resveratrol up-regulates the erythrocyte plasma membrane redox system and mitigates...

Resveratrol Up-Regulates the Erythrocyte PlasmaMembrane Redox System and Mitigates Oxidation-Induced

Alterations in Erythrocytes During Aging in Humans

Kanti Bhooshan Pandey and Syed Ibrahim Rizvi

Abstract

Reactive oxygen/nitrogen species (ROS/RNS)–mediated oxidative damage followed by disturbed cellular ho-meostasis is involved in aging and related consequences. Lipid peroxidation, post-translational modifications ofproteins, and an impaired defense system due to increased oxidative stress jeopardize cell fate and functions,resulting in cell senescence. Resveratrol, a natural stilbene, has extensively been reported to elicit a plethora ofhealth-promoting effects. The present study carried out on 97 healthy human subjects (62 males and 35 females) ofboth sexes provides experimental evidence that resveratrol confers ability to up-regulate the plasma membraneredox system (PMRS) along with ascorbate free radical reductase, a compensatory system operating in the cell tomaintain cellular redox state. Furthermore, resveratrol provided significant protection against lipid peroxidationand protein carbonylation and restored the cellular redox homeostasis measured in terms of glutathione (GSH) andsulfhydryl (–SH) group levels during oxidation injury in erythrocytes of different age groups in humans. Findingssuggest a possible role of resveratrol in retardation of age-dependent oxidative stress.

Introduction

Oxidative damage induced by generation of reactiveoxygen/nitrogen species (ROS/RNS) over a period of

time is acknowledged as one of the main determinants ofaging.1,2 A chronic state of oxidative stress due to imbalancebetween the pro-oxidants and anti-oxidants is assumed to bethe causative factor underlying the pathobiology of manyage-related manifestations.2,3

Studies performed on a large variety of aging systemshave demonstrated the accumulation of oxidation productsof lipids, nucleic acids, proteins, sugars, and sterols, ulti-mately causing cellular dysfunction and making the bodyprone to external deleterious agents.4 Lipids and proteinsare the vital molecules of cells providing structural integrityand playing key roles for specific cellular functions. Damage tothese biomolecules significantly alters the structure of mem-branes, resulting in altered fluidity, permeability, transport,and metabolic processes.5 There are reports relating the inti-mate involvement of lipid peroxidation and protein and nu-cleic acid oxidation in aging and age-associated events, such asstroke, Alzheimer disease, diabetes, and hypertension.6,7

Reduced glutathione (GSH), the main intracellular non-protein sulfhydryl (–SH), plays an important role in main-tenance of cellular proteins and lipids in their functional state

and provides major protection in oxidative injury by beingoxidized into glutathione disulfide (GSSG).8 The GSH/GSSGredox, per se, functions in redox signaling and control as wellas anti-oxidant protection. –SH groups in membranesmaintain the microelasiticity of the cell and thus balance thedeformability of the whole cell.

During the last decade, dietary supplementation of plant-derived polyphenols has emerged as a promising approachto counteract age-associated physiological dysfunctions.9

Resveratrol (3,5,4¢-trihydroxystilbene), present abundantly inthe skin of grapes and in red wine, has been shown to elicitsignificant protective effects in diabetes, cardiovascular dis-eases, hypertension, and neuro-disorders10 and to increaselife span in many organisms.11 However, the molecular tar-gets of resveratrol that mediate its diverse biological effectsremain speculative.10

Oxidative damage to erythrocytes has been studied in-tensely with regard to age-associated cellular consequencesand their possible interventions.7,12 It has been reported thathuman erythrocyte membrane possesses a group of oxido-reductases that transfer the electrons from intracellular do-nors (nicotinamide adenine dinucleotide [NADH] and/orascorbate [ASC]) to extracellular reactions to maintain thecellular redox status.13,14 The plasma membrane redox sys-tem (PMRS) incorporation with ascorbate free radical (AFR)

Department of Biochemistry, University of Allahabad, Allahabad, India.

REJUVENATION RESEARCHVolume 16, Number 3, 2013ª Mary Ann Liebert, Inc.DOI: 10.1089/rej.2013.1419

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reductases donates reducing equivalents to extracellularAFR, thereby regenerating ASC in plasma. Such a redoxsystem prevents depletion of ASC in plasma, enabling thecells to counteract oxidative processes. The PMRS is hy-pothesized as a compensatory/protective mechanism thatoperates to mitigate oxidative stress during aging.15,16

In view of our previous findings regarding the intracel-lular uptake of resveratrol by erythrocytes and providingelectrons to PMRS, 17 the aim of this study was to investigatethe efficacy of resveratrol to modulate PMRS during aging inhumans. We also report the effect of resveratrol on markersof oxidative stress in erythrocytes subjected to oxidativeconditions from young, middle-aged, and old humans.

Materials and Methods

The study was carried out on 97 normal healthy subjectsof both genders (62 males and 35 females) between the agesof 16 and 80 years. The selection criteria have already beenestablished and described in previous published reports.12,16

Briefly, the subjects were divided into three age groups—young ( < 40 years; n = 34), middle-aged (40–60 years; n = 32),and old ( > 60 years; n = 31). The body mass index (BMI) ofthe subjects ranged from 18.8 to 26.2 kg/m2. All volunteerswere screened for asthma, tuberculosis, diabetes mellitus, orany other major illness. None of the subjects were smokers orwere taking any medication. Care was also taken to excludethe volunteers that were taking or had taken any nutritionalsupplements during last 3 months. The elderly subjects wereliving at home, but were functionally independent withoutany cognitive impairment. All persons gave their informedconsent for the use of their blood samples for the study. Theprotocol of study was in conformity with the guidelines ofthe Allahabad University Ethical Committee.

Collection of blood, isolation of packed red blood cells,and preparation of red blood cell ghosts

Human venous blood was obtained by venipuncture inheparin and centrifuged at 4�C for 10 min at 800 · g. Afterremoval of plasma, buffy coat, and the upper 15% of eryth-rocytes, isolated erythrocytes were washed four to five timeswith 0.154 M NaCl, resulting in packed red blood cells(RBCs). The erythrocyte membranes from leukocyte-free redcells were prepared according to our previously publishedmethod,18 which involves the principle of osmotic shocktreatment with hypotonic and hypertonic buffers (pH 7.4).The protein content was measured by the method of Lowryet al.19 using bovine serum albumin (BSA) as the standard.

Determination of erythrocyte PMRS activity

The activity of erythrocyte PMRS was estimated by fol-lowing the reduction of ferricyanide as described earlier.16

Briefly, 0.2 mL of RBCs were suspended in phosphate-buffered saline (PBS) containing 5 mM glucose and 1 mMfreshly prepared potassium ferricyanide to a final volume of2.0 mL. The suspension were incubated for 30 min at 37�C andthen centrifuged at 1,800 · g at 4�C. The supernatant collectedwas assayed for ferrocyanide content using 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid disodium salt and measuringabsorption at 535 nm (e = 20,500 M - 1 cm - 1). Results are ex-pressed in lmol ferrocyanide/mL PRBC/30 min.

Determination of AFR reductase activity

The erythrocyte AFR reductase activity was assayed fol-lowing the method as described by May et al.20 The washederythrocytes were hemolysed and diluted 100% (vol/vol) byaddition of water, followed by centrifugation for 10 min inthe cold. AFR was generated in diluted hemolysates by in-cubating them at 37�C in PBS (pH 7.0), containing 1 mMascorbate, 5 units/mL ascorbate oxidase, and 0.1 mM ofNADH. The rate of NADH oxidation was measured spec-trophotometrically at 340 nm for 3 min at 37�C. The change inNADH concentration was calculated from the slope of theresulting line, using an extinction coefficient e = 6.22 mM - 1

cm - 1. The values were corrected in each experiment for therate observed with lysate and reduced nucleotide alone. AFRreductase activity is reported in terms of lmol NADH oxi-dized/min per mL PRBC.

Determination of malondialdehyde content

Erythrocyte malondialdehyde (MDA) level was measuredusing the method of Esterbauer and Cheeseman.21 Packederythrocytes (0.2 mL) were suspended in 3 mL of Krebs–Ringer phosphate buffer (pH 7.4). The lysate (1 mL) wasadded to 1 mL of 10% trichloroacetic acid, and the mixturewas centrifuged for 5 min at 1,000 · g. The supernatant (1 mL)was added to 1 mL of 0.67% thiobarbituric acid in 0.05 MNaOH, and boiled for 20 min at a temperature greater than90�C. The solution was cooled and read against a comple-mentary blank at 532 nm optical density (OD1) and 600 nm(OD2). The net OD was calculated by subtracting absorbanceat OD2 from that at OD1. The concentration of MDA inerythrocytes was determined from a standard plot, and wasexpressed as nmol/mL of packed RBCs.

Determination of reduced glutathione level

Erythrocyte reduced GSH content was measured follow-ing the method described earlier,22 based on the ability of the–SH group to reduce 5,5¢-dithiobis-(2-nitrobenzoic acid)(DTNB) and form a yellow-colored anionic product whoseOD is measured at 412 nm. Concentration of GSH is ex-pressed in mg/mL packed RBCs and was determined from astandard plot.

Determination of erythrocyte membraneprotein carbonyls

Erythrocyte membrane protein carbonyls were measuredaccording to procedure of Levine et al.23 Erythrocyte mem-brane samples (0.2 mL) in PBS were taken in two tubes as testand control samples. A total of 4.0 mL of 10 mmol$L - 1 2,4-dinitrophenylhydrazine (DNPH), prepared in 2 M HCl, wasadded to the test sample, and 4.0 mL of 2 M HCl alone wasadded to the control sample. The contents were mixedthoroughly and incubated for 1 hr in the dark at 37�C. Thetubes were shaken intermittently every 10 min to facilitatereactions with proteins. After shaking, 20% trichloroaceticacid (wt/vol) was added to both tubes, and the mixture wasleft in ice for 10 min. The tubes were then centrifuged at850 · g for 20 min to obtain the protein pellets. The super-natant was carefully aspirated and discarded. The proteinpellets were washed three times with ethanol–ethyl acetate(1:1, vol/vol) solution to remove unreacted DNPH and lipid

RESVERATROL AND OXIDATIVE STRESS DURING AGING 233

remnants. Finally, protein pellets were dissolved in 6 Mguanidine hydrochloride and incubated for 10 min at 37�C.The insoluble materials were removed by centrifugation.Carbonyl content was determined by measuring the absor-bance of the supernatant at 370 nm. Each sample was readagainst the blank. The carbonyl content was calculated usingthe molar extinction coefficient of DNPH, e = 22,000 M - 1cm - 1,and data were expressed in nmol/mg protein.

Determination of erythrocyte membrane –SH group

The erythrocyte membrane –SH group was estimated ac-cording to the Kitajima method.24 Erythrocyte ghosts(0.2 mL) in 0.1 M sodium phosphate buffer containing0.1 mM EDTA (pH 8.2) were dissolved in 10% sodium do-decyl sulfate (SDS) at room temperature. After 10 min ofincubation, the mixture was centrifuged and supernatantwas mixed with 0.1 mL of DTNB (0.1 M). The solution wasallowed to stand for 15 min to develop the color, and OD wasmeasured at 412 nm against reference blank. The concentra-tion of the –SH group in erythrocyte ghosts is expressed asnmol/mg protein.

Induction of oxidative stress and in vitro experimentswith resveratrol

Oxidative stress was induced in vitro by incubating wa-shed erythrocytes/erythrocyte ghosts of all three age groupswith 10 - 5 M tert-butylhydroperoxide (t-BHP) for 60 min at37�C. The effect of resveratrol at different doses (0.1–100 lM)was evaluated by co-incubating erythrocytes/erythrocyteghosts with t-BHP and resveratrol for 60 min at 37�C. Theconcentration and duration of t-BHP used in the presentstudy to induce oxidative stress in erythrocytes. The proce-dure of experiments with resveratrol was similar to alreadypublished reports.18,22

Statistical analysis

Statistical analyses were performed using the softwarePRISM 5 (Graphpad Software Inc., San Diego, CA). Theresults are reported as means – standard deviation (SD) of10–12 independent experiments and were considered to besignificant when p < 0.05.

Results

Treatment of resveratrol activated the erythrocyte PMRSand AFR reductase in all three age group erythrocytes in adose-dependent manner. Interestingly, resveratrol at verylow concentration (0.1 lM) activated both the parameters;however, the effects were significant ( p < 0.05) only in theyoung age human erythrocytes. The magnitude of activationof PMRS as well as AFR reductase by resveratrol was slightlyreduced in the old age group erythrocytes in comparison tothe middle-aged and young groups at the same concentra-tions (Fig. 1).

Induction of oxidative stress to the erythrocytes causedan increase in MDA levels in all three age groups; however,the erythrocytes of the old age group showed higher sus-ceptibility to oxidation. Incubation with t-BHP caused a 37%( p < 0.001) elevation in MDA levels, whereas in the middle-aged and young age group erythrocytes, it was 30%( p < 0.01) and 24% ( p < 0.05), respectively (Fig. 2). Treatmentof resveratrol provided significant ( p < 0.05) protectionagainst lipid peroxidation, as evidenced by decreased gen-eration of MDA in all age group erythrocytes in a dose-de-pendent manner. However, this effect was more pronouncedin the old age group because the effect of resveratrol wasinsignificant at 0.1 lM in young and middle-aged erythro-cytes (Fig. 2).

Similar to the lipid peroxidation, proteins of old age grouperythrocytes were severely affected by oxidative injury, withincreased generation of carbonyl derivatives in their mem-branes on induction of oxidative stress. In the old age group,membrane protein carbonyl group (PCO) generation was59% ( p < 0.001); it was 45% ( p < 0.001) in middle-aged and37%% ( p < 0.001) in young age group erythrocyte mem-branes. Presence of resveratrol in the medium attenuated themembrane protein oxidation significantly ( p < 0.01) in all agegroup erythrocytes (Fig. 3). Although the effect was con-centration dependent, it increased from 0.1 to 100 lM; how-ever, resveratrol at a concentration of 0.1 lM was protectiveonly in the old age group.

The increase in oxidative stress by t-BHP incubation re-sulted in depletion in GSH content; quantitatively, depletionwas 48% ( p < 0.001) in the old age group and was 27%( p < 0.05) in the middle-aged group. In the young age group,

FIG. 1. Concentration-dependent (0.1–100 lM) effect of resveratrol on the plasma membrane redox system (PMRS) (A) andascorbate free radical (AFR) reductase (B) activities in erythrocytes of different age group humans. Activities of PMRS andAFR reductase are expressed in lmol ferrocyanide/mL packed red blood cells (PRBC)/30 min and lmol nicotinamideadenine dinucleotide (NADH) oxidixed/min per mL of PRBC, respectively. (*) The effect was not significant in comparison torespective control.

234 PANDEY AND RIZVI

FIG. 2. Concentration-dependent (0.1–100 lM) effect of resveratrol (Res) treatment on malondialdehyde (MDA) content intert-butylhydroperoxide (t-BHP)-induced, oxidatively stressed erythrocytes of different age groups—young ( < 40 years,n = 34), middle-aged (40–60 years, n = 32), and old ( > 60 years, n = 31). Concentration of MDA is expressed in nmol/mLpacked red blood cells (PRBC) and values are expressed as mean – standard deviation (SD) of 10–12 independent experi-ments. Statistical differences were analyzed with the Student t-test and considered to be significant when p < 0.05. (*) Theeffect was not significant.

FIG. 3. Concentration-dependent (0.1–100 lM) effect of resveratrol (Res) treatment on generation of membrane proteincarbonyl groups (PCO) in tert-butylhydroperoxide (t-BHP)-induced, oxidatively stressed erythrocytes of different agegroups—young ( < 40 years, n = 34), middle-aged (40–60 years, n = 32), and old ( > 60 years, n = 31). Concentration of PCO isexpressed in nmol/mg protein, and values are expressed as mean – standard deviation (SD) of 10–12 independent experi-ments. Statistical differences were analyzed with the Student t-test and were considered to be significant when p < 0.05. (*)The effect was not significant.


erythrocytes depletion was 19% ( p < 0.05). Resveratrol dem-onstrated a protective effect against GSH oxidation, this ef-fect being more pronounced in erythrocytes of young agehumans ( p < 0.01) than old ( p < 0.05) (Fig. 4). Effect of re-sveratrol at 0.1 lM was not significant in any age group.

Subjecting erythrocytes to increased oxidative stresscaused a decline in the –SH group content in all age groupsand again the decline was more pronounced in the old agegroup 41% ( p < 0.001) than the middle-aged group 34%( p < 0.01) and the young age group 29% ( p < 0.01), suggestiveof an increased oxidative stress in higher age groups.The presence of resveratrol in the media protected –SHgroup oxidation significantly ( p < 0.01) in a concentration-dependent manner (Fig. 5). Interestingly resveratrol at verylow concentration (0.1 lM) elicited a significant protection( p < 0.05) against oxidation of erythrocyte membrane –SHgroups in all three age groups.

Discussion

PMRS regulates the cellular redox state in various ways,including transfer of reducing equivalent and energy me-tabolism.25 Indeed, PMRS activity has been linked to pro-cesses such as modulation of the cellular NAD + /NADHratio in response to fluctuations in energy demand, cellgrowth, differentiation, and survival.25,26 The complemen-tary role of activated PMRS has been discussed many timesduring aging and other age-related diseases.14,27

PMRS incorporation with AFR reductase is known to re-cycle ASC in the plasma.13 During oxidative stress/presenceof oxidants, ASC is oxidized first to AFR and then to

dehydroascorbate (DHA), which is unstable and undergoesirreversible hydrolysis to 2,3-diketo-l-gulonic acid, resultingin a decreased level of ASC in the body.28 Erythrocytes cantake up DHA from the plasma through the GLUT1 glucosetransporter, and inside the cell DHA can be recycled to ASCvia direct reduction by GSH/glutathione-dependent en-zymes such as glutaredoxin and protein disulfide isomer-ases, and by nicotinamide adenine dinucleotide phosphate(NADPH)-dependent thioredoxin reducatase (Fig. 6).16 ASCcan recycle a-tocopherol in low-density lipoprotein (LDL) inthe face of an oxidant stress and thus affords protectionagainst oxidation.29

In our pervious study,17 we have reported that resveratrolcan enter the cell and, once inside, it can achieve criticalhigher concentration at which it donates electrons to PMRS,thereby regenerating ASC in plasma. Our observation of anup-regulation of PMRS and AFR reductase by resveratrol inall age group erythrocytes provides a vital clue regardingthe importance of ASC regeneration and the role of ASC asthe primary anti-oxidant present in plasma contributing to thefirst line of protection against oxidative injury. The impor-tance of ASC lies in the fact that ASC performs importantfunctions in humans and other mammals serving as a co-factor in several important enzyme reactions, including thoseinvolved in the synthesis of catecholamines, carnitine, cho-lesterol, amino acids, and certain peptide hormones.29,30

Significantly, humans are unable to synthesize this vitalmolecule because of lack of functional l-gulonolactone oxi-dase.31 The lower efficiency of resveratrol in activation oferythrocyte PMRS in old age population in comparison withmiddle-aged and young populations may be explained by

FIG. 4. Concentration-dependent (0.1–100 lM) effect of resveratrol (Res) treatment on reduced glutathione (GHS) level intert-butylhydroperoxide (t-BHP)-induced, oxidatively stressed erythrocytes of different age groups—young ( < 40 years,n = 34), middle-aged (40–60 years, n = 32), and old ( > 60 years, n = 31). GSH content is expressed in mg/mL of packed redblood cells (PRBC). Values are expressed as mean – standard deviation (SD) of 10–12 independent experiments. Statisticaldifferences were analyzed with the Student t-test; and were considered to be significant when p < 0.05. (*) The effect was notsignificant.


the fact that the PMRS is already activated in old age in itscompensatory role of maintaining reduced plasma anti-oxidant potential.

Our proposed mechanism on up-regulation of PMRSalong with the AFR reductase by resveratrol from inside thecell is substantiated by the recent finding in which authorshave reported a much higher intracellular resveratrol levelcompared to ingested level.32 We propose that the activationof PMRS and AFR reductase by resveratrol mimics caloricrestriction (CR), because CR has also been shown to activatePMRS and associated enzymes in the brain during aging.33

Damage from ROS/RNS and depleted cellular anti-oxidant defenses manifests in several tissues, including thekidney, eye, nervous system, and heart, for which the endresult is aging and age-related events. Lipid peroxidation is achain reaction that disintegrates membranes and impairscellular function of a wide range of proteins involved insignaling, metabolism, and housekeeping enzymes. Even amoderate level of lipid peroxidation could have significantphysiological meaning for cell signaling and membrane re-modeling; however, the current understanding against anti-lipid peroxidation intervention is limited.5

MDA is the well-established index of lipid peroxidation andreferred to as potent biomarker of oxidative stress. It is highlyreactive itself in cellular systems; MDA may interact with andbind to the vital proteins, making them non-functional. MDAhas the capability to induce oxidative stress by targeting mi-tochondrial complexes I and II and thereby disrupting theproper flow of electrons through the electron transportchain.34,35 A decline in MDA generation due to treatment withresveratrol not only lowers the oxidative stress and resultingdamages, but also ensures the protection against future dele-terious biological consequences caused by MDA.

Oxidative stress–induced post-translational modificationof proteins has a great impact on the whole-cell physiology.Carbonylation and other oxidative stress–induced post-translational modification of proteins readily inactivate them,and thus malfunctioning enzymes. The accumulation of oxida-tive junk can jeopardize cellular functions and fate.36 All aminoacid residues are sensitive to oxidative injury, causing oxidationof their side chains and protein–protein cross linkage.37 Directoxidation of lysine, arginine, proline, and threonine residuesgenerates carbonyl derivatives, which due to easy detection andcomparatively higher stability are most often used as the mar-ker of oxidative stress.37 It is interesting that carbonyl moietiescan also be formed by reaction of proteins with MDA and otheraldehydes or by glycation with glucose.6,36

Because the erythrocyte membrane is comparable to theplasma membrane of most eukaryotic cells in terms of cellmembrane structure and function,38 protection of proteinsfrom oxidation during aging by resveratrol in other cells mayalso be explained by the sirtuin1 (SIRT1) activation effect ofresveratrol.11,39 It has been reported that age-associated ox-idized proteins could be a result of the impaired proteasomesystem, a decreased protein turnover that increases their lifespan and thus a lengthened time for ROS/RNS-mediatedprotein damage.40 The acetylation related lack of ubiquiti-niation could increase the life span of proteins, resulting inincreased carbonylation. Deceased acetylation via activationof SIRT1 might increase the rate of degradation because in-tervention to prevent age-associated decrease in proteasomeactivity and maintain the level of protein degradation appearto be useful in retarding aging and onset of other age-specificdiseases.5,41

GSH levels in tissue reflect the dynamic equilibrium be-tween its synthesis and utilization. Several reports indicate

FIG. 5. Concentration-dependent (0.1–100 lM) effect of resveratrol (Res) treatment on membrane sulfhydryl (–SH) groupcontent in tert-butylhydroperoxide (t-BHP)-induced, oxidatively stressed erythrocytes of different age groups—young ( < 40years, n = 34), middle-aged (40–60 years, n = 32), and old ( > 60 years, n = 31). –SH content is expressed in nmol/mg protein.Values are expressed as mean – standard deviation (SD) of 10–12 independent experiments. Statistical differences wereanalyzed with the Student t-test and were considered to be significant when p < 0.05.


that tissue injury, induced by various stimuli, is coupled withthe GSH depletion.42 GSH with ASC in the cell has manyfunctions, including protection of –SH groups being oxi-dized, function of glutathione peroxidase (GPx), and detox-ification of hydrophobic substances in reactions catalyzed byglutathione S-transferase (GST). GSH, GPx, glutathione re-ductase (GR), and NADH form the anti-oxidant system ofGSH in which NADH and GR are necessary for GSH re-generation.8 GR reduces GSSG to GSH with the help ofelectrons donated from NADH, enabling GSH to protectprotein –SH groups from oxidative injury and prevent pro-tein cross linking.7,8 Impairment in the oxidative stability ofthe cell may be due to degenerated –SH groups concomitantwith an increase in oxidative stress.7,43 Protection of GSHand –SH group oxidation by resveratrol during increasedoxidative stress in all age group erythrocytes not only en-hances the inherent anti-oxidant system of the cell but alsomaintains homeostasis, because changes in homeostasis canalso potentiate the accumulation of advanced glycation end-products, resulting in defects in protein processing andfunction as well as a further increase in inflammation.44

Furthermore, resveratrol may also prevent the depletion of

ASC in the cell, because a reduced amount of GSH is be-lieved to cause decreased reduction of DHA to ACS in thecell.45 A more pronounced effect in young age group eryth-rocytes in comparison to middle-aged and old erythrocytessignifies the inherent lower level of anti-oxidant defense inold age.

Resveratrol has also been reported to inhibit suicidalerythrocyte death, a major deleterious event induced byoxidative stress.46 Oxidative stress and/or energy depletionis reported to stimulate red cell membrane scrambling andcell shrinkage fallowed by suicidal erythrocyte death, or er-yptosis, leading finally to anemia, a clinical condition un-derlying many age-related diseases.47,48

Concentrations of resveratrol (0.1–100 lM) used in ourstudy assume significance because it has been reported thatplasma concentration of resveratrol levels after intake of aresveratrol-rich diet are in the micromolar range.49,50 It hasalso been reported that three servings, approximately 450 mLof wine, are more than sufficient to achieve plasma levels offree trans-resveratrol within the rage of 100 nM to 1 lM.51

Accumulation of resveratrol has been reported in many tis-sues, such as heart, liver, lungs, and kidney, after oral

FIG. 6. Schematic representation of involvement of resveratrol in maintaining the intra- and extracellular redox state inhuman erythrocytes. Under normal conditions, the plasma membrane redox system (PMRS) and ascorbate free radical (AFR)reductase function to transfer reducing equivalents from intracellular electron donors to plasma that are used to reduce the AFRto reduced ascorbate (ASC). This is a compensatory mechanism operating in the cell to protect against increased oxidative stressduring aging. We report that in addition to ASC and nicotinamide adenine dinucleotide (NADH), resveratrol can also donatethe electrons and activate the PMRS along with AFR reductase, and thus helps in increased ASC recycling during aging. DHA,dehydroascorbate; Glut, glucose transporter; RBC, red blood cell. Color images available online at www.liebertpub.com/rej


administration.52 It is of significance to mention the role ofPMRS in platelets, where it provides a mechanism to regu-late thrombus growth and cell–cell interaction in addition toplaying a crucial role in counterbalancing oxidative stress.53

However, unlike erythrocytes, platelets possess mitochon-dria, which may generate ROS leading to mitochondrialDNA damage.54 The resveratrol-driven activation of PMRS/AFR reductase in platelets may thus not only provide de-fense against oxidative stress but may also regulate the bloodcoagulation process and platelet–leukocyte cross-talk.

Involvement of oxidative stress in aging has been reportedin many knockout models either lacking or having a com-promised anti-oxidant defense system. In Drosophila melano-gaster, the absence of CuZn superoxide dismutase (Sod1)increases sensitivity to oxidative stress and diminishes lifespan by 80%.55,56 Mice lacking Sod1 (Sod - / - ) showed ex-tremely elevated oxidative stress relative to wild-type mice.In these mice, the level of lipid peroxidation, protein, andDNA damage was elevated about two- to three-fold withreference to control animals.57 The beneficial effects of CRhave been largely attributed to reduction in oxidative stress.

Our observation on the protection of oxidative alterationsin erythrocytes by resveratrol during aging is an effect thatopens the possibility of resveratrol to be considered as ananti-aging molecule. Despite the promise, the study ispregnant with limitations, including lack of experimentsshowing a cause-and-effect relationship and the use ofpharmacological inhibitors of suspected pathways, compar-ison with any reference compound and/or other polyphe-nols, and the reversibility of the observed effect. To the bestof our knowledge, this is the first report on the role of re-sveratrol in protection of cellular oxidative damage in RBCsand plasma during aging in humans.

Conclusion

Resveratrol significantly attenuated the deleterious effectof oxidative stress in erythrocytes of all age groups; however,the molecular targets of action remain speculative. The up-regulation of housekeeping compensatory mechanisms andprotection of vital cellular biomolecules from oxidation maycontribute to the anti-aging effect of resveratrol.

Acknowledgments

The work is supported by Council of Scientific and In-dustrial Research (CSIR), New Delhi, India in the form ofResearch Associateship to KBP.

Author Disclosure Statement.

No competing financial interests exist.

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Address correspondence to:Syed Ibrahim Rizvi

Department of BiochemistryUniversity of Allahabad

Allahabad 211002India

E-mail: [email protected]

Received: February 17, 2013Accepted: March 28, 2013


resveratrol up-regulates the erythrocyte plasma membrane redox system and mitigates...

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