INTRODUCTIONInjury is a leading cause of death in

people under the age of 45 and approxi-mately 15% to 20% of deaths due totrauma are preventable (1,2). Hemor-rhage is reported as the most commoncause of preventable death followingtrauma (3,4). Severe hemorrhage resultsin whole body oxygen and nutrient dep-rivation that may lead to organ dysfunc-tion and death. An early and effectivehemorrhage control is a determining fac-tor in the outcome following severe hem-orrhage (2). In humans, although a vari-ety of fluid resuscitation methods areemployed, an optimal resuscitative ap-proach to hemorrhagic shock is still un-settled (5,6). The use of agents that may

extend life of cells under adverse condi-tions such as hypoxia and decreased nu-trient availability following hemorrhagemay also play an important role inchanging the outcome following severehemorrhage (7).

Studies using animal models of hem-orrhagic injury (HI) have demonstratedincreased systemic inflammation, mito-chondrial dysfunction and reduced en-doplasmic reticulum stress with con-comitant decrease in organ functionincluding cardiac function, despite fluidresuscitation (8–11).

The hypoxic condition following hem-orrhage as well as tissue reperfusion fol-lowing resuscitation are conducive to theproduction of oxidative damage in most

organs (12–15). Mitochondria is likely tobe a major focal point for the generationof reactive oxygen species (16–19). In ad-dition, oxygen insufficiency results in de-creased mitochondrial function resultingin altered cellular energetics. Consistentwith these physiological changes follow-ing ischemia and reperfusion, antioxi-dants and agents that preserve mitochon-drial function have been shown toreduce organ functional decline and re-store cellular homeostasis in such in-juries (20–22). Our laboratory and that ofothers have demonstrated that the natu-ral antioxidant resveratrol improvesorgan function following severe hemor-rhage (23–25). Resveratrol has beenshown to enhance mitochondrial func-tion as well as biogenesis, and has beenimplicated in the extension of life span inseveral species (26–29). However, deter-mining the effect of resveratrol in reduc-ing mortality or prolonging survival fol-lowing hemorrhagic shock is importanttoward initiating clinical trials to use thisagent as an adjunct to resuscitation fluidfollowing severe hemorrhage.

Our study tests, for the first time,whether resveratrol can improve survival

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Resveratrol Improves Survival and Prolongs Life FollowingHemorrhagic Shock

Ahmar Ayub,1 Ninu Poulose,1 and Raghavan Raju1,2,3

Departments of 1Laboratory Sciences, 2Surgery and 3Biochemistry and Molecular Biology, Georgia Regents University, Augusta,Georgia, United States of America

Resveratrol has been shown to potentiate mitochondrial function and extend longevity; however, there is no evidence to sup-port whether resveratrol can improve survival or prolong life following hemorrhagic shock. We sought to determine whether (a)resveratrol can improve survival following hemorrhage and resuscitation and (b) prolong life in the absence of resuscitation. Usinga hemorrhagic injury (HI) model in the rat, we describe for the first time that the naturally occurring small molecule, resveratrol,may be an effective adjunct to resuscitation fluid. In a series of three sets of experiments we show that resveratrol administrationduring resuscitation improves survival following HI (p < 0.05), resveratrol and its synthetic mimic SRT1720 can significantly prolonglife in the absence of resuscitation fluid (<30 min versus up to 4 h; p < 0.05), and resveratrol as well as SRT1720 restores left ven-tricular function following HI. We also found significant changes in the expression level of mitochondria- related transcription fac-tors Ppar-α and Tfam, as well as Pgc-1α in the left ventricular tissues of rats subjected to HI and treated with resveratrol. The resultsindicate that resveratrol is a strong candidate adjunct to resuscitation following severe hemorrhage.Online address: http://www.molmed.orgdoi: 10.2119/molmed.2015.00013

Address correspondence to Raghavan Raju, CB2601, Sanders Building, Georgia Regents

University, 1479 Laney Walker Boulevard, Augusta, GA 30912. Fax: 815-346-3455; Phone:

706-723-4138; E-mail: RRaju@gru.edu.

Submitted January 14, 2015; Accepted for publication April 13, 2015; Published Online

(www. molmed. org) April 13, 2015.

following hemorrhagic shock in a ratmodel.

MATERIALS AND METHODS

AnimalsMale Sprague Dawley rats (250–350g)

were obtained from Charles River Labo-ratory (Wilmington, MA, USA). The ex-periments performed in this study in-cluding surgical procedures wereapproved by the Institutional AnimalCare and Use Committee (IACUC) atGRU. All animals were housed in GRUanimal facility during the experiments.

Hemorrhage ProcedureThe animals were fasted overnight

(water allowed ad libitum) prior to thesham or hemorrhagic injury (HI) proce-dure. Hemorrhagic procedure was as de-scribed before with some modification(23). The animals were anesthetized with2. 5% isoflurane (Henry Schein, Dublin,OH, USA) and were placed on Plexiglasplate in a supine position. Isoflurane wasadministered in oxygen using an anes-thetic vaporizer. Midline laparotomy(5 cm) was performed in both sham andHI animals to induce soft tissue trauma.Incision was closed aseptically in two lay-ers with sutures. Two femoral arteries andone femoral vein were cannulated (PE-50tubing), one artery was hooked up to aBlood Pressure Analyzer (Digi-Med;Micro-Med Inc., Louisville, KY, USA) tomonitor mean arterial pressure (MAP)and bleeding was performed through theother artery. Femoral vein was cannulatedto administer fluids. Surgical sites werebathed with bupivacaine. Sham animalswere not subjected to bleeding or resusci-tation. Upon awakening, the animals inHI groups were bled rapidly within thefirst 10 min to a MAP of 40 ± 5 mmHg.The bleeding was continued for 45 min,maintaining the low MAP, until 60% ofcirculatory blood volume was withdrawn.The animals were maintained in the stateof shock by maintaining MAP at 40 ± 5for another 45 min. During this time notmore than 40% of the shed blood volumein the form of Ringer lactate was given to

maintain the blood pressure. Resuscita-tion was performed with Ringer lactatefor one hour on animals in the respectivegroups. Vehicle (dimethyl sulfoxide[DMSO]; 120 μL/mouse) or drug was ad-ministered at 10 min from the onset of re-suscitation or immediately after shock pe-riod when there was no resuscitation.

10-d Survival StudyIn this group of animals, resuscitation

was carried out by Ringer lactate, fourtimes the volume of shed blood and therats were observed for 10 d. Resveratrol(10 mg/kg body weight) or vehicle(DMSO) was administered at 10 minafter the start of resuscitation. Weightwas monitored every day and left ven-tricular function was determined bymeasuring positive and negative maxi-mal dP/dt in animals that survived untilthe end of the study. One animal inresveratrol treated group was inadver-tently euthanized at d 9.

4-h Survival StudyThe animals in this group did not re-

ceive resuscitation fluid following theshock period. Resveratrol (10 mg/kg bodyweight), SRT1720 (2 mg/kg body weight)or vehicle (DMSO) was administered in-travenously at the end of the shock period.As death cannot be an endpoint as per theIACUC policy, animals were euthanizedwhen their mean arterial pressure dippedbelow 30 mmHg. This allowed us to col-lect blood from these animals before eu-thanasia, to test lactate and pH levels.

2-h StudyThe animals in this group were sub-

jected to a less aggressive resuscitationregimen by administering Ringer lactate,two times the volume of shed blood.Resveratrol (10 mg/kg body weight),SRT1720 (2 mg/kg body weight) or vehi-cle was administered 10 min followingthe start of resuscitation. After resuscita-tion, the animals were observed for 2 h,at which point the left ventricular func-tion was measured. The animals werethen euthanized and left ventricular tis-sue was removed for molecular analysis.

Cardiac ContractilityCardiac maximum and minimum con-

tractility were assessed by measuringpositive and negative dP/dt. Briefly, ani-mals were anesthetized in supine posi-tion with isoflurane (2. 5%). The surgicalsite was bathed in bupivacaine. The rightcarotid artery was exposed and a PE-50tubing was inserted into the left ventriclethrough the right carotid artery. Positiveand negative maximum dP/dt (+dP/dtand –dP/dt) were assessed using a HeartPerformance Analyzer (HPA) (Digi-Med).

Blood AnalysisAnalysis of hematocrit, hemoglobin,

lactate and pH were measured using aportable blood gas analysis instrument(Opti-Med, Atlanta, GA, USA) and ap-propriate cartridge as per the manufac-turer’s directions; 150 μL blood was usedfor each measurement.

Real-time Polymerase Chain ReactionThe TaqMan primer/probe set for

real-time PCR analysis of transcriptionfactor A, mitochondrial (Tfam)(Rn00580051_m1; cat. #4331182), peroxi-some proliferator-activated receptor α(Ppar-α) (Rn00566193_m1; cat. #4331182),Ppar-γ, coactivator 1α (Pgc-1α)(Rn01453111_m1; cat. #4331182) andβ-actin (Rn00667869_m1; cat. #4331182)were Life Technologies (Thermo FisherScientific Inc., Waltham, MA, USA) prod-ucts. Total RNA was isolated from hearttissue using Total RNA isolation mini kit(Agilent Technologies, Santa Clara, CA,USA) according to the manufacturer’sprotocol (Qiagen Inc, Valencia, CA, USA)and purity verified by measurement ofabsorbance ratios 260/280 nm in Take3Micro-Volume plate, (BioTek InstrumentsInc., Winooski, VT, USA). 200 ng of totalRNA isolated was reverse transcribed tocDNA using SuperScript VILO cDNAsynthesis kit (Life Technologies [ThermoFisher Scientific]). Quantitative real-timePCR was performed using Agilent Tech-nologies Stratagene Mx3000P real-timePCR machine with the TaqMan primer/probe set for Tfam, Ppar-α, and Pgc-1α,and normalized to β-actin. The 20 μL reac-

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tion mixture constituted 10 μL 2× mastermix containing ROX reference dye, 1 μLprimer/probe set, 8 μL RNase-DNase freewater and 1 μL cDNA as PCR template.The thermal cycling conditions were 95°Cfor 10 min followed by 40 cycles of 95°Cfor 15 s and 60°C for 1 min. As a negativecontrol, cDNA synthesis was carried outwithout reverse transcriptase enzyme inthe RNA sample, thereby precluding anyamplification due to contaminating ge-nomic DNA. Results are expressed afternormalizing to the values obtained forsamples in sham group.

Western Blot AnalysisThe heart tissues were homogenized in

Pierce RIPA lysis buffer (cat. #89901;Thermo Scientific [Thermo Fisher Scien-tific]) containing 25 mmol/L Tris-HCl pH 7.6, 150 mmol/L NaCl, 1% NP-40, 1%sodium deoxycholate, 0.1% SDS and pro-tease inhibitor cocktail (cat. #P2714;Sigma Aldrich, St. Louis, MO, USA) in aD1000 handheld homogenizer (Bench-mark Scientific, Sayreville, NJ, USA). Tis-sue lysates were centrifuged at 14,000gfor 10 min and the supernatant saved forprotein estimation and analysis. Proteinaliquots were combined with 4×Laemmli buffer (cat. #161-0747; Bio-Rad,Hercules, CA, USA) and resolved on a10% SDS polyacrylamide gel, transferredto PVDF membrane, blocked using 5%(w/v) nonfat dried milk or 5% BSA inTris-buffered saline containing 25 mmol/L Tris-HCl (pH 7. 4), 0.13 mol/L NaCl, 0.0027 mol/L KCl and0.1% Tween 20 for 1 h at room tempera-ture (RT) and then incubated with re-spective antibodies overnight at 4°C orfor 1 h at RT. The membranes wereprobed with antibodies to Ppar-α (cat.#ab8934; Abcam, Cambridge, MA, USA),Tfam (cat. #sc-23588; Santa Cruz Biotech-nology, Dallas, TX, USA), Pgc-1α (cat.#sc-13067; Santa Cruz Biotechnology)and Gapdh (cat. #2118; Cell SignalingTechnology, Danvers, MA, USA). Themembranes were subsequently washedand incubated with horseradish peroxi-dase conjugated secondary antibody for1 h at RT and developed using enhanced

chemiluminescence (cat. #34080; ThermoScientific [Thermo Fisher Scientific]). Pro-tein bands developed on X-ray filmswere quantified using the ImageJ soft-ware (Wayne Rasband, NIH, Rockville,MD, USA).

StatisticsSurvival analysis was performed by

Kaplan-Meier plot and significance be-tween survival curves was determinedusing GraphPad software (La Jolla, CA).Multigroup comparisons were carried outand significance determined by one-wayANOVA followed by Tukey test usingGraphPad software. Two-group compar-isons for significance were done by un-paired t test using GraphPad software.

All supplementary materials are availableonline at www.molmed.org.

RESULTS

Resveratrol Improves SurvivalFollowing Hemorrhagic Shock

Rats were subjected to soft tissuetrauma, hemorrhage and resuscitation.

Ten minutes into resuscitation eitherresveratrol or vehicle was administeredto the animals and monitored for 10 d.As shown in Figure 1A, mortality ratewas significantly reduced in animals thatreceived resveratrol. There was a signifi-cant improvement in MAP and heart rateat 2 h following resuscitation in the ani-mals that received resveratrol (Supple-mentary Figures 1A, B). Seven of the 12animals in the control group died, com-pared to one of the eight animals died inthe resveratrol-treated group. Most of theanimals that died following hemorrhagein the control group did not survive be-yond 24 h.

In addition to accounting for mortal-ity, we also monitored the weight gain ofall the animals in each of the groups forthe duration of the study. As shown inFigure 1B, there was a significant differ-ence in weight gain in the resveratrol-treated animals as compared with thecontrol animals, among those that sur-vived till the end of the study. It is note-worthy that the curve of means of theweight of the two groups of animals re-mained separated till the tenth day. In

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Figure 1. Resveratrol (RSV) improves survival after HI. (A) Kaplan-Meier survival curves forHI + Veh (n = 12) and HI + RSV (n = 8) groups. p < 0.05. (B) Weight gain/loss for HI + Veh(n = 5) and HI + RSV (n = 7), Mean ± SE; * indicates p < 0.05 compared with weight inthe Veh treated group on the respective day. (C) dP/dt 10 d following HI; (n = 4-6); Barsrepresent mean ± SE. *indicates p < 0.05 compared with the Veh control.

the survived animals, a significantlyhigher left ventricular function as mea-sured by dP/dt and heart rate was ob-served in the resveratrol-treated groupwhen compared with vehicle treatedgroup (Figure 1C and SupplementaryFigure 1C).

Resveratrol Prolongs Life in theAbsence of Resuscitation Fluid

To determine whether resveratrol is ef-fective in prolonging survival in the ab-sence of resuscitation, we conducted a 4-h survival study without performing resuscitation. In this study, in a subset ofanimals, we administered either resvera-trol or vehicle with 500-600 μL of Ringerlactate immediately after the shock pe-riod. No fluid was given thereafter. Theanimals that received resveratrol sur-

vived longer (Figures 2A–C). We found a sustained deterioration of MAP in the rats that received only vehicle,whereas the rats that received resveratrolhad a prolonged maintenance of MAP(Figures 2B, C).

SRT1720 Extends Life after SevereHemorrhage

Based upon the published knowledgethat one mechanism by which resveratrolexerts its metabolic action is by activa-tion of the NAD+-dependent enzymeSIRT1, we tested the effect of SRT1720, aspecific activator of SIRT1. SRT1720 wasadministered in a manner similar to thatof resveratrol and found a significantdelay in mortality, though not to the ex-tent seen in the rats that received resver-atrol (Figures 2A–C). This indicates that

SRT1720 target SIRT1 may be importantin the restoration of organ function fol-lowing hemorrhagic shock.

SRT1720 Improved CardiacContractility

To further verify the effect of SRT1720,we used a less aggressive resuscitationmodel than the one we used for 10-d sur-vival study to test whether SRT1720 mayimprove cardiac function as measured by+ and –dP/dt. SRT1720 or resveratrolwas administered 10 min into resuscita-tion and the rats were euthanized 2 hafter the end of resuscitation. Consistentwith our previous observation, resvera-trol also demonstrated significant im-provement in + and –dP/dt. We ob-served improved MAP, heart rate, leftventricular function and blood lactate in

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Figure 2. Resveratrol (RSV) and SRT1720 prolongs life after HI without resuscitation. (A) Kaplan-Meier survival curves for HI + Veh, HI + RSVand HI + SRT1720 (n = 5-6). *p < 0.05 versus HI + Veh, both curves. (B) Mean arterial pressure (MAP) at maximum bleed out (MBO), treat-ment (TX) with Veh, RSV or SRT1720, 5 min after TX, 10 min after TX and 15 min after TX in HI + Veh, HI + RSV and HI + SRT1720 (n = 5-6). Barsrepresent mean ± SE. *p < 0.05 versus HI + Veh. (C) MAP from the onset of hemorrhage to immediately prior to euthanasia in individualanimals in HI + Veh, HI + RSV and HI + SRT1720 groups. (n = 5-6). Black lines: HI + Veh; blue lines: HI + SRT1720; red lines: HI + RSV.

animals treated with resveratrol orSRT1720 (Figures 3A–D).

Molecular Mediators in the HeartWe tested the gene expression and pro-

tein expression of Ppar-α and Tfam byreal-time PCR and Western blot. Therewas a significant decrease in the tran-script level of both Ppar-α and Tfam fol-lowing HI, and the levels were restoredby resveratrol (Figures 4A, B). The West-ern blots and their semiquantitationdemonstrated a marked increase in bothPpar-α and Tfam proteins in the hearttissue of resveratrol treated animals com-pared with vehicle-treated animals asshown in Figures 5A, B and D, E, respec-tively. However, in our experiments,Tfam protein levels did not decrease fol-lowing HI. As Pgc-1α is a cofactor forPpar-α, we tested the change in mRNAtranscript (Figure 4C) and protein level(Figures 5C, F) of Pgc-1α and found sig-nificant alteration following HI andrestoration in resveratrol treated rats.

DISCUSSIONHemorrhagic injury is among the major

causes of death due to trauma. Severehemorrhage leads to whole body hypoxiaand nutrient deprivation. One of themajor cellular processes affected by severehemorrhage is mitochondrial function.Cells rely on mitochondrial oxidativephosphorylation for most of their energyneeds. The reduced tissue availability ofoxygen in hemorrhagic shock adverselyaffects oxidative phosphorylation and cel-lular energetics. We hypothesized thatagents that improve mitochondrial func-tion may promote cellular function andextend life following hemorrhagic injury.In previous studies, our laboratory andothers have observed improved organfunction in rats treated with resveratrolfollowing hemorrhagic injury (23,30).Resveratrol, a phytochemical, is a natu-rally occurring antioxidant and free radi-cal scavenger. During the past decade, in-vestigators have demonstrated that thesirtuin family of proteins, especially sir-tuin 1 (SIRT1), are among the intracellulartargets for resveratrol (31), although other

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Figure 3. Hemodynamics at 2 h after resuscitation: mean arterial pressure (MAP) (A) andheart rate (HR) (B) at the start of resuscitation and at 2 h after resuscitation in HI + Veh, HI +resveratrol (RSV) and HI + SRT1720; n = 5-6; bars represent mean ± SE; * indicates p < 0.05compared with the respective value at resuscitation. (C) ±dP/dt in sham group and at 2 hafter resuscitation in HI + Veh, HI + RSV and HI + SRT1720 groups; (n = 4-5); bars representmean ± SE. p < 0.05 compared with sham (*) or HI + Veh (#). (D) Lactate levels before HIand at 2 h after resuscitation in HI + Veh, HI + RSV and HI + SRT1720; n = 5-6; bars representmean ± SE; p < 0.05 versus HI + Veh; * indicates p < 0.05 compared with preresuscitation.

Figure 4. Effect of resveratrol (RSV) and SRT1720 on Tfam, Ppar-α and Pgc-1α expressionfollowing HI. HI down regulated the mRNA expression of Ppar-α (A) mitochondrial tran-scription factor Tfam (B) and Pgc-1α (C) in the heart and the levels restored by RSV treat-ment. The values are normalized to sham; n = 4-6. Bars represent mean ± SE. p < 0.05 com-pared with sham (*) or HI + Veh (#).

targets are also described (32,33). Our pre-viously published study demonstratedaugmented expression of SIRT1 in ratssubjected to trauma and severe hemor-rhage and treated with resveratrol (23).

Though resveratrol has been shown toimprove organ function and mitochondr-ial function, determining the effect ofresveratrol on survival following HI mayresult in better strategies in the manage-ment of hemorrhagic shock and has notbeen investigated before. In our first ex-periment in this study, young rats weresubjected to soft tissue trauma and se-vere hemorrhage, and resveratrol or ve-hicle was administered at 10 min follow-ing the start of resuscitation. We found asignificantly increased mortality in therats that were not treated with resvera-trol, six of the 12 rats in this group diedwithin 24 h and one died on the d 3,whereas only one of the eight resveratroltreated rats died during the 10-day ob-servation period. We also observed a sig-nificant weight gain in the rats that re-

ceived resveratrol and survived for theduration of the study, demonstrating thebeneficial effect of an early interventionwith this naturally occurring small mole-cule. The results show a profound influ-ence of resveratrol on survival when ad-ministered as a single dose as an adjunctto the resuscitation fluid. We had previ-ously shown that rats that were adminis-tered resveratrol along with resuscitationfluid following hemorrhagic injury hadsignificantly elevated ATP and decreasedcytosolic cytochrome c levels in the leftventricular tissue and improved cardiaccontractility, indicating a positive influ-ence on mitochondrial homeostasis andorgan function (23).

In our 10-day survival experiment,most of the mortality observed in ani-mals that were not treated with resvera-trol was found to be within 24 h. The leftventricular contractility measured in sur-viving animals at the end of the studyshowed a persisting effect on hemody-namics as observed by decreased dP/dt

and heart rate in this group of animals.This once again demonstrates the signifi-cance of early intervention following HIin providing a lasting effect not only forsurvival, but also for organ function.

We further wanted to test whetherresveratrol can extend survival in the ab-sence of resuscitation. One of the objec-tives of this experiment was to determinethe effectiveness of resveratrol in themaintenance of life in a field setting fol-lowing hemorrhage, in the absence of re-suscitation. In clinical situations whereblood transfusion or resuscitation is notpossible, whether resveratrol can extendlife until medical help becomes availablewill be of significant clinical advantage.Secondly we wanted to determine thephysiological effect of resveratrol in theabsence of resuscitation fluid, to furtherunderstand whether this molecule canpreserve hemodynamics for a prolongedperiod in the absence of exogenous fluidsupplementation. To address these ques-tions, we administered resveratrol imme-diately after shock period in a subset ofrats and found that the rats that receivedresveratrol survived for a significantlylonger period than those that receivedonly vehicle. It is noteworthy that all theresveratrol-treated animals had distinctlylonger survival time than the longest sur-vived animal in the untreated group (Fig-ure 2C). The difference in mean arterialpressure between the two groups was ev-ident from five minutes after the admin-istration of resveratrol (Figure 2B). At thetime of euthanasia there was a significantimprovement in both lactate and bloodpH in resveratrol-treated rats (Supple-mentary Figure 2). It is not clear whetherthis is a direct or indirect (prolonged sur-vival and therefore more time to reachmetabolic balance) effect of resveratrol.

Various studies have shown that SIRT1,an NAD+-dependent deacetylase, is a mo-lecular target of resveratrol (27,34,35). Wetherefore tested whether administrationof SRT1720, a small molecule activator ofresveratrol, mimics the life-extending ef-fect of resveratrol in nonresuscitated rats.The administration of SRT1720 resultedin a significant improvement in survival

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Figure 5. Effect of resveratrol (RSV) and SRT1720 (SRT) on Tfam, Ppar-α and Pgc-1α proteinlevel following HI. HI significantly downregulated Ppar-α (A,D) and Pgc-1 expressions (C,F)and the levels were restored significantly with RSV. RSV and SRT augmented Tfam expres-sion levels (B,E). Western blots from three experiments were used in the semiquantitation.Bars represent mean + SE. n = 4–6. p < 0.05 compared with sham (*) or HI + Veh (#).

time, though the effect seemed to be lessthan that of resveratrol. These results in-dicate that at least part of the effect ofresveratrol is through activation of SIRT1.

In our first experiment, we followed awell standardized and well describedsoft tissue trauma and hemorrhage pro-cedure with an aggressive resuscitationmethod (8,36,37) and found that resvera-trol can improve survival following HI.In the second experiment, we demon-strated that resveratrol can prolong lifein the absence of resuscitation andSIRT1720 can partially mimic the effectof resveratrol. In the third experiment,the effect of SRT1720 on organ functionfollowing the hemorrhage was furtherconfirmed. In the third experiment, weused a less aggressive resuscitationmodel compared with the model usedfor the 10-d study. There was significantimprovement in left ventricular functionat the end of 2 h following resuscitationin rats that received resveratrol as well asSRT1720. As we have done in a previousstudy (23) in a small group of three rats,we administered sirtinol, a known SIRT1inhibitor, five minutes prior to resvera-trol administration and found a markeddrop in left ventricular performance(+dP/dt = 6432 ± 1294 and –dP/dt =3104 ± 307; mean ± SE) as compared tothat obtained in resveratrol-treated ani-mals. These experiments demonstratethat activation of SIRT1 is an importantmolecular step in restoring organ func-tion following hemorrhagic shock.

It may be speculated that the salutaryeffect of SRT1720 is due to its effect onmitochondrial function through SIRT1(38,39). One of the mechanisms by whichresveratrol improves mitochondrial func-tion is likely due to allosteric activation ofSIRT1 (31). SIRT1, an NAD+-dependentdeacetylase, plays a significant role incellular physiology by deacetylating tar-get proteins. Among the target proteinsof SIRT1 are transcription factors such asFOXO3, p53 and NFκb, and the transcrip-tional cofactor Pgc-1α. The proteins Tfamand Ppar-α are transcription factors criti-cal to mitochondrial biogenesis and func-tion, and their expression level in left

ventricular tissue was restored by resver-atrol and markedly by SRT1720, followingHI. We also found that the level of Ppar-αand Tfam was restored or augmented, respectively, following resveratrol treat-ment (Figures 4, 5). Pgc-1α is a cofactorfor Ppar-α and the decreased level ofPgc-1α following HI and the increase withtreatment show the possible downregu-lation of the Pgc-1α–Ppar-α–mediatedpathway following HI and ameliorationwhen treated with resveratrol. This isone of the critical pathways in the home-ostasis of cellular energetics. Previously,we have shown that resveratrol can re-store total ATP and decrease cytosolic cy-tochrome C levels in the heart followingHI (23). We, and subsequently others,have also shown a declining SIRT1 levelin the heart following HI and restorationby resveratrol (23) or estradiol (40). Fur-ther studies using genetically modifiedmice might address the role of these factors in isolation, in a reductionist ap-proach, in resveratrol-induced restora-tion of cellular homeostasis. Neverthe-less, the results from these studies andthose reported in the present study pro-vide a mechanistic basis for the salutaryeffect of resveratrol on survival pre-sented in this report emphasizing thesignificance of mitochondrial function inrestoring organ function and improvingsurvival following HI.

In a previous study by Kentner et al.,administration of Tempol early duringthe resuscitation phase significantly im-proved survival in Sprague Dawley rats(41). The 3-d survival study also showedthat when the antioxidant was adminis-tered during the later part of resuscita-tion, the benefit was markedly less. Thestudy therefore demonstrated the benefi-cial effect of early intervention in hemor-rhagic shock (41). On the basis of the re-sults of this study, we may speculatethat the time of administration of resver-atrol also played an important role insurvival advantage observed in ourstudy.

Oxidative stress is largely recognized asa pathogenic hallmark of hemorrhagic/reperfusion shock (42,43). Therefore the

effect of resveratrol as an antioxidant isvery pertinent in this model. In additionto resveratrol and tempol, several otheragents that are known to reduce oxida-tive stress have also been tested in hem-orrhagic shock/resuscitation models, inshorter term studies (24,44,45). In variousmodels of oxidant stress, it has been re-ported that resveratrol inhibits the ex-pression and activity of NADPH oxidase,enhances activity of several antioxidantenzymes, including that of the SOD andscavenges reactive oxygen free radicals(ROS) (46). Juan et al. reported thatresveratrol augments the expression ofheme oxygenase-1 (HO-1) in human aor-tic smooth muscle cells, reducing ROS(47). The effect of resveratrol on lipidperoxidation, NADPH oxidase and xan-thine oxidase are also well studied in dif-ferent models (48,49). In view of these re-sults, the complex hit involved inhemorrhagic shock involves alterationsin multiple different pathways that leadto cellular dysfunction and cell deathleading to organ dysfunction.

CONCLUSIONIn summary, for the first time, we

show that when resveratrol is used as anadjunct to resuscitation fluid followingHI, there is significant improvement insurvival. In addition, in the absence ofresuscitation, resveratrol extended lifesignificantly making this small moleculea strong candidate in the treatment of se-vere hemorrhagic condition.

ACKNOWLEDGMENTSRR acknowledges financial support

from the National Institute of GeneralMedical Sciences (R01 GM 101927) andlaboratory startup assistance from theGeorgia Regents University, Augusta,Georgia, United States of America.

DISCLOSUREThe authors declare they have no

competing interests as defined by Molec-ular Medicine, or other interests thatmight be perceived to influence the re-sults and discussion reported in thispaper.

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R E S V E R A T R O L E X T E N D S L I F E A F T E R H E M O R R H A G I C S H O C K

Cite this article as: Ayub A, et al. (2015) Resveratrolimproves survival and prolongs life following hemorrhagic shock. Mol. Med. 21:305–12.