Research Article

Apocynin improves endothelial function and prevents the development of hypertension in fructose fed rat Banappa S. Unger, Basangouda M. Patil1

ABSTRACT

BBackgroundackground andand Objectives:Objectives: Exaggerated production of and inactivation of Department of Pharmacology nitric oxide have been implicated in pathogenesis of hypertension. NAD(P)H oxidase is and Toxicology, KLES’s College of one of the major source of in vasculature. In the present study, we Pharmacy, J N Medical College aimed to determine the effect of chronic administration of Apocynin an NAD(P)H oxidase Campus, Nehru Nagar, inhibitor on endothelial function and hypertension in fructose-fed rat. Belgaum - 590 010, MMaterialsaterials andand Methods:Methods: Endothelial function, vascular superoxide, and nitric oxide 1 KLES’s College of Pharmacy, production/bioavailability in aortas from fructose-fed rats and age-matched controls Vidya nagar, Hubli - 580 031, treated with or without apocynin were assessed using isometric tension studies in organ Karnataka, India chambers. Systolic blood pressure was measured by the tail cuff method. RReceived:eceived: 02.02.2009 RResults:esults: In fructose-fed rats, acetylcholine-induced relaxation was impaired, vascular RRevised:evised: 06.05.2009 superoxide production was increased, and nitric oxide bioavailability was decreased along AAccepted:ccepted: 26.10.2009 with an increase in systolic blood pressure compared to controls. Apocynin treatment prevented the increased generation of superoxide, decreased nitric oxide bioavailability, DDOI:OI: 10.4103/0253-7613.58508 impaired acetylcholine-induced relaxation, and elevation of systolic blood pressure. CConclusion:onclusion: Chronic administration of apocynin improves the endothelial function by CCorrespondenceorrespondence to:to: reducing oxidative stress, improving NO bioavailability, and prevents the development Dr. Basangouda M. Patil hypertension in fructose-fed rat. E-mail: [email protected]

KKEYEY WWORDS:ORDS: Superoxide, nitric oxide, NAD(P)H oxidase, apocynin, endothelial dysfunction, hypertension

Introduction aimed to determine the effect of chronic administration of apocynin, an NAD(P)H oxidase inhibitor, on endothelial Hypertension, a component of metabolic/insulin resistance function and development of hypertension in fructose-fed rats. syndrome, is an important risk factor for cardiovascular disease contributing to the increased morbidity and mortality. Materials and Methods Oxidative stress has emerged as an important pathogenic factor in the development of hypertension.[1-5] Exaggerated Animals and experimental design Male SD rats (175-200 g) were procured from National production of superoxide (O2 ) by the vascular wall has been observed in different animal models of hypertension including Center for Lab Animal Sciences, National Institute of Nutrition, fructose-fed rat.[6-9] Growing evidence supports the possibility Hyderabad, India. They were acclimatized for laboratory that increased oxidative inactivation of nitric oxide (NO) by an conditions for 7 days and randomly divided into four groups excess superoxide may account for the decrease in availability each having seven animals. (1) Control (C): Fed with standard of nitric oxide and endothelial dysfunction contributing to chow diet; (2) fructose fed (F): Fed with high fructose diet elevation of blood pressure.[10,11] A fructose-fed rat, an animal (60% of fructose); (3) control apocynin (CA): Fed with model of insulin resistance state has been shown to exhibit standard chow diet plus apocynin (1.5 mM) in drinking water; hypertension and endothelial dysfunction.[12,13] Recently, (4) fructose apocynin (FA): Fed with high fructose diet plus increased superoxide production in vascular tissue, mediated apocynin (1.5 mM) in drinking water. The animals were housed through NAD(P) H oxidase has been noted.[9] We therefore under standard laboratory conditions and maintained under a postulate that increased vascular superoxide production by 12-h light-dark cycle and had free access to drinking water and NAD(P)H oxidase is responsible for endothelial dysfunction, diet for 8 weeks. The experiment was carried out according which in turn may contribute to the development of hypertension to guidelines of Committee for the Purpose of Control and in insulin-resistance state. Hence, in the present study, we Supervision of Experiments on Animals (CPCSEA), Ministry of

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Social Justice and Empowerment, Govt. of India. Institutional submaximal precontraction of the vessel with phenylephrine Animal Ethical Committee approved all the procedures. Systolic (1 106 M) as previously described.[14] To prevent synthesis of blood pressure was measured once in a week. At the end of the prostaglandins, we performed the experiment in the presence experimental period, the rats were killed and thoracic aorta was of 10 mM indomethacin. The seven number of aortic ring isolated for isometric tension studies in the organ chamber. preparation each isolated from seven animals per group were Measurement of systolic blood pressure used for the experiment (i.e. N 7). Systolic blood pressure was measured indirectly in a Drugs and chemicals conscious, pre-warmed, and slightly restrained rat by the tail L-NAME, tempol, acetylcholine, sodium nitroprusside, cuff method (Harvard rat-tail blood pressure monitor, USA). L-phenyephrine, and apocynin/acetovanillone were purchased An average of eight consecutive readings was noted. For these from Sigma Chemical Company Inc., St Louis, MO, USA. measurements, rats were trained adequately before the study. Indomethacin was obtained as a gift sample from Sun Pharma, Chennai, India. All other chemicals used were analytical grade Isometric tension studies in organ chamber purchased from Himedia Laboratories Ltd, Mumbai, India. Aortic ring preparation Data analysis Rats were killed by cervical dislocation under mild ether Data in the manuscript are expressed as Mean SEM. anesthesia and thoracic aorta was isolated and cut into ring Comparisons between groups were made using one-way ANOVA. segments of approximately 4 mm length and mounted in When significance was indicated, a Student-Newman-Keuls post organ chambers for isometric tension recording. The vessel hoc analysis was used. Statistical significance was assumed rings were mounted onto two parallel stainless-steel pins at P 0.05. through the lumen and placed in a chamber containing a 20 ml Krebs-Henseleit buffer (pH 7.4) solution, which was maintained Results at 37C and gassed with 5% CO and 95% O . Over a period of 1 h 2 2 Systolic blood pressure equilibration period, the resting tension was gradually increased Fructose-fed rats exhibit elevation in systolic blood pressure to 2 g. The vessels were left at this resting tension throughout within 1 week (14.41%, P 0.05) but became significantly the remainder of the study. Isometric tension developed in high by the second week (18.8% P 0.001) and continued to vasculature was recorded by using a force transducer and a increase till eighth week (24% P 0.001) compared to chow-fed computerized data acquisition system (Biopac Systems Inc., CA). control [Figure 1]. Interestingly, this increase was completely Separate aortic ring preparation (n 7) was used for each of prevented in apocynin-treated fructose-fed rats [Figure 1]. the following experiments such as endothelium-dependent However, apocynin had no effect on systolic blood pressure in and -independent relaxation (procedure described in the next chow-fed control rats. section), superoxide production (procedure described in section Vascular reactivity (endothelium-dependent and -independent Measurement of vascular superoxide production) and NO relaxation) bioavailability (procedure described in section Measurement The endothelium-dependent vasodilator acetylcholine of vascular NO bioavailability). (Ach) and endothelium-independent vasodilator (SNP) elicited Measurement of vascular reactivity (endothelium-dependent the concentration-dependent relaxation of phenylephrine and -independent relaxation) preconstricted isolated aortic rings of all animals Concentration-response curves to acetylcholine (endo [Figures 2 and 3]. Aortic rings of fructose-fed rat had a markedly thelium-dependent vasodilator) and sodium nitroprusside impaired endothelium-dependent relaxation as compared (endothelium-independent vasodilator) were performed after to control [Figure 2]. As shown in Figure 2, the maximum submaximal precontraction of the vessel with phenyephrine (1 106 M). The seven number of aortic ring preparation Figure 1: Systolic blood pressure in control and fructose fed-animals each isolated from seven animals per group were used for treated with or without apocynin. Control, control apocynin, fructose fed, fructose apocynin. **P 0.01 F vs. C, **P 0.01 FA vs. F experiment (i.e. N 7). Measurement of vascular superoxide production A main factor limiting the bioavailability of NO is the superoxide anion (O2 ). We therefore assessed O2 formation in the aorta. Super oxide level was measured as Tempol (1 104 M, SOD mimetic) induced relaxation of isolated rat aorta after submaximal precontraction of the vessel with phenylephrine (1 106 M) as previously described.[14] To prevent synthesis of prostaglandins, we performed the experiments in the presence of 10 mM indomethacin. The seven aortic ring preparations each isolated from seven animals per group were used for experiment (i.e., N 7). Measurement of vascular NO bioavailability Aortic nitric oxide bioavailability was measured as a NOS inhibitor [NG-nitro-L-arginine methyl ester (L-NAME, 1 104 M)] induced contraction of isolated rat aorta after

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relaxation response (Emax) induced by acetylcholine was revealed a marked increase in vascular O2 in aorta isolated significantly reduced in aortas isolated from high fructose from fructose-fed rats compared with levels in aorta isolated diet-fed rats when compared to chow-fed rats (47.42 8.69 from control. This increase was prevented in aortic rings vs. 78.26 5.91, P 0.05). Note, this impaired response to isolated from apocynin-treated fructose-fed rats [Figure 4]. Ach was normalized by treatment with the NAD(P)H oxidase However, in chow-fed control, apocynin had no significant inhibitor apocynin (68.56 7.79 vs. 47.42 8.69, P 0.05) effect on O2 level. [Figure 2]. The sensitivity of the aortae from all experimental Vascular NO bioavailability groups to acetylcholine was similar as indicated by no significant A significant decrease in L-NAME-induced contraction of difference in pD values among the groups. The sensitivity 2 isolated rat aorta preconstricted with phenylephrine revealed a (pD ) and maximum relaxation response (E ) induced by the 2 max marked decrease in vascular NO bioavailability in aorta isolated endothelium-independent vasodilator sodium nitroprusside from fructose-fed rats compared with control. This decrease were not significantly different among the groups [Figure 3]. was prevented in apocynin-treated fructose-fed rat [Figure 5]. Vascular superoxide production However, in chow fed control, apocynin had no significant effect A significant increase in tempol (SOD mimetic)-induced on NO level. relaxation of isolated rat aorta preconstricted with phenylephrine

Figure 2: Acetylcholine-induced relaxation of isolated rat aorta Figure 3: Sodium nitroprusside-induced relaxation of isolated rat aorta preconstricted with phenylephrine in control and fructose-fed animals preconstricted with phenylephrine in control and fructose-fed animals treated with or without apocynin. Control, control apocynin, fructose treated with or without apocynin. Control, control apocynin, fructose fed, fructose apocynin. *P 0.05 F vs. C, *P 0.05 FA vs. F fed, fructose apocynin

Figure 4: Super-oxide level measured as tempo-induced relaxation Figure 5: Nitric oxide level measured as L-NAME-induced contraction of isolated rat aorta preconstricted with phenylephrine in control of isolated rat aorta preconstricted with phenylephrine in control and fructose-fed animals treated with or without apocynin. Control, and fructose-fed animals treated with or without apocynin. Control, control apocynin, fructose fed, fructose apocynin. **P 0.01 control apocynin, fructose fed, fructose apocynin. *P 0.05 F vs. C, ** P 0.01 FA vs. F F vs. C, *P 0.05 FA vs. F

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Discussion and Conclusion from fructose-fed rat and endothelial dysfunction precedes the development of hypertension.[30,31] Thus, it is clearly possible In the present study, we demonstrate for the first time that that improved endothelial function as a result of inhibition of chronic administration of apocynin, a putative NAD(P)H oxidase NAD(P)H oxidase might contribute to decreased systolic BP in inhibitor, prevents the development of endothelial dysfunction apocynin-supplemented fructose-fed rat. and hypertension by inhibiting exaggerated vascular superoxide Fructose-fed rat, a widely used animal model of insulin production and increasing the bioavailability of nitric oxide, resistance syndrome, exhibits oxidative stress, endothelial supporting the hypothesis that reduced NO bioavailability due dysfunction, and hypertension. Treatment with antioxidants has to increased reactive oxygen species (ROS, superoxide) plays a been shown to reduce blood pressure in this model.[28,29] Recently, critical role in the development of endothelial dysfunction and it has been reported that vascular superoxide production is hypertension in fructose-fed rat. increased in fructose-fed rat, and in vitro incubation with apocynin Growing body of evidence supports the role of ROS in prevents this increase in vascular superoxide production.[9] pathogenesis of hypertension. This notion is supported by Further, it has been found that NAD(P)H oxidase expression and the observation that vascular ROS production is elevated in activity are increased in cardiovascular tissues in fructose-fed different experimental models of hypertension.[6-9] Increased animals.[9,30] However, there was lack of evidence to support O level is known to inactivate the vasodilator nitric oxide, 2 contribution of NAD(P)H oxidase-mediated superoxide production leading to endothelial dysfunction, a characteristic feature of to the development of endothelial dysfunction and hypertension in many vascular diseases, including hypertension.[15-17] In various fructose-fed rats. In the present study, we observed that apocynin animal models of hypertension, an antioxidant superoxide administration prevented the development of endothelial dismutase (SOD) and SOD mimetics were found to lower the dysfunction by inhibiting increased generation of superoxide blood pressure suggesting that the oxidative inactivation of NO and thus increasing the bioavailability of NO. These observations contributes to blood pressure elevation.[18-20] In the recent past, support the notion that activation of vascular NAD(P)H oxidase many studies have demonstrated that the major source of ROS plays a critical role in the development of endothelial dysfunction in the vasculature is NAD(P)H oxidase. NAD(P)H oxidase enzyme in fructose-fed rats. It has been reported that superoxide expression and activity are increased in cardiovascular tissues production is increased well before (first week) the development of animal models of hypertension.[21,22] of hypertension in aortae from fructose-fed rat and endothelial Apocynin, a methoxy-substituted catechol from the dysfunction precedes the development of hypertension.[30,31] Thus, herb Picrorhiza kurroa, a orally active reversible inhibitor of it is clearly possible that improved endothelial function as a result NAD(P)H oxidase, has been reported to inhibit the activation of of inhibition of NAD(P)H oxidase might contribute to decreased NAD(P)H oxidase by blocking the assembly of a functional NADPH systolic BP in apocynin-supplemented fructose-fed rat. oxidase complex.[23]Administration of apocynin reduces vascular O2 production and attenuates hypertension in hypertensive Conclusion rats.[24-26] Thus, there is compelling evidence to suggest a role for ROS mediated by NAD(P)H oxidase in the pathogenesis of This work demonstrates that NAD(P)H oxidase-mediated hypertension. Previous studies have demonstrated that apocynin excessive superoxide production may reduce the bioavailability inhibits the NAD(P)H oxidase in the vasculature, indicating of NO which may account for endothelial dysfunction and suitability, selectivity, and wide usage of this compound as a hypertension in fructose-fed rats. These findings may provide pharmacological tool for vascular NAD(P)H oxidase inhibition.[23-27] additional evidence/basis for therapeutic interventions aimed at Fructose-fed rat, a widely used animal model of insulin reducing superoxide-induced vascular dysfunctions associated resistance syndrome, exhibits oxidative stress, endothelial with insulin resistance and hypertension. dysfunction, and hypertension. Treatment with antioxidants References has been shown to reduce blood pressure in this model.[28,29] Recently, it has been reported that vascular superoxide 1. Touyz RM, Paravicini TM. Redox signaling in hypertension. 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