetized by an intraperitoneal injection of sodium pentobarbital. Blood samples were drawn from abdominal aorta, centrifuged to obtain serum, and kept at − 20 °C until assayed. Thoracic aortas of rats were carefully removed, cleaned of adhering tissue, and divided into two parts. One part contained the descending thoracic aorta which were cut into two transverse rings (3–4 mm in length) used for vascular reactivity experiments, and the other was quickly frozen in liquid nitrogen and stored −80 °C until processed. 2.4. Vascular reactivity studies Aortic rings were mounted between two steel hooks, randomly suspended in two tissue baths containing 10 mL Krebs solution at 37 °C, and continuously bubbled with carbogen. Changes in isometric tension were detected by JZ101 force transducers (Xinhang Electric Apparatus, Gaobeidian, China) and recorded via a MedLab U/8C polygraph (Medease Science and Technology, Nanjing, China). Preload (2 g) was applied to the rings, and the vessels were allowed to equilibrate for 60 min (with 4 washouts). The rings were stimulated with norepinephrine (3 × 10− 7 M) to evaluate their viability, then serially washed to baseline and equilibrated once again. Subsequently, in the first bath, the concentration-relaxation response curves to acetylcholine (10− 8 to 10− 4 M) and nitroprusside (10− 9 to 10− 6 M) were performed in rings, which were precontracted by phenylephrine (10− 6 M). The relaxant responses to acetylcholine and nitroprusside were calculated as a percentage of the response to phenylephrine. In the second bath, rings were also precontracted with 10− 6 M phenylephrine, and then incubated with 10− 4 M L-NAME. Under these conditions, the contraction induced by L-NAME resulting from a loss of physiological NO in aortas was observed. Thus, the plateau phase of L-NAME-induced contraction was measured and expressed as a percentage of the phenylephrine-induced contraction to reflect NO bioactivity. 2.5. Determination of lipid and malondialdehyde levels
Fig. 1. The structure of sesamin.
The serum levels of total cholesterol and triglyceride were determined by enzymatic colorimetric reaction with commercial diagnostic kits. Malondialdehyde, a degrading product of lipid peroxidation known as thiobarbituric acid reactive substances, was
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also determined in serum according to the thiobarbituric acid methods (Wang et al., 2009). 2.6. Western blot analysis Western blotting was performed in the aortas from each group to detect eNOS, p47phox and nitrotyrosine protein levels as previously described (Roberts et al., 2006; López-Sepúlveda et al., 2008). Proteins of aortic homogenates were separated by electrophoresis on a sodium dodecyl sulfate polyacrylamide gel. The proteins were transferred electrophoretically to polyvinylidene difluoride membranes, then incubated with primary polyclonal anti-eNOS (Boster Biotechnology, Wuhan, China), polyclonal anti-p47phox, and monoclonal anti-nitrotyrosine antibodies (SantaCruz Biotechnology, Santa Cruz, USA) overnight and with the correspondent secondary peroxidase conjugated antibodies. The blots were visualized using enhanced chemiluminescence kit (Pierce, Rockford, IL) and evaluated by densitometry. The intensity of the bands was normalized to that of β-actin detected by polyclonal antibody and values are presented as relative density. 2.7. Statistical analysis Data were expressed as mean ± S.E.M. For statistical analysis, we used one-way ANOVA followed by Newman–Keuls tests. P < 0.05 was considered statistically significant. 3. Results 3.1. Systolic blood pressure and lipid levels Before clipping the left renal artery, no significant difference in basal level of SBP was observed among all experimental groups. As shown in Table 1, SBP and serum lipid levels in the model group (2K1C rats on HFS diet) were significantly higher than those in the sham group. Daily oral administration of both 120 and 60 mg/kg sesamin induced a progressive reduction in SBP (reduced 17% and 11% at the end of 8 weeks, P < 0.01 versus model rats). Moreover, treatment with 120 mg/kg sesamin obviously attenuated the elevation of serum total cholesterol and triglyceride levels (P < 0.01 versus model rats). These results confirmed previous evidence that sesamin has hypolipidemic and antihypertensive effects. 3.2. In vitro vascular activity Acetylcholine elicited a concentration-dependent relaxation in phenylephrine-precontracted aortic rings from all experimental groups. Acetylcholine-induced vasorelaxation was significantly decreased (by 18, 23 and 28% for 10− 6, 10− 5 and 10− 4 M acetylcholine, respectively; P < 0.05 or P < 0.01) in the model group compared with the sham group. The aortic rings obtained from the 120 mg/kg sesamin treated group showed significant increase in vasodilatation induced by acetylcholine as compared to those from the model group
Table 1 Effects of sesamin on systolic blood pressure and lipid metabolism. Group
Sham Model Ses 120 Ses 60
n
8 8 7 7
SBP (mm Hg) 5 weeks
9 weeks
13 weeks
114 ± 6.4 174 ± 4.9d 172 ± 6.3d 171 ± 6.8d
115 ± 6.6 175 ± 8.0d 155 ± 6.7bd 164 ± 7.4ad
118 ± 4.8 179 ± 5.1d 142 ± 7.6bd 153 ± 9.0bd
TG (mmol/L)
TC (mmol/L)
1.03 ± 0.22 2.05 ± 0.52d 1.34 ± 0.33b 1.63 ± 0.23d
1.64 ± 0.44 3.42 ± 0.90d 2.41 ± 0.54ac 2.87 ± 0.46d
Values are expressed as mean ± S.E.M. aP < 0.05, bP < 0.01 vs. model group. cP < 0.05, d P < 0.01 vs. sham group. SBP: systolic blood pressure, TC: triglyceride, TG: total cholesterol.
(Fig. 2A). The endothelium-independent vasorelaxation induced by sodium nitroprusside was not different among all experimental groups (Fig. 2B). The addition of L-NAME to phenylephrine-precontracted aortic rings induced further vasoconstriction. The magnitude of L-NAMEinduced responses was diminished in 2K1C rats on HFS diet as compared to the sham group, indicating a reduced bioactive NO formation in the model group. A significant improvement of NO bioactivity was found in the sesamin 120 mg/kg treated group (Fig. 3). 3.3. Serum level of malondialdehyde As shown in Fig. 4, an increase of serum malondialdehyde content confirmed that oxidative damage had been induced in 2K1C rats on HFS diet. The level of malondialdehyde in the sesamin 120 mg/kg treated group was significantly lower than those in the model group (P < 0.05 versus model rats). 3.4. Protein expression of eNOS in rat aortas Compared with the sham rats, 2K1C rats on HFS diet exhibited a significant reduction of eNOS protein expression in aortic tissues. Treatment with 120 mg/kg sesamin was able to enhance protein expression of eNOS in aortas (Fig. 5). 3.5. Protein expression of nitrotyrosine and p47phox in rat aortas As shown in Fig. 6, p47phox and nitrotyrosine protein expression in aortic tissues were significantly higher in the model group compared with the sham group. These abnormalities were essentially reversed by treatment with 120 mg/kg sesamin for 8 weeks. 4. Discussion The major conclusions to be drawn from this study were that chronic treatment with sesamin significantly reduced SBP and improved acetylcholine-induced vasorelaxation in 2K1C rats on HFS diet. This effects seem to be related to restore NO bioactivity through upregulation of eNOS and suppression of p47phox and nitrotyrosine protein expression. Among the various endothelium-derived molecules, NO has been demonstrated to play a key role in the regulation of vascular tone and blood pressure. In rat aortas, specially in SHR and 2K1C, endotheliumdependent vasodilatation relies almost entirely on the endothelial release of NO (Rodriguez-Rodriguez et al., 2007; Callera et al., 2000; Kong et al., 2009). In the present study, 2K1C rats on HFS diet were accompanied by a reduced aortic relaxant response to acetylcholine. Indeed, aortic rings from the model group showed diminished bioactivity of NO, which was assessed on the basis of the magnitude of L-NAME (an inhibitor of NOS)-induced vasoconstriction. Furthermore, the relaxant response to the NO donor nitroprusside was comparable among the sham and the model groups. Taken together, these data indicated that endothelial dysfunction in 2K1C rats on HFS diet occurred and was characterized by impaired the endotheliumdependent relaxation and NO inactivation. In addition, we found that sesamin treatment improved the aortic endothelial dysfunction in 2K1C rats on HFS diet. It is of note that endothelial function plays an important role in the modulation of blood pressure. Thus, the rise in NO bioactivity and NO-mediated vasodilatation after sesamin treatment, could, in part, account for the observed amelioration of hypertension. These results confirmed and extended previous evidence about the improvement in endothelial function of sesamin in hypertensive rats (Nakano et al., 2003). Several potential mechanisms would be involved in the sesamin-induced increase of NO bioactivity, such as changes in the expression of eNOS, changes in the
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Fig. 2. Effects of sesamin on the vascular relaxant responses induced by acetylcholine (A) and nitroprusside (B) in aortic rings precontracted by 10− 6 M phenylephrine. Values are mean ± S.E.M. n = 5 to 6 rings from different rats. ⁎P < 0.05, ⁎⁎P < 0.01 vs. model group. †P < 0.05, ††P < 0.01 vs. sham group.
vascular levels of superoxide and thus superoxide-mediated NO inactivation. NO is generated within normal endothelium by eNOS from L-arginine. Similar to previous reports (Lee et al., 1999; Roberts et al., 2005), at the age of 13 weeks we have found lower eNOS protein expression levels in aortas from the model group when compared with the sham group. Long-term administration of sesamin was able to increase eNOS expression in this animal model. Several recent findings (Lee et al., 2004; Nakano et al., 2006), i.e., sesamin induced eNOS mRNA and protein expression and enhanced NOS activity in human umbilical vein endothelial cells, sesamin metabolites induced NOdependent vasorelaxation, and sesamin feeding had no antihypertensive action in chronically L-NOARG-treated rats or DOCA-salttreated eNOS−/− mice, supported this conclusion. The results suggested that the improvement of endothelial function and NO bioactivity after sesamin treatment might be attributed, at least in part, to a quantitative eNOS restoration, since the protein expression of eNOS were increased in aortas from 2K1C rats on HFS diet. Excessive release of oxygen radicals, if not controlled by the endogenous antioxidant systems, can lead to lipid peroxidation. Oxidant stress is evidenced by significantly higher malondialdehyde level in serum of 2K1C rats on HFS diet compared to the sham rats. The superoxide inactivates NO, thereby diminishing its halflife, and leads to generate peroxynitrite. Peroxynitrite, a highly reactive and cytotoxic radical attacks and denatures lipids, nucleic acids, and
proteins (Halliwell, 1997). Peroxynitrite reacts with tyrosine residues to produce nitrotyrosine, which is frequently used as a stable footprint of superoxide-mediated inactivation of NO. The elevation of aortic nitrotyrosine abundance in 2K1C rats on HFS diet was found herein and closely resembled the earlier findings in SHRSP on high-salt, highfat diet (Ma et al., 2001), SHR (Hong et al., 2000) and HFS diet-induced hypertensive rats (Roberts et al., 2000). Indeed, the increased nitrotyrosine protein expression and malondialdehyde level from the model group were tangible evidence for the presence of oxidative stress leading to enhanced NO inactivation. The NADPH oxidase has recently been characterized in several cell lines and shown to be one of the main sources of vascular superoxide (Ülker et al., 2003a,b). Upregulation of this oxidase, in particular p47phox (a cytoplasmic subunit of NADPH oxidase), can contribute to the pathogenesis of oxidative stress in several animal models of acquired and genetic hypertension (Vaziri and Ni, 2005; Sánchez et al., 2006; Yu et al., 2008; López-Sepúlveda et al., 2008). In our experimental conditions, we also found upregulation of p47phox protein expression in the vascular tissues of 2K1C rats on HFS diet. Taken together, the increased p47phox expression could raise superoxide production and thereby contributed to the elevation of nitrotyrosine expression and NO inactivation in this animal model. Several reports have recently suggested that sesamin feeding decreased aortic superoxide production and mRNA expression of NADPH oxidase subunits (p22phox, gp91phox, Nox1 and Nox4) in DOCA-salt hypertensive rats (Nakano
Fig. 3. Effects of sesamin on bioactive NO production in aortic rings. (A) Representative recording for 10− 4 M L-NAME-induced vasocontraction in phenylephrine (PE)-precontracted aortic rings from sham, model and sesamin 120 mg/kg treated rats, respectively. (B) Averaged values (mean ± S.E.M. n = 5 to 6 rings from different rats) were calculated as the percentage of increased tension induced by L-NAME vs. the plateau value induced by phenylephrine. ⁎P < 0.05 vs. model group. †P < 0.05, ††P < 0.01 vs. sham group.
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Fig. 4. Effects of sesamin on serum malondialdehyde concentration in rats from each group. Values are mean ± S.E.M. n = 7–8. ⁎P < 0.05 vs. model group. ††P < 0.01 vs. sham group. MDA: malondialdehyde.
et al., 2003; Nakano et al., 2008). In the present study, we showed that sesamin was effective to decrease serum malondialdehyde level and reduce expression of p47phox and nitrotyrosine in aortas from 2K1C rats on HFS diet. These results suggested that another cause of the effect of sesamin on improving the endothelial dysfunction and NO bioactivity is due to its antioxidative activity, which might have come from downregulation of p47phox expression and subsequent suppression of NO oxidative inactivation. In addition, oxidative stress results in depletion of the NOS cofactor tetrahydrobiopterin and in uncoupling of the NOS. The latter events, in turn, reduce NO production and promote superoxide generation by NOS (Roberts et al., 2006; Landmesser et al., 2003). Supplementation with tetrahydrobiopterin significantly reduced SBP in SHR which might be mediated through its direct antioxidant activity and/or decreasing oxygen free radical production from NOS (Hong et al., 2000). d'Uscio et al. (2003) reported that treatment with antioxidative vitamin C increased vascular tetrahydrobiopterin levels and NOS activity. Thus, the possibility of sesamin-induced increase in aortic NO bioactivity from protecting uncoupling of the NOS and its cofactor tetrahydrobiopterin from oxidative stress could be assumed. Nevertheless, further experiments should be carried out to evaluate these mechanisms. In our investigation, we also demonstrated that chronic treatment with 120 mg/kg sesamin decreased serum total cholesterol and triglyceride levels in rats. These effects were related to the sesamin properties of inhibiting lipid metabolism, such as desaturation in the biosynthesis of polyunsaturated fatty acid and cholesterol absorption (Shimizu et al., 1991; Hirose et al., 1991). The underlying mechanism is that sesamin could inhibit the HMG-CoA reductase (Hirose et al., 1991), which is the rate-limiting enzyme in the cholesterol biosynthetic pathway.
Fig. 5. Effects of sesamin on the protein expression of eNOS in aortic tissues by Western blotting. Histogram represents densitometric values normalized to the corresponding β-actin (n = 4). ⁎P < 0.05 vs. model group. †P < 0.05, ††P < 0.01 vs. sham group.
Fig. 6. Effects of sesamin on the protein expression of p47phox and nitrotyrosine in aortic tissues by Western blotting. Histograms represent densitometric values normalized to the corresponding β-actin (n = 4). ⁎P < 0.05, ⁎⁎P < 0.01 vs. model group. †P < 0.05, ††P < 0.01 vs. sham group.
In summary, our results demonstrated that orally administrated sesamin by gavage for 8 weeks reduced the elevated blood pressure and improved the vascular endothelial function in 2K1C rats fed with a HFS diet. These effects, at least in part, were associated with enhanced eNOS expression and reduced NO inactivation. Acknowledgments This work was supported by grants from the Key Program of Natural Science Foundation of Education Department of Anhui Province (No: 2006kj099A) and the Program of Universities Excellent Young Talent Foundation of Anhui Province (No: 2009SQRZ184). References Bourgoin, F., Bachelard, H., Badeau, M., Mélancon, S., Pitre, M., Larivière, R., Nadeau, A., 2008. Endothelial and vascular dysfunctions and insulin resistance in rats fed a high-fat, high-sucrose diet. Am. J. Physiol-heart C. 295, H1044–1055. Callera, G.E., Varanda, W.A., Bendhack, L.M., 2000. Impaired relaxation to acetylcholine in 2K-1C hypertensive rat aortas involves changes in membrane hyperpolarization instead of an abnormal contribution of endothelial factors. Gen. Pharmacol-vasc. S. 34, 379–389. d'Uscio, L.V., Milstien, S., Richardson, D., Smith, L., Katusic, Z.S., 2003. Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ. Res. 92, 88–95. García-Saura, M.F., Galisteo, M., Villar, I.C., Bermejo, A., Zarzuelo, A., Vargas, F., Duartel, J., 2005. Effects of chronic quercetin treatment in experimental renovascular hypertension. Mol. Cell. Biochem. 270, 147–155. Gu, J.Y., Wakizono, Y., Dohi, A., Nonaka, M., Sugano, M., Yamada, K., 1998. Effect of dietary fats and sesamin on the lipid metabolism and immune function of Sprague– Dawley rat. Biosci. Biotechnol. Biochem. 62, 1917–1924. Halliwell, B., 1997. What nitrates tyrosine? is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo? FEBS Lett. 411, 157–160. Hirose, N., Inoue, T., Nishihara, K., Sugano, M., Akimoto, K., Shimizu, S., Yamada, H., 1991. Inhibition of cholesterol absorption and synthesis in rats by sesamin. J. Lipid Res. 32, 629–638. Hong, H.J., Loh, S.H., Yen, M.H., 2000. Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase. Br. J. Pharmacol. 131, 631–637. Kita, S., Matsumura, Y., Morimoto, S., Akimoto, K., Furuya, M., Oka, N., Tanaka, T., 1995. Antihypertensive effect of sesamin. II. Protection against two-kidney, one-clip renal hypertension and cardiovascular hypertrophy. Biol. Pharm. Bull. 18, 1283–1285. Konan, A.B., Datté, J.Y., Yapo, P.A., 2008. Nitric oxide pathway-mediated relaxant effect of aqueous sesame leaves extract (Sesamum radiatum Schum. & Thonn.) in the guinea-pig isolated aorta smooth muscle. BMC Complement Altern. Med. 8, 23. Kong, X., Yang, J.R., Guo, L.Q., Zhou, Y., 2009. A comparing study of aortic function between renal hypertension rat and renal hypertensive–hyperlipidemia rat. Chin. Pharmacol. Bull. 25, 252–255. Landmesser, U., Dikalov, S., Price, S.R., McCann, L., Fukai, T., Holland, S.M., Mitch, W.E., Harrison, D.G., 2003. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J. Clin. Invest. 111, 1201–1209. Lee, J., Kang, D.G., Kook, H., Kim, I.K., Oh, B.S., 1999. Differentially-altered vascular guanylate cyclase isoforms in experimental hypertensive rats. J. Korean Med. Sci. 14, 386–392. Lee, C.C., Chen, P.R., Lin, S., Tsaia, S.C., Wanga, B.W., Chena, W.W., Tsaid, C.E., Shyua, K.G., 2004. Sesamin induces nitric oxide and decreases endothelin-1 production in HUVECs: possible implications for its antihypertensive effect. J. Hypertens. 22, 2329–2338.
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Roberts, C.K., Barnard, R.J., Sindhu, R.K., Jurczak, M., Ehdaie, A., Vaziri, N.D., 2005. A highfat, refined-carbohydrate diet induces endothelial dysfunction and oxidant/ antioxidant imbalance and depresses NOS protein expression. J. Appl. Physiol. 98, 203–210. Roberts, C.K., Barnard, R.J., Sindhu, R.K., Jurczak, M., Ehdaie, A., Vaziri, N.D., 2006. Oxidative stress and dysregulation of NAD(P)H oxidase and antioxidant enzymes in diet-induced metabolic syndrome. Metabolism 55, 928–934. Rodriguez-Rodriguez, R., Herrera, M.D., de Sotomayor, M.A., Ruiz-Gutierrez, V., 2007. Pomace Olive Oil improves endothelial function in spontaneously hypertensive rats by increasing endothelial nitric oxide synthase expression. Am. J. Hypertens. 20, 728–734. Sánchez, M., Galisteo, M., Vera, R., Villar, I.C., Zarzuelo, A., Tamargo, J., Pérez-Vizcaíno, F., Duarte, J., 2006. Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats. J. Hypertens. 24, 75–84. Shimizu, S., Akimoto, K., Shinmen, Y., Kawashima, H., Sugano, M., Yamada, H., 1991. Sesamin is a potent and specific inhibitor of delta 5 desaturase in polyunsaturated fatty acid biosynthesis. Lipids 26, 512–516. Ülker, S., McMaster, D., McKeown, P.P., Bayraktutan, U., 2003a. Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation. Cardiovasc. Res. 59, 488–500. Ülker, S., McKeown, P.P., Bayraktutan, U., 2003b. Vitamins reverse endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities. Hypertension 41, 534–539. Vaziri, N.D., Ni, Z., 2005. Expression of NOX-1, gp91phox, p47phox and P67phox in the aorta segments above and below coarctation. BBA-Gen. Subjects 1723, 321–327. Wang, J.Q., Li, J., Zou, Y.H., Cheng, W.M., Lu, C., Zhang, L., Ge, J.F., Huang, C., Jin, Y., Lv, X.W., Hu, C.M., Liu, L.P., 2009. Preventive effects of total flavonoids of Litsea coreana leve on hepatic steatosis in rats fed with high fat diet. J. Ethnopharmacol. 121, 54–60. Xu, P., Zhang, X.G., Li, Y.M., Yu, C.H., Xu, L., Xu, G.Y., 2006. Research on the protection effect of pioglitazone for non-alcoholic fatty liver disease (NAFLD) in rats. J. Zhejiang Univ. Sci. B 7, 627–633. Yamamoto, Y., Oue, E., 2006. Antihypertensive effect of Quercetin in rats fed with a high-fat high-sucrose diet. Biosci. Biotech. Bioch. 70, 933–939. Yu, Y., Fukuda, N., Yao, E.H., Matsumoto, T., Kobayashi, N., Suzuki, R., Tahira, Y., Ueno, T., Matsumoto, K., 2008. Effects of an ARB on endothelial progenitor cell function and cardiovascular oxidation in hypertension. Am. J. Hypertens. 21, 72–77.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Sesamin improves endothelial dysfunction in renovascular hypertensive rats fed with a high-fat, high-sucrose diet Xiang Kong, Jie-ren Yang ⁎, Li-qun Guo, Ying Xiong, Xiang-qi Wu, Kai Huang, Yong Zhou Department of Pharmacology, Third-Grade Pharmacology Laboratory of State Administration of Traditional Chinese Medicine, Wannan Medical College, 10 Weiliu Road, Wuhu 241001, Anhui Province, China
a r t i c l e
i n f o
Article history: Received 10 May 2009 Received in revised form 3 July 2009 Accepted 6 August 2009 Available online 20 August 2009 Keywords: Endothelial dysfunction Endothelial nitric oxide synthase NADPH oxidase Sesamin
a b s t r a c t The present study was designed to evaluate the possible in vivo protective effects of sesamin on hypertension and endothelial function in two-kidney, one-clip renovascular hypertensive rats fed with a high-fat, high-sucrose diet (2K1C rats on HFS diet). Sesamin was orally administered for 8 weeks in 2K1C rats on HFS diet. Then, the serum malondialdehyde level was determined. The protein expression of endothelial nitric oxide synthase (eNOS), nitrotyrosine and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47phox in aortas was detected by Western blotting. Vasorelaxation response to acetylcholine and nitroprusside, and functional assessment of nitric oxide (NO) bioactivity were also determined in aortic rings. Sesamin treatment reduced systolic blood pressure, improved vasodilatation induced by acetylcholine and enhanced NO bioactivity in the thoracic aortas. These changes were associated with increased eNOS, decreased malondialdehyde content, and reduced nitrotyrosine and p47phox protein expression. All these results suggest that chronic treatment with sesamin reduces hypertension and improves endothelial dysfunction through upregulation of eNOS expression and reduction of NO oxidative inactivation in 2K1C rats on HFS diet. © 2009 Elsevier B.V. All rights reserved.
1. Introduction It is well known that diminished nitric oxide (NO) production and/ or increased reactive oxygen species, in particular superoxideinduced oxidative inactivation may lead to decrease NO availability, contributing to endothelial dysfunction and maintenance of hypertension. Several studies have shown that endothelial nitric oxide synthase (eNOS) expression and/or NO concentration are significantly increased although endothelium-dependent vasorelaxation is decreased in spontaneously hypertensive (SHR), stroke-prone spontaneously hypertensive (SHRSP), and two-kidney, one-clip renovascular hypertensive rats (Hong et al., 2000; Ülker et al., 2003a,b; McIntyre et al., 1999; García-Saura et al., 2005). These results suggest that only inactivation of NO is responsible for endothelial dysfunction in these models. However, Ma et al. (2001) reported that severe endothelial dysfunction occurred and caused not only an increase in NO inactivation, but also a decrease in its production from eNOS in SHRSP supplemented with a high-salt, high-fat diet. In fact, a link between high-fat or high-fat, high-sucrose (HFS) diet mediated oxidative stress and a decrease in NO concentration has been recently demonstrated (Rodríguez et al., 2002; Yamamoto and Oue, 2006). Chronic consumption of HFS diet induces dyslipidemia and hyperten-
⁎ Corresponding author. Tel.: +86 553 3932464. E-mail address: [email protected] (J. Yang). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.08.023
sion in genetically normal rats. Endothelial dysfunction in this model is associated with NO inactivation and downregulation of eNOS expression (Roberts et al., 2005, 2006; Bourgoin et al., 2008). Thus, we hypothesized that NO inactivation in combination with decreased eNOS expression could contribute to endothelial dysfunction in twokidney, one-clip renovascular hypertensive rats fed with a HFS diet (2K1C rats on HFS diet). Sesamin, one of the major lignan in sesame seeds has received a great deal of interest. The literatures reported that sesamin has hypolipidemic (Gu et al., 1998) and antihypertensive effect (Matsumura et al., 1995, 1998; Kita et al., 1995). Recent works demonstrated that sesamin metabolites induced a vasorelaxation in vitro (Nakano et al., 2006) and sesamin feeding enhanced endothelium-dependent relaxation in deoxycorticosterone acetate (DOCA)-salt hypertensive rats (Nakano et al., 2003). Konan et al. (2008) reported that the aqueous extract of leaves from sesame induced dose-dependent vasorelaxation in guinea-pig aortas. Nevertheless, the underlying mechanisms of in vivo protective effects of sesamin on blood pressure and endothelial function are not completely understood. In a series of recent studies, Nakano et al. (2003, 2008) suggested that sesamin decreased superoxide production and suppressed mRNA expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in aortas from DOCA-salt hypertensive rats. Lee et al. (2004) reported that sesamin increased NO concentration and induced eNOS mRNA and protein expression in the medium of human umbilical vein endothelial cells. Moreover, sesamin metabolite-induced relaxations
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in isolated rat aortic rings were attenuated by pretreatment with NGnitro-L-arginine (L-NOARG, a NOS inhibitor) (Nakano et al., 2006). The aqueous extract of leaves from sesame caused relaxation was attenuated in the presence of the L-NOARG (Konan et al., 2008). These results suggested that the effects of sesamin on biosynthesis and/or reduced superoxide-mediated inactivation of NO may be involved in its protective action. Therefore, the purpose of this study was to evaluate the effects of sesamin in the development of hypertension and endothelial dysfunction in 2K1C rats on HFS diet. In addition, we further examined the roles of eNOS, NADPH oxidase subunit p47phox, and nitrotyrosine (the footprint of NO interaction with reactive oxygen species) expression in sesamin-mediated protection. 2. Materials and methods 2.1. Drugs and reagents Sesamin (>94% purity) was provided by Tianyi Lvbao Technology Co., Ltd. (Wuhu, China) with an invention patent number ZL 03113181.6 of China to extract sesamin. Its structure is shown in Fig. 1. Norepinephrine, phenylephrine, acetylcholine (an endothelium-dependent vasodilator), nitroprusside (an endothelium-independent vasodilator), and NG-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NOS) were purchased from Sigma (St. Louis, MO, USA). Total cholesterol and triglyceride assay kits were purchased from Rongsheng Biotechnology Co. (Shanghai, China). Malondialdehyde analysis kit was obtained from Jiancheng Institute of Biotechnology (Nanjing, China). The composition of Krebs solution was as follows (in mM): NaCl 118.3, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 11.1. 2.2. Animals, diet and surgery Thirty-six 6–7 week old (weighing 200–240 g) male Sprague– Dawley rats [Certificate No: SCXK(jing) 2007-0001] were purchased from Weitong Lihua Experimental Animal Co., Ltd. (Beijing, China). The rats were housed in individual cages at 24–26 °C with a 12h light–dark cycle, and acclimatized to these conditions for 1 week. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Rat chow [Certificate No: SCXK(jun) 2002-018] was purchased from the Experimental Animal Center at the Academy of Military Medical Sciences of PLA in Beijing. High-fat, high-sucrose (HFS) diet was prepared as described previously (Xu et al., 2006) with slight modification, and composed of 18% protein, 22% fat (containing 15% lard oil and 2% cholesterin), 46% carbohydrate (containing 25% sucrose), and standard vitamins and mineral mix. Specifically, the percent distribution of calories and caloric density for the two diets were as follows: 28% protein, 13% fat, 59% carbohydrate and 3.1 kcal/g for the standard diet, and 16% protein, 44% fat, 40% carbohydrate and 4.5 kcal/g for the HFS diet. Rats were anesthetized with 3% sodium pentobarbital (30 mg/kg, i. p.) after the acclimatization period. 2K1C renovascular hypertension was induced as described previously (Ono et al., 1989). Briefly, a Ushaped silver clip (0.25 mm internal diameter) was placed around the
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left renal artery in 28 rats through a midline laparotomy. Shamoperated rats underwent the same procedure, except that the clip was not applied. The wound was closed in layers, and penicillin G (100,000 U/kg) was administered intramuscularly for 3 days. 2.3. Experimental design Seven days after surgery, the 2K1C and Sham-operated rats received the HFS and standard diet, respectively. Systolic blood pressure (SBP) was measured in conscious rats using the tail-cuff method (ALC-NIBP, Alcott Biotech, Shanghai, China) after the animals were maintained in a warm chamber for about 10 min. Before the first measurement of SBP, rats were trained for 3 days to adapt to the procedure. SBP values were derived from an average of 5 measurements per animal. Five weeks after placement of the clip, 22 hypertensive rats (SBP greater than 160 mm Hg) were randomly assigned to three groups, namely a model group (2K1C on HFS, n = 8), and two treatment groups (Ses 120 and Ses 60 groups, received the same HFS diet, given sesamin by gavage at the daily dose of 120 or 60 mg/kg, n = 7 each). In addition, the sham-operated group (with standard diet, n = 8) was set up. Sesamin was suspended in 0.5% carboxymethylcellulose sodium and orally administrated at 9:00– 10:00 AM everyday for 8 weeks in two treatment groups. Untreated groups received an equal volume of carboxymethylcellulose sodium as control. At the end of the experiment, the rats were fasted overnight and anesthetized by an intraperitoneal injection of sodium pentobarbital. Blood samples were drawn from abdominal aorta, centrifuged to obtain serum, and kept at − 20 °C until assayed. Thoracic aortas of rats were carefully removed, cleaned of adhering tissue, and divided into two parts. One part contained the descending thoracic aorta which were cut into two transverse rings (3–4 mm in length) used for vascular reactivity experiments, and the other was quickly frozen in liquid nitrogen and stored −80 °C until processed. 2.4. Vascular reactivity studies Aortic rings were mounted between two steel hooks, randomly suspended in two tissue baths containing 10 mL Krebs solution at 37 °C, and continuously bubbled with carbogen. Changes in isometric tension were detected by JZ101 force transducers (Xinhang Electric Apparatus, Gaobeidian, China) and recorded via a MedLab U/8C polygraph (Medease Science and Technology, Nanjing, China). Preload (2 g) was applied to the rings, and the vessels were allowed to equilibrate for 60 min (with 4 washouts). The rings were stimulated with norepinephrine (3 × 10− 7 M) to evaluate their viability, then serially washed to baseline and equilibrated once again. Subsequently, in the first bath, the concentration-relaxation response curves to acetylcholine (10− 8 to 10− 4 M) and nitroprusside (10− 9 to 10− 6 M) were performed in rings, which were precontracted by phenylephrine (10− 6 M). The relaxant responses to acetylcholine and nitroprusside were calculated as a percentage of the response to phenylephrine. In the second bath, rings were also precontracted with 10− 6 M phenylephrine, and then incubated with 10− 4 M L-NAME. Under these conditions, the contraction induced by L-NAME resulting from a loss of physiological NO in aortas was observed. Thus, the plateau phase of L-NAME-induced contraction was measured and expressed as a percentage of the phenylephrine-induced contraction to reflect NO bioactivity. 2.5. Determination of lipid and malondialdehyde levels
Fig. 1. The structure of sesamin.
The serum levels of total cholesterol and triglyceride were determined by enzymatic colorimetric reaction with commercial diagnostic kits. Malondialdehyde, a degrading product of lipid peroxidation known as thiobarbituric acid reactive substances, was
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also determined in serum according to the thiobarbituric acid methods (Wang et al., 2009). 2.6. Western blot analysis Western blotting was performed in the aortas from each group to detect eNOS, p47phox and nitrotyrosine protein levels as previously described (Roberts et al., 2006; López-Sepúlveda et al., 2008). Proteins of aortic homogenates were separated by electrophoresis on a sodium dodecyl sulfate polyacrylamide gel. The proteins were transferred electrophoretically to polyvinylidene difluoride membranes, then incubated with primary polyclonal anti-eNOS (Boster Biotechnology, Wuhan, China), polyclonal anti-p47phox, and monoclonal anti-nitrotyrosine antibodies (SantaCruz Biotechnology, Santa Cruz, USA) overnight and with the correspondent secondary peroxidase conjugated antibodies. The blots were visualized using enhanced chemiluminescence kit (Pierce, Rockford, IL) and evaluated by densitometry. The intensity of the bands was normalized to that of β-actin detected by polyclonal antibody and values are presented as relative density. 2.7. Statistical analysis Data were expressed as mean ± S.E.M. For statistical analysis, we used one-way ANOVA followed by Newman–Keuls tests. P < 0.05 was considered statistically significant. 3. Results 3.1. Systolic blood pressure and lipid levels Before clipping the left renal artery, no significant difference in basal level of SBP was observed among all experimental groups. As shown in Table 1, SBP and serum lipid levels in the model group (2K1C rats on HFS diet) were significantly higher than those in the sham group. Daily oral administration of both 120 and 60 mg/kg sesamin induced a progressive reduction in SBP (reduced 17% and 11% at the end of 8 weeks, P < 0.01 versus model rats). Moreover, treatment with 120 mg/kg sesamin obviously attenuated the elevation of serum total cholesterol and triglyceride levels (P < 0.01 versus model rats). These results confirmed previous evidence that sesamin has hypolipidemic and antihypertensive effects. 3.2. In vitro vascular activity Acetylcholine elicited a concentration-dependent relaxation in phenylephrine-precontracted aortic rings from all experimental groups. Acetylcholine-induced vasorelaxation was significantly decreased (by 18, 23 and 28% for 10− 6, 10− 5 and 10− 4 M acetylcholine, respectively; P < 0.05 or P < 0.01) in the model group compared with the sham group. The aortic rings obtained from the 120 mg/kg sesamin treated group showed significant increase in vasodilatation induced by acetylcholine as compared to those from the model group
Table 1 Effects of sesamin on systolic blood pressure and lipid metabolism. Group
Sham Model Ses 120 Ses 60
n
8 8 7 7
SBP (mm Hg) 5 weeks
9 weeks
13 weeks
114 ± 6.4 174 ± 4.9d 172 ± 6.3d 171 ± 6.8d
115 ± 6.6 175 ± 8.0d 155 ± 6.7bd 164 ± 7.4ad
118 ± 4.8 179 ± 5.1d 142 ± 7.6bd 153 ± 9.0bd
TG (mmol/L)
TC (mmol/L)
1.03 ± 0.22 2.05 ± 0.52d 1.34 ± 0.33b 1.63 ± 0.23d
1.64 ± 0.44 3.42 ± 0.90d 2.41 ± 0.54ac 2.87 ± 0.46d
Values are expressed as mean ± S.E.M. aP < 0.05, bP < 0.01 vs. model group. cP < 0.05, d P < 0.01 vs. sham group. SBP: systolic blood pressure, TC: triglyceride, TG: total cholesterol.
(Fig. 2A). The endothelium-independent vasorelaxation induced by sodium nitroprusside was not different among all experimental groups (Fig. 2B). The addition of L-NAME to phenylephrine-precontracted aortic rings induced further vasoconstriction. The magnitude of L-NAMEinduced responses was diminished in 2K1C rats on HFS diet as compared to the sham group, indicating a reduced bioactive NO formation in the model group. A significant improvement of NO bioactivity was found in the sesamin 120 mg/kg treated group (Fig. 3). 3.3. Serum level of malondialdehyde As shown in Fig. 4, an increase of serum malondialdehyde content confirmed that oxidative damage had been induced in 2K1C rats on HFS diet. The level of malondialdehyde in the sesamin 120 mg/kg treated group was significantly lower than those in the model group (P < 0.05 versus model rats). 3.4. Protein expression of eNOS in rat aortas Compared with the sham rats, 2K1C rats on HFS diet exhibited a significant reduction of eNOS protein expression in aortic tissues. Treatment with 120 mg/kg sesamin was able to enhance protein expression of eNOS in aortas (Fig. 5). 3.5. Protein expression of nitrotyrosine and p47phox in rat aortas As shown in Fig. 6, p47phox and nitrotyrosine protein expression in aortic tissues were significantly higher in the model group compared with the sham group. These abnormalities were essentially reversed by treatment with 120 mg/kg sesamin for 8 weeks. 4. Discussion The major conclusions to be drawn from this study were that chronic treatment with sesamin significantly reduced SBP and improved acetylcholine-induced vasorelaxation in 2K1C rats on HFS diet. This effects seem to be related to restore NO bioactivity through upregulation of eNOS and suppression of p47phox and nitrotyrosine protein expression. Among the various endothelium-derived molecules, NO has been demonstrated to play a key role in the regulation of vascular tone and blood pressure. In rat aortas, specially in SHR and 2K1C, endotheliumdependent vasodilatation relies almost entirely on the endothelial release of NO (Rodriguez-Rodriguez et al., 2007; Callera et al., 2000; Kong et al., 2009). In the present study, 2K1C rats on HFS diet were accompanied by a reduced aortic relaxant response to acetylcholine. Indeed, aortic rings from the model group showed diminished bioactivity of NO, which was assessed on the basis of the magnitude of L-NAME (an inhibitor of NOS)-induced vasoconstriction. Furthermore, the relaxant response to the NO donor nitroprusside was comparable among the sham and the model groups. Taken together, these data indicated that endothelial dysfunction in 2K1C rats on HFS diet occurred and was characterized by impaired the endotheliumdependent relaxation and NO inactivation. In addition, we found that sesamin treatment improved the aortic endothelial dysfunction in 2K1C rats on HFS diet. It is of note that endothelial function plays an important role in the modulation of blood pressure. Thus, the rise in NO bioactivity and NO-mediated vasodilatation after sesamin treatment, could, in part, account for the observed amelioration of hypertension. These results confirmed and extended previous evidence about the improvement in endothelial function of sesamin in hypertensive rats (Nakano et al., 2003). Several potential mechanisms would be involved in the sesamin-induced increase of NO bioactivity, such as changes in the expression of eNOS, changes in the
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Fig. 2. Effects of sesamin on the vascular relaxant responses induced by acetylcholine (A) and nitroprusside (B) in aortic rings precontracted by 10− 6 M phenylephrine. Values are mean ± S.E.M. n = 5 to 6 rings from different rats. ⁎P < 0.05, ⁎⁎P < 0.01 vs. model group. †P < 0.05, ††P < 0.01 vs. sham group.
vascular levels of superoxide and thus superoxide-mediated NO inactivation. NO is generated within normal endothelium by eNOS from L-arginine. Similar to previous reports (Lee et al., 1999; Roberts et al., 2005), at the age of 13 weeks we have found lower eNOS protein expression levels in aortas from the model group when compared with the sham group. Long-term administration of sesamin was able to increase eNOS expression in this animal model. Several recent findings (Lee et al., 2004; Nakano et al., 2006), i.e., sesamin induced eNOS mRNA and protein expression and enhanced NOS activity in human umbilical vein endothelial cells, sesamin metabolites induced NOdependent vasorelaxation, and sesamin feeding had no antihypertensive action in chronically L-NOARG-treated rats or DOCA-salttreated eNOS−/− mice, supported this conclusion. The results suggested that the improvement of endothelial function and NO bioactivity after sesamin treatment might be attributed, at least in part, to a quantitative eNOS restoration, since the protein expression of eNOS were increased in aortas from 2K1C rats on HFS diet. Excessive release of oxygen radicals, if not controlled by the endogenous antioxidant systems, can lead to lipid peroxidation. Oxidant stress is evidenced by significantly higher malondialdehyde level in serum of 2K1C rats on HFS diet compared to the sham rats. The superoxide inactivates NO, thereby diminishing its halflife, and leads to generate peroxynitrite. Peroxynitrite, a highly reactive and cytotoxic radical attacks and denatures lipids, nucleic acids, and
proteins (Halliwell, 1997). Peroxynitrite reacts with tyrosine residues to produce nitrotyrosine, which is frequently used as a stable footprint of superoxide-mediated inactivation of NO. The elevation of aortic nitrotyrosine abundance in 2K1C rats on HFS diet was found herein and closely resembled the earlier findings in SHRSP on high-salt, highfat diet (Ma et al., 2001), SHR (Hong et al., 2000) and HFS diet-induced hypertensive rats (Roberts et al., 2000). Indeed, the increased nitrotyrosine protein expression and malondialdehyde level from the model group were tangible evidence for the presence of oxidative stress leading to enhanced NO inactivation. The NADPH oxidase has recently been characterized in several cell lines and shown to be one of the main sources of vascular superoxide (Ülker et al., 2003a,b). Upregulation of this oxidase, in particular p47phox (a cytoplasmic subunit of NADPH oxidase), can contribute to the pathogenesis of oxidative stress in several animal models of acquired and genetic hypertension (Vaziri and Ni, 2005; Sánchez et al., 2006; Yu et al., 2008; López-Sepúlveda et al., 2008). In our experimental conditions, we also found upregulation of p47phox protein expression in the vascular tissues of 2K1C rats on HFS diet. Taken together, the increased p47phox expression could raise superoxide production and thereby contributed to the elevation of nitrotyrosine expression and NO inactivation in this animal model. Several reports have recently suggested that sesamin feeding decreased aortic superoxide production and mRNA expression of NADPH oxidase subunits (p22phox, gp91phox, Nox1 and Nox4) in DOCA-salt hypertensive rats (Nakano
Fig. 3. Effects of sesamin on bioactive NO production in aortic rings. (A) Representative recording for 10− 4 M L-NAME-induced vasocontraction in phenylephrine (PE)-precontracted aortic rings from sham, model and sesamin 120 mg/kg treated rats, respectively. (B) Averaged values (mean ± S.E.M. n = 5 to 6 rings from different rats) were calculated as the percentage of increased tension induced by L-NAME vs. the plateau value induced by phenylephrine. ⁎P < 0.05 vs. model group. †P < 0.05, ††P < 0.01 vs. sham group.
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Fig. 4. Effects of sesamin on serum malondialdehyde concentration in rats from each group. Values are mean ± S.E.M. n = 7–8. ⁎P < 0.05 vs. model group. ††P < 0.01 vs. sham group. MDA: malondialdehyde.
et al., 2003; Nakano et al., 2008). In the present study, we showed that sesamin was effective to decrease serum malondialdehyde level and reduce expression of p47phox and nitrotyrosine in aortas from 2K1C rats on HFS diet. These results suggested that another cause of the effect of sesamin on improving the endothelial dysfunction and NO bioactivity is due to its antioxidative activity, which might have come from downregulation of p47phox expression and subsequent suppression of NO oxidative inactivation. In addition, oxidative stress results in depletion of the NOS cofactor tetrahydrobiopterin and in uncoupling of the NOS. The latter events, in turn, reduce NO production and promote superoxide generation by NOS (Roberts et al., 2006; Landmesser et al., 2003). Supplementation with tetrahydrobiopterin significantly reduced SBP in SHR which might be mediated through its direct antioxidant activity and/or decreasing oxygen free radical production from NOS (Hong et al., 2000). d'Uscio et al. (2003) reported that treatment with antioxidative vitamin C increased vascular tetrahydrobiopterin levels and NOS activity. Thus, the possibility of sesamin-induced increase in aortic NO bioactivity from protecting uncoupling of the NOS and its cofactor tetrahydrobiopterin from oxidative stress could be assumed. Nevertheless, further experiments should be carried out to evaluate these mechanisms. In our investigation, we also demonstrated that chronic treatment with 120 mg/kg sesamin decreased serum total cholesterol and triglyceride levels in rats. These effects were related to the sesamin properties of inhibiting lipid metabolism, such as desaturation in the biosynthesis of polyunsaturated fatty acid and cholesterol absorption (Shimizu et al., 1991; Hirose et al., 1991). The underlying mechanism is that sesamin could inhibit the HMG-CoA reductase (Hirose et al., 1991), which is the rate-limiting enzyme in the cholesterol biosynthetic pathway.
Fig. 5. Effects of sesamin on the protein expression of eNOS in aortic tissues by Western blotting. Histogram represents densitometric values normalized to the corresponding β-actin (n = 4). ⁎P < 0.05 vs. model group. †P < 0.05, ††P < 0.01 vs. sham group.
Fig. 6. Effects of sesamin on the protein expression of p47phox and nitrotyrosine in aortic tissues by Western blotting. Histograms represent densitometric values normalized to the corresponding β-actin (n = 4). ⁎P < 0.05, ⁎⁎P < 0.01 vs. model group. †P < 0.05, ††P < 0.01 vs. sham group.
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