protective effect of e uterpe oleracea mart (açaí) extract on programmed changes in the adult...

Protective effect of Euterpe oleracea Mart (açaí) extract onprogrammed changes in the adult rat offspring caused bymaternal protein restriction during pregnancyGraziele Freitas de Bema, Cristiane Aguiar da Costaa, Paola Raquel Braz de Oliveiraa,Viviane Silva Cristino Cordeiroa, Izabelle Barcellos Santosa, Lenize Costa Reis Marins de Carvalhoa,Marcelo Augusto Vieira Souzaa, Dayane Texeira Ognibeneb, Julio Beltrame Dalepranec,Pergentino José Cunha Sousad, Angela Castro Resendea and Roberto Soares de Mouraa

aDepartment of Pharmacology, Institute of Biology, cDepartment of Basic and Experimental Nutrition, Institute of Nutrition, Rio de Janeiro StateUniversity, bLaboratory of Technology in Drug Production, State University of the West, Rio de Janeiro and dDepartment of Pharmacy, FederalUniversity of Pará, Belém, Brazil

KeywordsEuterpe oleracea Mart.; hypertension; lowprotein; oxidative stress; renal failure

CorrespondenceAngela de Castro Resende, Departamento deFarmacologia e Psicobiologia, I.B.,Universidade do Estado do Rio de Janeiro, Av.28 de Setembro, 87, Rio de Janeiro, BrasilE-mail: [email protected]

Received December 9, 2013Accepted March 2, 2014

doi: 10.1111/jphp.12258

Abstract

Objectives This study examined the effect of açaí (Euterpe oleracea Mart.) seedextract (ASE) on cardiovascular and renal alterations in adult offspring, whosemothers were fed a low-protein (LP) diet during pregnancy.Methods Four groups of rats were fed: control diet (20% protein); ASE(200 mg/kg per day); and LP (6% protein); LP + ASE (6% protein + ASE) duringpregnancy. After weaning, all male offspring were fed a control diet and sacrificedat 4 months old. We evaluated the blood pressure, vascular function, serum andurinary parameters, plasma and kidney oxidative damage, and antioxidant activityand renal structural changes.Key findings Hypertension and the reduced acetylcholine-induced vasodilationin the LP group were prevented by ASE. Serum levels of urea, creatinine and frac-tional excretion of sodium were increased in LP and reduced in LP + ASE. ASEimproved nitrite levels and the superoxide dismutase and glutathione peroxidaseactivity in LP, with a corresponding decrease of malondialdehyde and protein car-bonyl levels. Kidney volume and glomeruli number were reduced and glomerularvolume was increased in LP. These renal alterations were prevented by ASE.Conclusions Treatment of protein-restricted dams with ASE provides protectionfrom later-life hypertension, oxidative stress, renal functional and structuralchanges, probably through a vasodilator and antioxidant activity.

Introduction

Numerous experimental and epidemiological studies havehighlighted the association between small body size at birthand later cardiovascular disease and its biological riskfactors.[1] This association is explained by the concept of‘developmental programming’ that establishes a relation-ship between an adverse intra-uterine or early postnatalnutritional environment and permanent changes in theactivity of functional systems, predisposing the develop-ment of diseases in later life.[2] There is growing evidencethat maternal dietary composition and intake are important

determinants of fetal growth and development, includingaspects specific to reproduction and pregnancy outcomes,in both animal and human.[3] It has been shown that theexperimental model of perinatal protein restriction in ratshas several features in common with the observations inhumans. In particular, offspring of rats that were proteinrestricted during pregnancy have lower than normal birthweight, develop hypertension in adulthood,[4] endothelialdysfunction,[5] oxidative stress[6] and have reduced nefronnumber.[7] These indicate that maternal undernutrition

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Research Paper

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–•• 1

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correlates with later disease, implying that fetal nutritionaldeprivation is a strong programming stimulus.

Over the years, natural products have been shown to bean important source of molecular diversity leading to drugdiscovery currently used in modern medicine. The plantEuterpe oleracea Mart. belongs to the family Arecaceae and ispopular known as ‘açaí’. It is widely diffused in the Amazonregion of Brazil, and the fruit has always been an importantfood source for indigenous population. It is largely con-sumed in northern Brazil and has been considered one ofthe most important medicinal plants of the Amazon[8] by itsbeneficial effect in the treatment of fever, pain, inflamma-tion and anaemia.[9,10]

Epidemiological studies have shown an inverse correla-tion between polyphenol-enriched diet and reduced risks ofcardiovascular diseases.[11] Chemical studies have shownthat fruits of E. oleracea Mart. are rich in anthocyanins(cyanidin 3-O-glucoside, and cyanidin 3-O-rutinoside),pro-anthocyanidins (polymerers) and other flavonoids suchas epicatechin, catechin homoorientin, orientin, isovitexinand taxifolin deoxyhexose.[12,13]

Recently, we have reported that hydroalcoholic extract ofaçaí seeds (ASE) has a potent endothelium-dependent vaso-dilator effect and induces nitric oxide (NO) release fromendothelial cells in culture.[14] We also observed that chronictreatment with ASE prevents the development of hyperten-sion, endothelial dysfunction, vascular structural changesand oxidative damage in experimental renovascular hyper-tension.[15,16] Thus, ASE presents an attractive pharmaco-logical profile (efficacious antihypertensive and antioxidantproperties without side effects), which suggests its use inpathological cardiovascular and renal conditions. However,there is no report about the activity of ASE on cardiovascu-lar and renal disorders of adult offspring of an experimentalmodel of developmental programming. Hence, this studyaimed to test the hypothesis that the beneficial effects ofASE on cardiovascular control of dams would pass on to themale offspring as adults; that is, the cardiovascular andrenal derangements in offspring caused by maternal proteinrestriction during gestation could be ameliorated if damsare supplemented with ASE.

Therefore, experiments were undertaken to determinethe effects of ASE on cardiovascular and renal program-ming with focus on blood pressure and endothelial func-tion, oxidative status, and renal functional and structuralchanges observed in adult male offspring.

Materials and Methods

Reagents and animals

2,4-Dinitrophenylhydrazine, naphthylenediamide dihydro-chloride, acetylcholine (ACh), eosin, haematoxylin, nicoti-namide adenine dinucleotide phosphate (NADPH),

‘noradrenaline’ (NE), nitroglycerin (NG), oxidizedglutathione, reduced glutathione, paraplast plus, phos-phoric acid, sodium nitrite, sulfanilamide andthiobarbituric acid were purchased from Sigma Chemical(St Louis, MO, USA). Ethanol, formalin and hydrogen per-oxide were purchased from Vetec (Duque de Caxias, Brazil).The rats were obtained from the facilities of the Rio deJaneiro State University. All the experiments on animalswere reviewed and approved by the Ethics Committee forExperimental Animals Use and Care (CEUA) of Instituto deBiologia Roberto Alcântara Gomes/Universidade do Estadodo Rio de Janeiro (protocol: CEUA/021/2010). The CEUAfollow guidelines from Intramural Animal Care and Useprogram of the National Institutes of Health.

Preparation of extract from E. oleraceaMart (açaí)

Euterpe oleracea Mart. fruits were obtained from theAmazon Bay (Pará State, Brazil) voucher specimen MG205222 Goeldi Museum – Belém do Pará. Hydroalcoholicextracts were obtained from decoction of the seeds of thefruits, as previously described.[16] Typically 100 g of seedsyielded approximately 5 g of lyophilized extract. Thecontent of polyphenols in ASE, measured by analysing fortotal phenol by Folin–Ciocalteu procedure,[17] was around265 mg/g of extract. The protein chemical composition ofthe ASE, analysed according to the official methods ofanalysis,[18] was 0.4 g of protein/100 g of extract. Recently,an analysis of the composition of ASE was performed byHPLC (Figure 1).[19] Briefly, ASE was analysed on an RP-18column (250 mm × 4 mm, 5 m particles; Shimadzu, Kyoto,Japan), and elution was made with solvents A (0.2% (v/v)phosphoric acid) and B (82% (v/v) acetonitrile, 0.04% (v/v)phosphoric acid). Flow rate was 1 ml/min. DAD UV-visabsorption spectra were recorded online during HPLCanalysis. The HPLC elution profile observed is stronglyindicative of the presence of pro-anthocyanidins. The peakeluting at 37.2 min corresponds to catechin as confirmed byco-injection of a standard and by comparison of the ultra-violet absorption spectrum. The late elution (at 54.7 min)and UV spectrum of the main peak were consistent with thepresence of polymeric pro-anthocyanidins.

Experimental groups

Virgin female Wistar rats aged 3 months were caged withone male rat at a proportion of 3 : 1. After mating, deter-mined by vaginal smear, each female was placed in an indi-vidual cage. During the pregnancy period (21 days), thedams had free access to standard diet (American Institute ofNutrition (AIN) 93) or low-protein (LP) diet 6% protein.The control group corresponding to male offspring from

Graziele Freitas de Bem et al.ASE improves vascular and renal changes

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••2

dams fed the standard diet and allowed access to water(control group: 20% protein) or ASE (ASE group,200 mg/kg per day, intragastric gavage) during pregnancy.Two other groups were fed the LP diet with access to water(LP group: 6% protein) or ASE (LP + ASE group;200 mg/kg per day, intragastric gavage) during pregnancy(Table 1). At birth, the number of male pups was set as sixper litter, and dams were fed a control diet. After weaning,the animals were fed a normal diet and sacrificed at 4months old. The body weight (BW) was recorded weeklyfrom 1 day of age until animals were fully grown at 120 daysold. The diets (Table 1) were balanced by Rhoster (Rhoster,São Paulo, Brazil) in accordance with the standard

recommendations for rodents in the maintenance state ofAIN (AIN-93G).[20]

Arterial pressure measurement and vascularperfusion studies

Systolic blood pressure (SBP) was measured in consciousrats by using tail-cuff plethysmopraphy (Letica 5000 device,Panlab, Cornellà, Spain).

The perfusion pressure of isolated mesenteric arterialbeds (MABs) of the rats from the different groups wasmeasured as previously described.[16] The dose-dependentresponses to ACh (1–1000 pmol) and NG (1–1000 nmol)were performed in preconstricted MABs with NE (10–30 μmol/l), and the vasodilator effect of the drugs wasexpressed as the percentage decrease of the pressor effect ofNE.[16]

Determination of oxidative damage:malondialdehyde and carbonylprotein assay

The lipid membrane damage was determined by for-mation of products of lipid peroxidation (malondialde-hyde – MDA) concentration from plasma and kidneyhomogenates using the thiobarbituric acid reactivesubstances method as previously described.[21] Proteincarbonylation was determined according to the methoddescribed by Levine et al.[22]

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Figure 1 The HPLC elution profile observed is strongly indicative of the presence of pro-anthocyanidins. The peak eluting at 37.2 min correspondsto catechin as confirmed by co-injection of a standard and by comparison of the ultraviolet absorption spectrum. The late elution (at 54.7 min) andUV spectrum of the main peak are consistent with the presence of polymeric pro-anthocyanidins. Further evidences on the structure of the com-pounds present in açaí (Euterpe oleracea Mart.) seed extract were provided by positive mode electrospray mass spectrometry. The sodiated ionsdetected at m/z 601, 889, 1177, 1965, 1753 and 1893 are indicative of pro-anthocyanidins with 2, 3, 4, 5, 6 and 7 flavan units.

Table 1 Composition of the experimental diet

Nutrients

Diets

Normalprotein (%)

Lowprotein (%)

Carbohydrates 54 68Protein 20 6.0Ether extract 7.2 7.0Fibrous matter 4.1 5.0Mineral matter 2.8 2.8Calcium 0.56 0.5Phosphorus 0.26 0.3

Vitamins and mineral mix following the AIN-93G recommendation forrodents.

Graziele Freitas de Bem et al. ASE improves vascular and renal changes


Nitrite assay and superoxide dismutase,catalase and glutathione peroxidase activity

Nitrite levels in plasma and kidney homogenates weredetermined by a method based on the Griess reaction.[23]

Superoxide dismutase (SOD) activity was assayed by meas-uring the inhibition of adrenaline auto-oxidation as absorb-ance at 480 nm.[24] Catalase (CAT) activity was measured interms of the rate of decrease in hydrogen peroxide at240 nm.[25] Glutathione peroxidase (GPx) activity wasmeasured by monitoring the oxidation of NADPH at340 nm in the presence of hydrogen peroxide.[26]

Serum and plasma assays

Blood was collected from the rats at 120 days old. Theanimals were fasted for 12 h, and blood samples were thencollected by thoracic aorta puncture in anaesthetizedanimals. Serum albumin, creatinine and urea levels weremeasured by a colorimetric assay (Bioclin, Belo Horizonte,Brazil). Serum sodium and potassium concentrations weremeasured by flame photometry (Laborlife, Clinical Analysis,Rio de Janeiro, Brazil). Plasma renin level was measuredby radioimmunoassay (Active Renin IRMA kit, BeckmanCoulter, Brea, CA, USA).

Urine measurement

Rats were placed individually in plastic metabolic cages,and urine was collected for 24 h. Urinary protein excretionwas measured by the Bradford method and urinary sodiumconcentrations by flame photometry. Renal clearance wascalculated by a standard formula (C = urinary creatinine ×urinary flow/serum creatinine). Fractional sodium excre-

tion was calculated by a standard formula (FENa+ =urinary Na+ × serum creatinine × 100/serum Na+ × urinarycreatinine).

Light microscopy and stereology

The left kidneys were longitudinally divided in two halves,which were embedded face down in Paraplast plus (Sigma,São Paulo, Brazil), then serially sectioned at a nominalthickness of 5 μm and stained with H&E. Both cortex andmedulla volumes were estimated according to Cavalieri’smethod, glomeruli volume density (Vv(glom)) by thepoint-counting method, and fractionator method was usedto estimate the glomeruli number in a slice (starting with arandom number for each 10th section). The total numberof glomeruli per kidney (N(glom)) was estimated consider-ing the analysed fraction of the kidney corrected to theentire organ.[27]

Statistical analyses

Data are shown as mean ± standard error of the mean. Sta-tistical differences were evaluated by a two-way analysis ofvariance, with Bonferroni and Tukey post-hoc test. P-valuesless than or equal to 0.05 were accepted as statisticallysignificant.

Results

Effect of ASE on body weight

At the end of gestation, there was no change in the maternalBW (g) among groups (control group: 256.4 ± 11.9, ASE:256.6 ± 8.9, LP: 246.3 ± 8.1, LP + ASE: 255.3 ± 7.9). At 1 dayof life, offspring BW of LP group was lower (P < 0.05)

Table 2 Effects of ASE on body weight (BW), plasma, serum and urinary parameters of 120-day-old offspring from dams fed LP diet duringpregnancy

Parameter Control ASE LP LP + ASE

BW (g) 1 day old 6.6 ± 0.1 6.1 ± 0.1 4.6 ± 0.1*,** 5.8 ± 0.1**,***BW (g) 120 day old 363.6 ± 16.0 359.2 ± 14.9 345.3 ± 5.5 348.3 ± 4.9Renin (pg/ml) 1.5 ± 0.2 1.5 ± 0.3 2.9 ± 0.5*,** 0.9 ± 0.3***Urea (mg/dl) 7.2 ± 1.8 4.3 ± 1.1 26.2 ± 6.9*,** 7.2 ± 2.7***Na (mM) 129.6 ± 4.1 121.8 ± 1.4 127.0 ± 6.9 137.0 ± 3.6K (mM) 3.6 ± 0.2 3.3 ± 0.1 4.1 ± 0.2** 3.6 ± 0.1Creatinine (mg/dl) 0.66 ± 0.08 0.91 ± 0.05 1.42 ± 0.05*,** 0.85 ± 0.09***Cr/Cl per 100 g (ml/min) 0.12 ± 0.03 0.18 ± 0.05 0.03 ± 0.005 0.09 ± 0.03Albumin (mg/dl) 3.03 ± 0.05 3.09 ± 0.08 2.37 ± 0.04*,** 3.11 ± 0.04***Proteinuria (μg/ml) 0.53 ± 0.32 0.35 ± 0.28 2.70 ± 0.97*,** 0.94 ± 0.1Urinary volume (ml) 13.1 ± 4.0 16.6 ± 5.9 8.1 ± 0.8 10.6 ± 1.7EFNa+ (%) 3.2 ± 0.5 2.5 ± 0.9 15.3 ± 3.6*,** 7.0 ± 2.0***

ASE, açaí (Euterpe oleracea Mart.) seed; EFNa+, fractional sodium excretion; LP, low protein. Data are means ± standard error of the mean, n = 5–8.*Significantly different (P < 0.05 analysis of varianc) from control; **significantly different (P < 0.05) from ASE; ***significantly different (P < 0.05)from LP.



compared with controls (Table 2). Treatment of LP-feddams with ASE during pregnancy prevented (P < 0.05) off-spring BW reduction (Table 2). At 120 days of age, therewas no difference in offspring BW among groups.

Effects of ASE on blood pressure andvascular function

SBP was increased (P < 0.05) in LP group at 120 days ofage compared with control groups. Treatment of LP-feddams with ASE during pregnancy prevented SBP eleva-tion (Figure 2). The reduced ACh-induced vasodilation(P < 0.05) in MAB from LP group was restored by treat-ment with ASE (Figure 2). Endothelium-independentresponse to NG was not different among groups (Figure 2).

Effects of ASE on oxidative damage

MDA and carbonyl protein levels in plasma and kidneysamples were higher (P < 0.05) in the LP group than in thecontrol one (Figure 3). Treatment with ASE reduced(P < 0.05) plasma and kidney protein carbonyl levels, butalso plasma MDA levels in LP + ASE group. MDA levelsfrom kidney homogenates of LP + ASE rats showed no sig-nificant difference from those of LP group.

Effects of ASE on plasma and kidneynitrite content

LP group showed lower plasma and kidney nitrite levelscompared with the control groups (P < 0.05, Figure 3). ASEprevented kidney nitrite levels decrease (P < 0.05) with nochange in reduced plasma nitrite levels.

Effects of ASE on plasma and kidneyantioxidant enzymes (SOD, CAT and GPx)

SOD and GPx activity were lower in plasma of the LPgroup (P < 0.05, Figure 4) as compared with controls, andtreatment with ASE recovered the enzymatic activity(P < 0.05, Figure 4). The decreased kidney SOD activity inthe LP group (P < 0.05) was not restored by ASE. Treatmentof control group with ASE increased kidney SOD activity(P < 0.05). Kidney CAT and GPx activity, as well as plasmaCAT activity were not significantly different among groups.

Effects of ASE on serum creatinine,albumin, urea and plasma renin levels

Creatinine, urea and renin plasma levels were higher(P < 0.05) in the LP group compared with controls(Table 2). ASE treatment normalized creatinine, urea andrenin plasma levels (Table 2). The reduced albumin concen-trations (P < 0.05) in the LP group Ecompared with con-trols was prevented by ASE (P < 0.05; Table 2).

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Figure 2 Effect of açaí (Euterpe oleracea Mart.) seed extract(200 mg/kg per day) on (a) the systolic blood pressure (mmHg), andvasodilator effects of (b) acetylcholine and (c) nitroglycerin (c) inmesenteric arterial bed of 120-day-old adult offspring of control orlow-protein fed dams during pregnancy. Data are mean ± standarderror of the mean, n = 8 for all groups. *Significantly different(P < 0.05) from control; #significantly different (P < 0.05) from açaí(Euterpe oleracea Mart.) seed extract; +significantly different (P < 0.05)from low protein.



Effects of ASE on serum andurine parameters

Urinary protein excretion (proteinuria) and serum potas-sium were increased in the LP group compared with con-trols (P < 0.05; Table 2), and treatment with ASE did notalter these parameters. The increased FENa+ excretion in LPgroup (P < 0.05) was normalized by ASE (P < 0.05), and theserum sodium, renal clearance and urinary volume werenot different among groups (Table 2).

Effects of ASE on kidney structure

Total glomeruli number and kidney volume were decreasedin LP group compared with controls (P < 0.05). The

decrease in those parameters was reduced by ASE (P < 0.05;Figure 5). Glomerular volume was increased in the LPgroup compared with controls (P < 0.05), and treatmentwith ASE normalized this parameter (P < 0.05; Figure 5).

Discussion

The metabolic programming can be defined as a uterineevent that has permanent effects on the physiology, struc-ture and metabolism of the individual.[28] Undernutritionduring pregnancy impairs maternal haemodynamic adapta-tions, such as, systemic vasodilatation, increase in basaloxygen consumption and alveolar ventilation resulting ininsufficient rise in cardiac output, plasma volume and


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Figure 3 Effects of açaí (Euterpe oleracea Mart.) seed extract (200 mg/kg per day) on (a and b) protein carbonylation, (c and d) malondialdehydeand (e and f) nitrite levels in the plasma and kidney of 120-day-old adult offspring of control or low-protein fed dams during pregnancy. Data aremean ± standard error of the mean, n = 8 for all groups. *Significantly different (P < 0.05) from control; #significantly different (P < 0.05) from açaí(Euterpe oleracea Mart.) seed extract; +significantly different (P < 0.05) from low protein.



uteroplacental flow.[29] Several studies have shown that aprotein restriction diet during pregnancy can ‘programme’functional and structural alterations in adult offspring, suchas hypertension,[4] endothelial dysfunction[5] and decreasednefron number.[7] In this study, we demonstrated, forthe first time, the preventive action of ASE on the cardiovas-cular and renal function, as well as renal structural changesobserved in offspring from dams fed a LP diet duringpregnancy.

Exposure to an LP diet during pregnancy was associatedwith an increase in SPB of adult offspring, which is inaccordance with previous findings in the same experimentalmodel.[4] The mechanism involved in the development of

hypertension observed in offspring from dams fed a LPdiet is, at the moment, not established. Probably, oxidativestress, endothelial dysfunction and renin–angiotensinsystem may play a pathogenic role in programmed hyper-tension in adulthood. In this study, we found increasedlevels of renin in the LP group. Evidence has shown thatrenin and angiotensin converting enzyme (ACE) activity areelevated in rats exposed to an LP diet in the uterus, andbrief administration of an ACE inhibitor lowered bloodpressure in both neonates and young adults.[30]

Treatment of LP-fed dams during pregnancy with ASEprevented the increase in renin plasma levels contributingfor maintenance of normal blood pressure levels of adult






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Figure 4 Effects of açaí (Euterpe oleracea Mart.) seed extract (200 mg/kg per day) on (a and b) superoxide dismutase, (c and d) glutathioneperoxidase and (e and f) catalase activity in the plasma and kidney of 120-day-old adult offspring of control or low-protein fed dams during preg-nancy. Data are mean ± standard error of the mean, n = 8 for all groups. *Significantly different (P < 0.05) from control; #significantly different(P < 0.05) from açaí (Euterpe oleracea Mart.) seed extract; +significantly different (P < 0.05) from low protein.



offspring. The antihypertensive effect of ASE has previouslybeen demonstrated by our group in different models ofhypertension,[15,16] but the present result suggests that theconsumption of ASE early during pregnancy confers pro-tection against the insult of a LP diet in adult offspring.This antihypertensive effect of ASE, rich in polyphenols, islikely to be mediated by endothelium-dependent vasodila-tion because our previous findings showed that ASEinduces vasodilation of mesenteric arteries of the rat

mediated by NO in combination with endothelium-dependent hyperpolarizing factor.[14]

We found in this study a reduced endothelium-dependent vasodilation induced by ACh in the LP group,characterizing an endothelial dysfunction in this model. Inline with this finding, a reduction of endothelial responsive-ness to ACh[31,32] has been demonstrated in mesentericarteries from adult offspring whose dams were fed LP dietduring pregnancy. Additionally, placental factors may

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Figure 5 Photomicrographs of the renal cortex (400×). (a) Control, (b) açaí (Euterpe oleracea Mart.) seed extract, (c) low protein and (d) lowprotein + açaí (Euterpe oleracea Mart.) seed extract groups. Effects of açaí (Euterpe oleracea Mart.) seed extract (200 mg/kg per day) on (e) glo-meruli number per area, (f) glomeruli number per kidney, (g) glomerular volume and (h) kidney volume in 120-day-old adult offspring of control orlow-protein fed dams during pregnancy. Data are mean ± standard error of the mean, n = 5 for all groups. *Significantly different (P < 0.05) fromcontrol; #significantly different (P < 0.05) from açaí (Euterpe oleracea Mart.) seed extract; +significantly different (P < 0.05) from low protein.



induce endothelium-derived contracting factors, such asendothelin that has been demonstrated to induce increasedvasoconstriction in resistance vessels from adult offspring ofthe same model.[32] These findings suggest that maternalundernutrition alone is at least detrimental to offspringendothelial function. There are a number of possible expla-nations for this, including an oxidative insult that could beinduced in the neonatal period and perpetuated throughoutadulthood,[33] the attenuation in vascular endothelial nitricoxide synthase (eNOS) or in antioxidant protection.[31]

Metabolic changes, linked with the LP diet during preg-nancy, can also influence the vascular function. Theseinclude a reduction in the availability of L-arginine, whichcould adversely influence endothelial NO synthesis.[34]

Growing evidence suggests that the placental nitrergicsystem is involved in epigenetic fetal programming.[35]

Although the arginine levels are not reduced in the plasmaand kidney of adult offspring from a maternal caloricrestriction rat model, the asymmetric dimethylarginine(ADMA), an endogenous NOS inhibitor, is increased inplasma,[36] which could also induce endothelial dysfunction.In the same model, increased levels of ADMA are associatedwith oxidative stress and decreased renal neuronal NOS,resulting in chronic kidney disease progression.[36]

Recently, it was demonstrated that eNOS deficiencyreduces uterine blood flow, spiral artery elongation and pla-cental oxygenation in pregnant mice.[37] Furthermore, NOrelease induced by vascular endothelial growth factor isreduced in uterine arteries from protein-restricted dams,which may contribute to the decrease in uterine artery vaso-dilator potential,[37] demonstrating an important role of NOon fetal placental homeostasis.

There is evidence that polyphenols present in fruits areable to modulate the production of NO in vascularendothelium, contributing to the prevention of endothelialdysfunction.[38] We found that treatment with ASE pre-vented the endothelial dysfunction and increased the nitritelevels in adult offspring from LP-fed dams. The beneficialeffects of ASE in dams fed LP diet may be related toincreased bioavailability of NO in fetal-placental circulationduring pregnancy, as our previous findings[14] showed thatASE induces NO production in cultured endothelial cells.The increased NO bioavailability could also counterbalancethe increased vasoconstriction observed in resistance vesselsfrom adult offspring of this model.[32]

Oxidative stress has been suggested as causative agent inhuman pregnancy-related disorders, such as embryonicresorption, recurrent pregnancy loss, pre-eclampsia andintra-uterine growth restriction. Nutritional deficiencies inprotein and micronutrient antioxidant vitamins and traceminerals may impair cellular antioxidant capacities becauseproteins provide the amino acids needed for the synthesis ofantioxidant enzymes.[39] Some studies indicate that maternal

protein restriction during pregnancy and lactation inducesoxidative damage in the offspring by increasing mem-brane lipid peroxidation and decreasing the antioxidantdefence.[40] In this study, oxidative damage, assessed byMDA and protein carbonyl protein levels in plasma andkidney homogenates, was increased in the LP group. Inaccordance with previous findings in experimental model ofmaternal protein restriction,[41] the adult offspring of LPgroup showed a reduction in antioxidant enzyme acti-vity (SOD and GPx) in kidney and plasma homogenates.Therefore, the increased MDA and carbonyl protein levels,associated with decreased nitrite levels and antioxidantactivity in plasma and kidney may also contribute todecrease NO bioavailability, which can explain in part theendothelial dysfunction and early increase in blood pressurebecause it is largely known that NO contributes to themaintenance of SBP. We found that treatment with ASEduring pregnancy reduced MDA and protein carbonyllevels, and increased antioxidant enzyme activity in theadult offspring, suggesting that its antioxidant actionduring developmental intrauterine period may be associ-ated with the beneficial effects of ASE. The antioxidantaction of ASE was previously demonstrated by our group indifferent experimental models of hypertension.[15,16]

Therefore, our results indicate that the beneficial effectsof ASE in dams fed LP diet during pregnancy may pass toadult offspring and is probably associated with theimprovement of the endothelial function in fetal-placentalcirculation. The increased NO production in endothelialcells[14] and antioxidant effects produced by ASE mayincrease NO bioavailability, leading to the improvement ofuterine artery vasodilatation, blood flow oxygen and nutri-ents supply to the fetus.

LP diet in dams during pregnancy induces disturbancesin the fetal placental homeostasis that can explain thereduction of BW,[7] glomeruli number and increase ofthe glomerular volume in the offspring.[42] These resultsare associated with altered renal parameters such ashipoalbunaemia, proteinuria, hypertension and increasedlevels of urea, creatinine and %FENa+ of animals from theLP group. The exact mechanism involved in the reducednumber of nephrons due to maternal protein restrictionremains unclear, but increased evidence shows thatapoptosis in the developing kidney and suppression ofrenin–angiotensin system in newborns could inhibitnephrogenesis, reducing the number of nephrons leading toa fall in the rate of total glomerular filtration.[43] Alterationsin glomerular volume density could be mainly the result ofan average glomerular volume, an event that has beenproposed as a key stage in the pathogenesis of secondaryrenal lesions, including arterial hypertension, whichrepresents a common final pathway for the eventualdevelopment of glomerulosclerosis.[42] Treatment with ASE



during pregnancy improved the renal structural changes,normalized BW, urea, creatinine, %FENa+ and serumalbumin levels in the adult offspring, probably by its anti-oxidant action,[15,16] contributing to improvement ofnephrogenesis, renal function and protecting against thedevelopment of renal disease. Further studies should becarried out to elucidate the oxidant and renin–angiotensinsystem-regulating mechanisms by ASE.

Conclusion

In conclusion, this study provides further support for theearly postnatal environment playing a critical role in pro-gramming later life hypertension, endothelial dysfunction,renal functional, and structural disorders and oxidativestress. These data emphasize the importance of a balanceddiet during pregnancy and the consequences on chronicdisease incidence if an unhealthy diet is consumed duringthis period. We suggest that the administration of ASE inrats fed an LP diet during pregnancy could contribute touterine artery vasodilation, blood flow oxygen and nutrients

supply to the fetus. These beneficial effects may be related toincreased bioavailability of NO and to their antioxidantactions, contributing to the improvement of the parametersobserved in this study. Thus, the administration of ASE mayoffer a promising natural and safe new trend for treatmentand prevention of changes caused by maternal proteinrestriction.

Declarations

Conflicts of interest

Roberto Soares de Moura is inventor of a patent PCT/BR2007/000178 that may support the development of aproduct. The other authors state no conflict of interest.

Funding

This work was supported by the National Council of Scien-tific and Technological Development (CNPq, Protocol473514/2011-7) and Rio de Janeiro State Research Agency(FAPERJ; Protocol E-26/102.920/2011).

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protective effect of e uterpe oleracea mart (açaí) extract on programmed changes in the adult...

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