Male SD rats (175-200 g) were procured from National Center for Lab Animal Sciences, National Institute of Nutrition, Hyderabad, India. They were acclimatized for laboratory conditions for 7 days and randomly divided into four groups each having seven animals. (1) Control (C): Fed with standard chow diet; (2) fructose fed (F): Fed with high fructose diet (60% of fructose); (3) control + apocynin (CA): Fed with standard chow diet plus apocynin (1.5 mM) in drinking water; (4) fructose + apocynin (FA): Fed with high fructose diet plus apocynin (1.5 mM) in drinking water. The animals were housed under standard laboratory conditions and maintained under a 12-h light-dark cycle and had free access to drinking water and diet for 8 weeks. The experiment was carried out according to guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Govt. of India. Institutional Animal Ethical Committee approved all the procedures. Systolic blood pressure was measured once in a week. At the end of the experimental period, the rats were killed and thoracic aorta was isolated for isometric tension studies in the organ chamber.

Systolic blood pressure was measured indirectly in a conscious, pre-warmed, and slightly restrained rat by the tail cuff method (Harvard rat-tail blood pressure monitor, USA). An average of eight consecutive readings was noted. For these measurements, rats were trained adequately before the study.

Aortic ring preparation

Rats were killed by cervical dislocation under mild ether anesthesia and thoracic aorta was isolated and cut into ring segments of approximately 4 mm length and mounted in organ chambers for isometric tension recording. The vessel rings were mounted onto two parallel stainless-steel pins through the lumen and placed in a chamber containing a 20 ml Krebs-Henseleit buffer (pH 7.4) solution, which was maintained at 37?C and gassed with 5% CO₂ and 95% O₂. Over a period of 1 h equilibration period, the resting tension was gradually increased to 2 g. The vessels were left at this resting tension throughout the remainder of the study. Isometric tension developed in vasculature was recorded by using a force transducer and a computerized data acquisition system (Biopac Systems Inc., CA). Separate aortic ring preparation (n = 7) was used for each of the following experiments such as endothelium-dependent and -independent relaxation (procedure described in the next section), superoxide production (procedure described in section Measurement of vascular superoxide production) and NO bioavailability (procedure described in section Measurement of vascular NO bioavailability).

Concentration-response curves to acetylcholine (endo thelium-dependent vasodilator) and sodium nitroprusside (endothelium-independent vasodilator) were performed after submaximal precontraction of the vessel with phenyephrine (1 ? 10^?6 M). The seven number of aortic ring preparation each isolated from seven animals per group were used for experiment (i.e. N = 7).

A main factor limiting the bioavailability of NO is the superoxide anion radical (O₂^?). We therefore assessed O₂^? formation in the aorta. Super oxide level was measured as Tempol (1 ? 10^?4 M, SOD mimetic) induced relaxation of isolated rat aorta after submaximal precontraction of the vessel with phenylephrine (1 ? 10^?6 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).

Aortic nitric oxide bioavailability was measured as a NOS inhibitor [NG-nitro-L-arginine methyl ester (L-NAME, 1 ? 10^?4 M)] induced contraction of isolated rat aorta after submaximal precontraction of the vessel with phenylephrine (1 ? 10^?6 M) as previously described.[¹⁴] To prevent synthesis of prostaglandins, we performed the experiment in the presence of 10 mM indomethacin. The seven number of aortic ring preparation each isolated from seven animals per group were used for the experiment (i.e. N = 7).

Drugs and chemicals

L-NAME, tempol, acetylcholine, sodium nitroprusside, L-phenyephrine, and apocynin/acetovanillone were purchased from Sigma Chemical Company Inc., St Louis, MO, USA. Indomethacin was obtained as a gift sample from Sun Pharma, Chennai, India. All other chemicals used were analytical grade purchased from Himedia Laboratories Ltd, Mumbai, India.

Data analysis

Data in the manuscript are expressed as Mean ? SEM. Comparisons between groups were made using one-way ANOVA. When significance was indicated, a Student-Newman-Keuls post hoc analysis was used. Statistical significance was assumed at P < 0.05.

Fructose-fed rats exhibit elevation in systolic blood pressure within 1 week (14.41%, P > 0.05) but became significantly high by the second week (18.8% P < 0.001) and continued to increase till eighth week (24% P < 0.001) compared to chow-fed control [Figure 1]. Interestingly, this increase was completely prevented in apocynin-treated fructose-fed rats [Figure 1]. However, apocynin had no effect on systolic blood pressure in chow-fed control rats.

Vascular reactivity (endothelium-dependent and -independent relaxation)

The endothelium-dependent vasodilator acetylcholine (Ach) and endothelium-independent vasodilator (SNP) elicited the concentration-dependent relaxation of phenylephrine preconstricted isolated aortic rings of all animals [Figures 2 and 3]. Aortic rings of fructose-fed rat had a markedly impaired endothelium-dependent relaxation as compared to control [Figure 2]. As shown in Figure 2, the maximum relaxation response (E_max) induced by acetylcholine was significantly reduced in aortas isolated from high fructose diet-fed rats when compared to chow-fed rats (47.42 ? 8.69 vs. 78.26 ? 5.91, P < 0.05). Note, this impaired response to Ach was normalized by treatment with the NAD(P)H oxidase inhibitor apocynin (68.56 ? 7.79 vs. 47.42 ? 8.69, P < 0.05) [Figure 2]. The sensitivity of the aortae from all experimental groups to acetylcholine was similar as indicated by no significant difference in pD₂ values among the groups. The sensitivity (pD₂) and maximum relaxation response (E_max) induced by the endothelium-independent vasodilator sodium nitroprusside were not significantly different among the groups [Figure 3].

A significant increase in tempol (SOD mimetic)-induced relaxation of isolated rat aorta preconstricted with phenylephrine revealed a marked increase in vascular O₂^? in aorta isolated from fructose-fed rats compared with levels in aorta isolated from control. This increase was prevented in aortic rings isolated from apocynin-treated fructose-fed rats [Figure 4]. However, in chow-fed control, apocynin had no significant effect on O₂^? level.

A significant decrease in L-NAME-induced contraction of isolated rat aorta preconstricted with phenylephrine revealed a marked decrease in vascular NO bioavailability in aorta isolated from fructose-fed rats compared with control. This decrease was prevented in apocynin-treated fructose-fed rat [Figure 5]. However, in chow fed control, apocynin had no significant effect on NO level.