Pharmacological Research 58 (2008) 356–363

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Pharmacological Research

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Benfotiamine attenuates nicotine and uric acid-induced vascular endothelialdysfunction in the rat

Pitchai Balakumar ∗, Ramica Sharma, Manjeet SinghCardiovascular Pharmacology Division, ISF College of Pharmacy, Moga 142001, Punjab, India

a r t i c l e i n f o

Article history:Received 24 August 2008Received in revised form24 September 2008Accepted 25 September 2008

Keywords:NicotineUric acidOxidative stressNitric oxideVascular endothelial dysfunctionBenfotiamine

a b s t r a c t

The study has been designed to investigate the effect of benfotiamine, a thiamine derivative, in nico-tine and uric acid-induced vascular endothelial dysfunction (VED) in rats. Nicotine (2 mg kg−1 day−1, i.p.,4 weeks) and uric acid (150 mg kg−1 day−1, i.p., 3 weeks) were administered to produce VED in rats. Thedevelopment of VED was assessed by employing isolated aortic ring preparation and estimating serum andaortic concentration of nitrite/nitrate. Further, the integrity of vascular endothelium was assessed usingthe scanning electron microscopy (SEM) of thoracic aorta. Moreover, the oxidative stress was assessedby estimating serum thiobarbituric acid reactive substances (TBARS) and aortic superoxide anion gener-ation. The administration of nicotine and uric acid produced VED by impairing the integrity of vascularendothelium and subsequently decreasing serum and aortic concentration of nitrite/nitrate and attenu-ating acetylcholine-induced endothelium dependent relaxation. Further, nicotine and uric acid producedoxidative stress, which was assessed in terms of increase in serum TBARS and aortic superoxide generation.However, treatment with benfotiamine (70 mg kg−1 day−1, p.o.) or atorvastatin (30 mg kg−1 day−1 p.o., astandard agent) markedly prevented nicotine and uric acid-induced VED and oxidative stress by improv-ing the integrity of vascular endothelium, increasing the concentration of serum and aortic nitrite/nitrate,enhancing the acetylcholine-induced endothelium dependent relaxation and decreasing serum TBARS

and aortic superoxide anion generation. Thus, it may be concluded that benfotiamine reduces the oxida-tive stress and consequently improves the integrity of vascular endothelium and enhances the generation

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. Introduction

Endothelium is an innermost layer of blood vessels. A healthyndothelium possesses antiatherogenic, antiplatelet and antipro-iferative properties to regulate the vascular tone and maintainhe free flow of blood in vessels [1,2]. Nitric oxide (NO), a potentasodilatory substance, is generated from l-arginine by endothe-ial nitric oxide synthase (eNOS) in the presence of cofactors suchs Ca++-calmodulin, reduced nicotinamide adenine dinucleotidehosphate (NADPH), flavin adenine dinucleotide (FAD) and tetrahy-robiopterin (BH4) in endothelial cells [3,4]. Vascular endothelialysfunction (VED) is characterized by suppression of endothe-

ium dependent vasorelaxation caused by reduced generation and

ioavailability of nitric oxide (NO) due to reduced activity ofNOS and increased oxidative stress in the vessel wall, which inurn impair the regulation of vascular homeostasis [5–7]. VEDas been associated in the pathogenesis of atherosclerosis [8],

∗ Corresponding author. Tel.: +91 9815557265; fax: +91 1636236564.E-mail address: pbala2006@gmail.com (P. Balakumar).

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043-6618/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.phrs.2008.09.012

ne and uric acid-induced experimental VED.© 2008 Elsevier Ltd. All rights reserved.

ypertension [9], diabetes mellitus [2,10], coronary artery diseases11] and stroke [12]. Various risk factors including hyperuricemiand cigarette smoking are implicated in the vascular pathogen-sis [13,14]. Hyperuricemia, a condition of increased serum uriccid, has been implicated in pathogenesis of VED [14,15]. Uriccid is the major product formed from purine metabolism. Gout,s an inflammatory disorder characterized by increased uric acidevel and has been reported to produce vascular pathogenesis14–16]. Hyperuricemia has been noted to decrease the vasculareneration of NO through inactivation of eNOS [16]. Further, hyper-ricemia has been shown to produce reactive oxygen species (ROS)xcessively through activation of NADPH oxidase and xanthine oxi-ase [17–20]. The C-reactive protein (CRP) level was noted to be

ncreased in hyperuricemia-induced VED [21]. Hyperuricemia haseen regarded as an independent risk factor for various cardio-ascular disorders such as atherosclerosis [22], hypertension [23]

nd heart failure [24]. Nicotine exposure via cigarette smoking haseen implicated in pathogenesis of cardiovascular disorders liketherosclerosis [25] and hypertension [26]. Moreover, nicotine haseen noted to produce VED by downregulating the expression ofNOS, increasing the generation of ROS and upregulating asymmet-

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ic dimethylarginine (ADMA), an endogenous inhibitor of eNOS andhus impair the endothelial production of NO [13,27,28]. Nicotineas been noted to upregulate the expression of various proteinsuch as basic fibroblast growth factor, tumor necrosis factor-�TNF-�) and plasminogen activator inhibitor-1 [29]. In addition,icotine induces mononuclear leukocyte adhesion and expressionf adhesion molecules such as vascular cell adhesion molecule-1VCAM-1) and intracellular adhesion molecule (ICAM) in endothe-ial cells [30]. No satisfactory therapeutic option is available to treaticotine and uric acid-induced vascular pathogenesis. Thus, theresent study has been aimed to explore the possible therapeu-ic strategy to prevent nicotine and uric acid-induced dysfunctionf vascular endothelium. Benfotiamine, S-acyl thiamine derivative,as been shown to reduce the formation of advanced glycationnd products (AGE) by activating transketolase [31,32]. Benfoti-mine has been noted to possess clinical efficacy in the treatment ofiabetic cardiomyopathy [33], diabetic nephropathy [34] and dia-etic neuropathy [35,36]. Moreover, benfotiamine has been showno reduce the oxidative stress through a mechanism unrelated toGE formation [37]. Activation of Akt (protein kinase B) has beenemonstrated to stimulate eNOS activity, increase the bioavailabil-

ty of NO and reduce the generation of ROS [38]. Benfotiamine haseen reported to improve the function of endothelium by activatingkt and subsequently stimulating eNOS and inhibiting the gener-tion of ROS [39,40]. Thus, the present study has been designedo investigate the effect of benfotiamine in nicotine and uric acid-nduced experimental vascular endothelial dysfunction.

. Materials and methods

The experimental protocol used in the present study waspproved by the Institutional Animal Ethical Committee. Ageatched young male Wistar rats weighing about 200–250 g were

mployed in the present study. They were fed on standard chowiet and water ad libitum. They were acclimatized in the institu-ional animal house and were exposed to normal light and darkycle of the day.

.1. Assessment of vascular endothelial dysfunction

.1.1. Isolated rat aortic ring preparationThe rat was decapitated, thoracic aorta was removed, cut into a

ing of 3–4 mm in length and mounted in an organ bath contain-ng Krebs–Henseleit solution (NaCl, 119 mM; KCl, 4.7 mM; NaHCO3,5 mM; MgSO4, 1.0 mM; glucose, 11.1 mM; KH2PO4, 1.2 mM andaCl2, 2.5 mM) of pH 7.4, bubbled with carbonated oxygen (95% O2nd 5% CO2) and maintained at 37 ◦C. The preparation was allowedo equilibrate for 90 min under 1.5 g tension. The isometric contrac-ions were recorded with a force-transducer (Ft-2040) connectedo Physiograph (INCO, Ambala, India). The aortic ring preparationas primed with 80 mM of KCl to check its functional integrity

nd to improve its contractility. The cumulative dose responsesf acetylcholine (Ach; 10−8 to 10−4 M) or sodium nitroprussideSNP; 10−8 to 10−4 M) were recorded in phenylephrine (3 × 10−6 M)recontracted preparation with intact or denuded endothelium,espectively [41]. The intimal layer of the aortic ring was rubbedently with a moistened filter paper for 30 s to obtain endotheliumree preparation [14,42]. Loss of Ach (1 × 10−6 M)-induced relax-tion confirmed the absence of vascular endothelium.

.1.2. Scanning electron microscopy (SEM)The scanning electron microscopic study was performed to

xamine the integrity of vascular endothelium [2,43,44]. 3–4 mmongitudinal strips of thoracic aorta were fixed in 3% glutaralde-yde phosphate-buffer (pH 7.4) and subsequently dehydrated in a

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l Research 58 (2008) 356–363 357

eries of acetone solution (50% for 20 min, 70% for 20 min, 80% for0 min, 90% for 20 min, 100% for 50 min) and isoamylacetate (100%):cetone (100%) solutions (1.1) for 20 min followed by isoamylac-tate (100%) for 20 min. Arterial segments were dried further usingour flushes of liquid CO2 with 100 psi pressure in critical pointrier. Then, the segments were mounted on aluminium stubs andoated with gold palladium (JFC-1100) and were viewed using JOELSM 6100 scanning electron microscope to observe the integrity ofascular endothelium.

.1.3. Estimation of serum or aortic nitrite/nitrate concentrationThe aortic tissue was homogenized in phosphate-buffer saline

f pH 7.4 and centrifuged at 10,000 × g for 20 min. The supernatantas used to estimate the aortic concentration of nitrite/nitrate

nd protein content. 400 �L of carbonate buffer (pH 9.0) wasdded to 100 �L of serum sample or 100 �L of supernatant fromomogenized aortic samples, followed by addition of small amount0.15 g) of copper–cadmium alloy. The tubes were incubated atoom temperature for 1 h to reduce nitrate to nitrite. The reac-ion was stopped by adding 100 �L of 0.35 M sodium hydroxide.ollowing this, 400 �L of zinc sulphate solution (120 mM) wasdded to deproteinate the samples. The samples were allowedo stand for 10 min and then centrifuged at 4000 × g for 10 min.reiss reagent (250 �L of 1.0% sulfanilamide and 250 �L of 0.1%-naphthylethylenediamine) was added to aliquots (500 �L) oflear supernatant and nitrite/nitrate concentration was measuredpectrophotometrically (Thermo Double Beam Spectrophotometer,hermo Electron Corporation, UK) at 545 nm. The standard graphf sodium nitrite (0.5–40 �M) was plotted to calculate concentra-ion of serum nitrite/nitrate (�M) and aortic nitrite/nitrate (�M/mgf protein) [14,45,46]. The protein concentration in homogenizedortic preparation was estimated by Lowry’s method [47].

.2. Assessment of oxidative stress

.2.1. Estimation of serum thiobarbituric acid reactive substancesTBARS)

1 mL of 20% trichloroacetic acid was added to 100 �L of serumnd 1 mL of thiobarbituric acid reagent and then mixed and incu-ated at 100 ◦C for 30 min. After cooling on ice, samples wereentrifuged at 1000 × g for 20 min. Serum concentration of TBARSas measured spectrophotometrically at 532 nm. A standard graphsing 1, 1, 3, 3 tetramethoxypropane (1–50 �M) was plotted to cal-ulate the concentration of TBARS [14,48].

.2.2. Estimation of superoxide anionAorta was cut into transverse rings of 6 mm in length and placed

n 5 mL of Krebs–Henseleit solution buffer containing 100 �M ofitroblutetrazolium (NBT) and incubated at 37 ◦C for 1.5 h. NBTeduction was stopped by adding 5 mL of 0.5 N HCl. The rings wereinced and homogenized in a mixture of 0.1 N sodium hydroxide

NaOH) and 0.1% sodium dodecyl sulphate (SDS) in water con-aining 40 mg/L of diethylenetriamine pentaacetic acid (DTPA). The

ixture was centrifuged at 20,000 × g for 20 min and the resul-ant pellets were resuspended in 1.5 mL of pyridine and kept at0 ◦C for 1.5 h to extract formazan. The mixture was centrifuged at0,000 × g for 10 min and the absorbance of formazan was deter-ined spectrophotometrically at 540 nm [14,49]. The amount of

f reduced NBT = A × V/(T × Wt × ε × l), where A is the absorbance,the volume of solution (1.5 mL), T the time for which aortic ringsere incubated with NBT (90 min), Wt the blotted wet weight of

ortic rings, ε the extinction coefficient (0.72 L mmol−1 mm−1) andis the length of light path (10 mm).

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.3. Estimation of serum uric acid

Serum uric acid was estimated spectrophotometrically using aommercial available kit (Transia Bio-medical Ltd., Daman, India).0 �L of serum sample was added to 1000 �L of working uric acideagent to prepare test sample. 20 �L of standard uric acid solu-ion (6 mg dL−1) was added to 1000 �L of working uric acid reagento prepare standard sample. 20 �L of distilled water was added to000 �L of working uric acid reagent to prepare blank. The test,tandard and blank samples were incubated at 37 ◦C for 5 min. Thebsorbances of test and standard samples were measured againstlank spectrophotometrically at 510 nm. The amount of serum uriccid level was calculated using the following formula.

Concentration of serum uric acid (mg/dL)

= absorbance of testabsorbance of standard

× Concentration of standard (6 mg/dL)

.4. Experimental protocol

Eight groups were employed in the present study and eachomprised of 7 rats. The uric acid and atorvastatin were dissolvedn 0.5% w/v of carboxy methyl cellulose (CMC). The benfotiamineas dissolved in 1% w/v of CMC. Group I (Normal Control), ratsere maintained on standard food and water and no treatmentas given. Group II (Nicotine Control), rats were administeredicotine (2 mg kg−1 day−1, i.p.) for 4 weeks. Group III (Uric Acidontrol), rats were administered uric acid (150 mg kg−1 day−1, i.p.)or 3 weeks. Group IV (Benfotiamine per se), rats were adminis-ered benfotiamine (70 mg kg−1 day−1, p.o.) dissolved in 1% w/vf CMC for 4 weeks. Group V (Benfotiamine Treated Nicotine),ats administered nicotine (2 mg kg−1 day−1, i.p., 4 weeks) werereated with benfotiamine (70 mg kg−1 day−1, p.o.) and the treat-

ent was started 3 days before the administration of nicotine andt was continued for 4 weeks from the day of administration oficotine. Group VI (Benfotiamine Treated Uric Acid), rats admin-

stered uric acid (150 mg kg−1 day−1, i.p., 3 weeks) were treatedith benfotiamine (70 mg kg−1 day−1, p.o.) and the treatment was

tarted 3 days before the administration of uric acid and it wasontinued for 3 weeks from the day of administration of uriccid. Group VII (Atorvastatin Treated Nicotine), rats administeredicotine (2 mg kg−1 day−1, i.p., 4 weeks) were treated with ator-astatin (30 mg kg−1 day−1, p.o.) as mentioned in Group V. GroupIII (Atorvastatin Treated Uric Acid), rats administered uric acid

150 mg kg−1 day−1, i.p., 3 weeks) were treated with atorvastatin30 mg kg−1 day−1, p.o.) as mentioned in Group VI.

.5. Statistical analysis

All values were expressed as mean ± S.D. Data for isolated aorticing preparation were statistically analyzed using repeated mea-ures of ANOVA followed by Newman Keul’s test. The data for serumnd aortic levels of nitrite/nitrate, serum TBARS, aortic superoxidenion generation and serum uric acid were statistically analyzedsing one-way ANOVA followed by Tukey’s multiple comparisonest. The p < 0.05 was considered to be statistically significant.

.6. Drugs and chemicals

Acetylcholine hydrochloride and l-phenylephrine were pur-hased from Sigma–Aldrich Ltd., St. Louis, USA. Diethyl triamineentaacetic acid, nitroblue tetrazolium and uric acid wereurchased from Sanjay Biologicals Amritsar, India. 1,1,3,3 tetram-thoxypropane and carboxymethyl cellulose were purchased from

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l Research 58 (2008) 356–363

. K Chemicals, India. Nicotine was obtained from Nicosulf Indiavt. Ltd., Dakor, Gujarat, India. Atorvastatin was obtained fromr. Reddy’s Laboratory Ltd., Hyderabad, India. Benfotiamine wasbtained from Orchid Healthcare Ltd., Chennai. The uric acid esti-ation kit was purchased from Transia Bio-medical Ltd., Daman,

ndia.

. Results

The treatment with benfotiamine (70 mg kg−1 day−1, p.o.) ortorvastatin (30 mg kg−1 day−1, p.o.) to normal rats did not produceny significant per se effects on various parameters performed inhe present study such as endothelium dependent relaxation andndependent relaxation, serum and aortic level of nitrite/nitrate,BARS and aortic superoxide anion generation and serum uric acidevel. Less than 5% of mortality rate was observed in nicotine andric acid administered rats with or without treatments.

.1. Effect of pharmacological interventions on endotheliumependent and independent relaxation

Ach and SNP were noted to produce endothelium dependent andndependent relaxation, respectively in phenylephrine (3 × 10−6 M)recontracted isolated rat aortic ring preparation in a dose depen-ent manner. Administration of nicotine (2 mg kg−1 day−1, i.p., 4eeks) and uric acid (150 mg kg−1 day−1, i.p., 3 weeks) signifi-

antly attenuated Ach-induced endothelium dependent relaxation;ut their administration did not affect SNP-induced endothe-

ium independent relaxation. The treatment with benfotiamine70 mg kg−1 day−1, p.o.) or atorvastatin (30 mg kg−1 day−1, p.o.) sig-ificantly prevented nicotine and uric acid-provoked attenuationf Ach-induced endothelium dependent relaxation. Marked Ach-nduced endothelium dependent relaxation was noted in uric aciddministered rats treated with atorvastatin as compared with ben-otiamine treated rats (Figs. 1 and 2).

.2. Effect of pharmacological interventions on integrity ofascular endothelium

The integrity of vascular endothelium was noted to be impairedn thoracic aorta of nicotine (2 mg kg−1 day−1, i.p., 4 weeks) and uriccid (150 mg kg−1 day−1, i.p., 3 weeks) administered rats. However,reatment with benfotiamine (70 mg kg−1 day−1, p.o.) or atorvas-atin (30 mg kg−1 day−1, p.o.) markedly improved the integrity ofascular endothelium in nicotine and uric acid administered ratsFig. 3).

.3. Effect of pharmacological interventions on serum and aorticitrite/nitrate concentration

The serum and aortic concentration of nitrite/nitrate was notedo be reduced in nicotine (2 mg kg−1 day−1, i.p., 4 weeks) andric acid (150 mg kg−1 day−1, i.p., 3 weeks) administered ratshen compared with normal rats. Moreover, marked reduction in

erum and aortic nitrite/nitrate occurs in rats administered uriccid as compared to nicotine-administered rats. However, treat-ent with benfotiamine (70 mg kg−1 day−1, p.o.) or atorvastatin

30 mg kg−1 day−1, p.o.) significantly attenuated nicotine and uric

cid-induced decrease in serum and aortic nitrite/nitrate concen-ration. Atorvastatin produced marked restoration of decreasederum and tissue nitrite/nitrate concentration in rats adminis-ered uric acid when compared with benfotiamine treated ratsFigs. 4 and 5).

logical Research 58 (2008) 356–363 359

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.4. Effect of pharmacological interventions on serum TBARS andortic superoxide anion generation

The increase in serum TBARS concentration and aortic super-xide anion generation was noted in animals administeredith nicotine (2 mg kg−1 day−1, i.p., 4 weeks) and uric acid

150 mg kg−1 day−1, i.p., 3 weeks). Moreover, marked increasen serum TBARS and aortic superoxide anion generation wasoted in rats administered uric acid as compared to nico-ine administered rats. However, treatment with benfotiamine70 mg kg−1 day−1, p.o.) or atorvastatin (30 mg kg−1 day−1, p.o.) sig-ificantly attenuated nicotine and uric acid-induced increase inerum TBARS concentration and aortic superoxide anion generationFigs. 6 and 7).

.5. Effect of pharmacological interventions on serum uric acidoncentration

The concentration of serum uric acid was noted to be markedlyncreased in uric acid (150 mg kg−1 day−1, i.p., 3 weeks) adminis-ered rats when compared with normal rats. The treatment with

enfotiamine (70 mg kg−1 day−1, p.o.) has no effect on serum uriccid level; but treatment with atorvastatin (30 mg kg−1 day−1, p.o.)arkedly reduced the serum concentration of uric acid in rats

dministered uric acid (150 mg kg−1 day−1, i.p., 3 weeks) (Fig. 8).

ig. 1. Effect of benfotiamine on acetylcholine-induced endothelium dependentelaxation. Responses are expressed as percentage of maximum contraction inducedy phenylephrine (3 × 10−6 M). All values are represented as mean ± S.D. a = p < 0.05s Normal Control; b = p < 0.05 vs Nicotine Control; c = p < 0.05 vs Uric Acid Control;= p < 0.05 vs Benfotiamine Treated Uric Acid.

Fig. 2. Effect of benfotiamine on sodium nitroprusside (SNP)-induced endothe-lcm

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. Discussion

The impairment in integrity of vascular endothelium, decreasen serum and aortic nitrite/nitrate concentration and reduction inch-induced endothelium dependent vasodilation have been doc-mented to be an index of experimental VED [14,42,50]. In theresent study, administration of nicotine (2 mg/kg) for 4 weeks andric acid (150 mg/kg) for 3 weeks in rats impaired the integrity ofascular endothelium, decreased the serum and aortic concentra-ion of nitrite/nitrate and consequently reduced the Ach-inducedndothelium dependent vasodilation. It suggests that both nico-ine and uric acid produced VED, which is consistent with ourecent studies and reports by others [14,15,51,52]. Nicotine has beenocumented to reduce the activity of eNOS and decrease the gen-ration and bioavailability of NO [53,54]. Further, nicotine has beenoted to upregulate ADMA, which is an endogenous eNOS inhibitor28]. Hyperuricemia has been shown to decrease the generationnd bioavailability of NO by inactivating eNOS and damaging thentegrity of vascular endothelium [14,16]. Thus, the observed nico-ine and uric acid-induced VED may be due to downregulation ofNOS and subsequent reduction in generation of NO in the vesselall. This contention is supported by the results obtained in theresent study that administration of nicotine and uric acid signifi-antly reduced the serum and aortic nitrite/nitrate levels.

In the present study, rats administered uric acid produced VED

arkedly as compared to nicotine administered rats. The oxidative

tress has been documented to play a major role in the progres-ion of VED [55,56]. Elevated level of serum TBARS and aorticuperoxide generation are regarded as an index of development

360 P. Balakumar et al. / Pharmacological Research 58 (2008) 356–363

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ig. 3. The scanning electron microscopic study was performed to examine the intehe impairment in integrity of vascular endothelium was noted in thoracic aorta ofarkedly prevented nicotine and uric acid-induced impairment in integrity of vasc

f oxidative stress [14,57,58]. Administration of nicotine and uriccid increased serum TBARS and aortic superoxide anion genera-ion, which indicate the development of oxidative stress. Nicotineas been reported to generate ROS through activation of NADPH59]. Hyperuricemia has been reported to produce ROS excessivelyy activating NADPH oxidase and xanthine oxidase [18]. Thus, theoted marked induction of VED in uric acid administered rats maye due to the development of high degree of oxidative stress and

onsequent inactivation of NO. This contention is strongly sup-orted by the fact that in the present study, uric acid administeredats showed high degree of oxidative stress and marked reductionn serum and aortic nitrite/nitrate concentration as compared toicotine administered rats.

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f vascular endothelium using JOEL JSM 6100 scanning electron microscope (800×).ne or uric acid administered rats. The treatment with benfotiamine or atorvastatinndothelium.

The pharmacological treatment with benfotiamine, a lipophilichiamine derivative [60] has been noted to prevent nicotine and uriccid-induced VED by improving the integrity of vascular endothe-ium, increasing the serum and aortic nitrite/nitrate level andnhancing the Ach-induced endothelium dependent relaxation.enfotiamine has been reported to reduce the generation of ROShrough the mechanism unrelated to AGE formation [37] and pre-ent the process of apoptosis [61]. Benfotiamine has been reported

o activate Akt/Protein kinase B [39,40]. Activation of Akt/Proteininase B has been noted to further activate eNOS [62] and reduces

he oxidative stress [63]. Activation of eNOS increases the gener-tion and bioavailability of NO and thus improves the functionf vascular endothelium [62,64]. Thus, the observed beneficial

P. Balakumar et al. / Pharmacological Research 58 (2008) 356–363 361

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ig. 4. Effect of benfotiamine on serum concentration of nitrite/nitrate (�M). Allalues are represented as mean ± S.D. a = p < 0.05 vs Normal Control; b = p < 0.05 vsicotine Control; c = p < 0.05 vs Uric Acid Control; d = p < 0.05 vs Nicotine Control;= p < 0.05 vs Benfotiamine Treated Nicotine.

ffect of benfotiamine in preventing nicotine and uric acid-inducedxperimental VED may be due to activation of eNOS throughkt/Protein kinase B, reduction of ROS generation and consequentpregulation of synthesis and bioavailability of NO. This con-ention is supported by the results obtained in the present study

hat benfotiamine increases the serum and aortic concentrationf nitrite/nitrate and reduces the oxidative stress by decreasingerum TBARS and aortic superoxide anion generation. Atorvastatinas been well noted to improve the function of endothelium byarkedly reducing the oxidative stress [14,42,65]. Further, ator-

ig. 5. Effect of benfotiamine on aortic concentration of nitrite/nitrate (�M/mgf protein). All values are represented as mean ± S.D. a = p < 0.05 vs Normal Con-rol; b = p < 0.05 vs Nicotine Control; c = p < 0.05 vs Uric Acid Control; d = p < 0.05 vsicotine Control; e = p < 0.05 vs Benfotiamine Treated Uric Acid.

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ig. 6. Effect of benfotiamine on serum concentration of TBARS (�M). All values areepresented as mean ± S.D. a = p < 0.05 vs Normal Control; b = p < 0.05 vs Nicotineontrol; c = p < 0.05 vs Uric Acid Control; d = p < 0.05 vs Nicotine Control; e = p < 0.05s Benfotiamine Treated Uric Acid.

astatin has been reported to upregulate eNOS and improve thentegrity of vascular endothelium [14,42]. Therefore, in the presenttudy, atorvastatin has been employed as a standard drug to com-are the potential of benfotiamine in preventing VED. The beneficial

een observed to be almost similar to the effect produced by ator-astatin. But, the vascular protective effect of atorvastatin wasoted to be slightly superior in attenuating the uric acid-induced

ig. 7. Effect of benfotiamine on superoxide anion generation as assessed bystimating reduced nitroblutetrazolium (NBT) (pmol/min/mg). All values are repre-ented as mean ± S.D. a = p < 0.05 vs Normal Control; b = p < 0.05 vs Nicotine Control;= p < 0.05 vs Uric Acid Control; d = p < 0.05 vs Nicotine Control; e = p < 0.05 vs Ben-

otiamine Treated Uric Acid.

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ED as compared to benfotiamine. Atorvastatin has been reportedo have uricosuric effect as it increases the excretion of uric acid66]. Thus, the additional uricosuric property of atorvastatin may beesponsible to markedly attenuate the uric acid-induced VED. Thisontention is supported by the fact that the serum uric acid levelas noted to be markedly reduced by treatment with atorvastatin.

On the basis of the above discussion, it may be concludedhat benfotiamine reduces the oxidative stress and consequentlymproves the integrity of vascular endothelium and enhances theeneration of nitric oxide to prevent nicotine and uric acid-inducedxperimental vascular endothelial dysfunction.

cknowledgement

We wish to express our gratitude to Shri. Parveen Garg, Hon-urable Chairman, ISF College of Pharmacy, Moga, Punjab, India foris praiseworthy suggestion and constant support for this study.

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