research article radioprotective effect of grape seed ...week-old male balb/c mice (chinese academy...

Research ArticleRadioprotective Effect of Grape Seed ProanthocyanidinsIn Vitro and In Vivo

Yijuan Huang1 Hainan Zhao2 Kun Cao2 Ding Sun2 Yanyong Yang2 Cong Liu2

Jianguo Cui2 Ying Cheng2 Bailong Li2 Jianming Cai2 and Fu Gao2

1Department of Radiology The First Hospital of Jiaxing 1882 Zhonghuan South Road Jiaxing Zhejiang 314000 China2Department of Radiation Medicine Faculty of Naval Medicine Second Military Medical University800 Xiangyin Road Shanghai 200433 China

Correspondence should be addressed to Bailong Li libailong2013163com Jianming Cai runnersmmu2011163comand Fu Gao gaofusmmu163com

Received 21 January 2016 Accepted 25 February 2016

Academic Editor Ciprian Tomuleasa

Copyright copy 2016 Yijuan Huang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We have demonstrated that grape seed proanthocyanidins (GSPs) could effectively scavenge hydroxyl radical (∙OH) in a dose-dependent manner Since most of the ionizing radiation- (IR-) induced injuries were caused by ∙OH this study was to investigatewhether GSPs would mitigate IR-induced injuries in vitro and in vivo We demonstrated that GSPs could significantly reduceIR-induced DNA strand breaks (DSBs) and apoptosis of human lymphocyte AHH-1 cells This study also showed that GSPs couldprotect white blood cells (WBC) from IR-induced injuries speed up the weight ofmice back and decrease plasmamalondialdehyde(MDA) thus improving the survival rates of mice after ionizing radiation It is suggested that GSPs have a potential as an effectiveand safe radioprotective agent

1 Introduction

Ionizing radiation (IR) produces large amount of reactiveoxygen species (ROS) such as hydroxyl radical (∙OH) super-oxide (O

2

minus) and hydrogen peroxide (H2O2) among which

∙OH contributes 60ndash70 to IR-induced injuries [1] IR exertsimmediate biological effects by rapid reaction of ROS withcellular macromolecules like DNA lipids and proteins

The sulphydryl compound amifostine which is the onlyradioprotectant approved to be used in humans by FDAhas shown effective radioprotective ability [2] However theobvious side effects limit its clinical use such as hypertensionnausea and vomiting [3] Considering the urgent require-ments we need to bring progress in the development of safeand effective therapeutic agents

Previously we have demonstrated that grape seed proan-thocyanidins (GSPs) could effectively scavenge hydroxylradical (∙OH) in a dose-dependentmanner [4] In the currentstudy we investigated whether GSPs exerted radioprotectiveeffect in vitro and in vivo

2 Materials and Methods

21 Cell Culture and GSPs Treatment Human lymphocyteAHH-1 cells (American Type Culture Collection ManassasVA) were cultured in RPMI 1640 (Invitrogen CA) with 10foetal bovine serum (FBS) and 1 penicillin-streptomycin-glutamine at 37∘C in a 5CO

2humidified chamber Cells

were treated with different concentrations of GSPs-rich PBSand then radiated with different doses of 120574-ray immediatelyaccording to the need of the present study

22 Radiation The resource of 60Co radiation facility wasprovided by the Radiation Center in Faculty of NavalMedicine SecondMilitaryMedical University China AHH-1 cells were radiated with 8Gy at a dose rate of 1 GyminMicewere exposed to different doses of radiation according to theneed of the present study

23 Cell Viability AHH-1 cells were cultured in 96-well platesfor 24 hrs and pretreated with or without GSPs-rich PBS at

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 5706751 7 pageshttpdxdoiorg10115520165706751

2 Oxidative Medicine and Cellular Longevity

5mins before radiation and further cultured for 24 hrs afterradiation Cell viability was determined by Cell Counting Kit(Dojindo Laboratories Kumamoto Japan)

24 Apoptosis Assays for Cultured Cells The level of theapoptosis was determined by Apoptosis Detection Kit (BipecBiopharma Massachusetts MA) Treated cells were analysedby flow cytometry Alternatively apoptosis was determinedby Hoechst 33258 fluorescein diacetate (FDA) and PI stain-ing as described previously [5]

25 Comet Assay TheDNA strand breaks (DSBs) were mea-sured by using single-cell gel electrophoresis (comet assay)based on the method of Dubner et al [6]

26 Animals andGSPs Treatment All the protocols of animalexperiment were consistent with the Guide for Care andUse of Laboratory Animals published by the US NIH Six-week-old male BALBc mice (Chinese Academy of SciencesChina) were used in the experiments The animal housewas kept with unwavering temperature (24∘C) and the micewere fed ad libitum About 1 h before radiation grape seedproanthocyanidins were administered to mice by gavagewhich were provided by Pure-one Bio Technology Co Ltd(Shanghai China) GSPs were maintained through drinkingfor 1 week after radiation

27 Survival Assays Survival rates of the mice were recordeddaily for 30 days after radiation with 8Gy at a dose rate of1 Gymin

28 Body Weight All mice were weighed by an electronicscale (GH-202 AND Japan) at the same time of the day

29 White Blood Cell Caudal vein WBC was counted in cellcount plate at the same time of the day as described previously[7]

210 MDA Assay Arterial blood samples (06mL) of themice which were collected 12 hrs after radiation were takenfor MDA assay with the method defined by Ohkawa et al[8]

211 Statistical Analysis Statistics were expressed as meansplusmn SEM for each experiment and analysed by using One-WayAnalysis of Variance Between groups a two-tailed Studentrsquos119905-test was used A 119875 value less than 005 was considered to bestatistically significant

3 Results

31 GSPs-Rich PBS Increases Cell Viability of Radiated AHH-1Cells Figure 1(a) showed that GSPs inhibited cellular prolif-eration and diminished the viability of AHH-1 cells when theconcentration of GSPs was more than 80 120583gmL In additionwe demonstrated that pretreatment of AHH-1 cells with5ndash40 120583gmL GSPs-rich PBS before radiation significantly

increased cell survival rates as compared to cells treated withradiation alone at all examined doses (up to 10Gy) and theradioprotective effect of GSPs was dose-dependent (Figures1(b) and 1(c))

32 GSPs-Rich PBS Attenuated Apoptosis in Radiated AHH-1 Cells The early apoptotic cells decreased when AHH-1cells were pretreated with 40 120583gmL GSPs-rich PBS as com-pared to cells pretreated with PBS alone in flow cytometryassay (Figure 2(a) 151 versus 342 resp) These findingswere corroborated by the morphology of dying cells usingHoechst 33258 fluorescein diacetate and propidium iodidestaining Radiated AHH-1 cells pretreated with GSPs-richPBS had a reduced apoptosis of 284 as compared to 573in PBS pretreated radiated cells (Figure 2(b)) These datasuggest that GSPs attenuate apoptosis in radiated AHH-1cells

33 Comet Assay The radiated mice resulted in an increasein the levels of all comet parameters (comet tail lengthand tail DNA) whereas pretreatment of GSPs before radi-ation inhibited the increase of these parameters significantlyindicating the protective effect of GSPs on IR-induced DNAdamage (Figure 3)

34 Survival Rates of Mice 70 of radiated mice withoutGSPs treatment died by the 15th day after radiation while50 of the mice survived when treated with 200mgkgof the body weight GSPs and 80 survived when treatedwith 400mgkg of the body weight GSPs (Figure 4(a))Figure 4(b) showed the different effects of the three strategiesof GSPs (400mgkg of the body weight) delivery includingtherapy (after radiation survival 40) prevention (beforeradiation survival 50) and the combination of therapy andprevention medication (after and before radiation survival80) We found that the combined medication improved thesurvival rate of the mice most

35 Effect of GSPs on Body Weight of Mice after Radiation(5 Gy) Weight of mice fell sharply within 4 days after radi-ation and GSPs cannot slow down its decrease (Figure 5(a))However in the subsequent time period weight of micebegan to increase and GSPs significantly speeded up theweight back

36 Effect of GSPs on WBC Count after Radiation (5 Gy)GSPs slowed down the decrease ofWBC count after radiationand speeded up the recovery of WBC count from the 4th dayafter radiation while WBC count remained at lower levelsuntil the 11th day after radiation in the radiation alone group(Figure 5(b))WBC count was back up to the peak at the 24thday after radiation and then fell to a stable level HoweverWBC count remained at higher levels in the GSPs-treatedgroup than in the radiation alone group at the end of theexperiment

37 Changes in the Levels of Plasma MDA The plasma MDAconcentrations were measured at 12 hrs after 5Gy radiation

Oxidative Medicine and Cellular Longevity 3

00

02

04

06

08

10

12C

ell s

urvi

val f

ract

ion

5 10 20 40 800GSPs treatment (120583gmL)

lowast

(a)

00

02

04

06

08

Cell

surv

ival

frac

tion

(8G

y)

5 10 20 400GSPs treatment (120583gmL)

lowast

lowast

lowast

(b)

00

02

04

06

08

10

12

Cel

l sur

viva

l fra

ctio

n

2 4 6 8 100Radiation dose (Gy)

lowast lowast lowastlowast

GSPs (+)GSPs (minus)

(c)Figure 1 Grape seed proanthocyanidins (GSPs) mitigated radiation injuries on AHH-1 cells (a) The cytotoxicity of different concentrationsof GSPs against AHH-1 cells (b) Dose-dependent effect of GSPs on cell viability induced by 8Gy gamma radiation (c) Variation of cellsurvival fractions pretreated with 40120583gmL GSPs before different doses of radiation lowast119875 lt 005 119899 = 6

Ann

exin

V-p

ositi

ve ce

lls (

of t

otal

)

0

10

20

30

40

80Radiation dose (Gy)


lowast

(a)

Apop

totic

cells

( o

f tot

al)

0

20

40

60

80


lowast


(b)Figure 2 GSPs-rich PBS attenuated radiation-induced apoptosis in AHH-1 cells (a) A bar graph of Annexin V-positive cells expressed as apercentage of total cells Treated cells were collected 24 hrs after radiation stained with Annexin V-APC and propidium iodide and analysedby flow cytometry (b) A bar graph of apoptotic cells expressed as a percentage of total cells Cells were stained with FDA Hoechst 33258 andPI 24 hrs after radiation and apoptotic cells were counted in multiple randomly selected fields lowast119875 lt 005 119899 = 6


(a)

0

20

40

60

80

100

Com

et (

)


lowast


(b)

0

20

40

60

80

Tail

leng

th (120583

m)


lowast


(c)

0

10

20

30

Tail

DN

A (

)



lowast

(d)

Figure 3 Effect of GSPs in AHH-1 cells on IR-induced DNA strand breaks assayed by comet assay (a) Representative micrographs (b)Comet (c) Tail length (d) Tail DNA lowast119875 lt 005 119899 = 6

(Figure 6) Plasma MDA concentrations in the GSPs-treatedgroupwere significantly lower than that in the radiation alonegroup Changes in the levels of MDA indicate the antioxidantpotential of GSPs

4 Discussion

An immediate effect of radiation is the generation of reactiveoxygen species (ROS) that can result in oxidative deteriora-tion of DNA lipids and proteins [9] ROS are also generatedby inflammatory cells through induction of a cascade ofcytokine activity [10] and from damaged mitochondria vialeakage from the electron transport chain [11] The factthat antioxidants were effective in ameliorating ionizingradiation-induced oxidative injuries has been demonstrated[12 13] Previously we have assessed that GSPs exhibitedsignificant free radical scavenging ability in a cell-free Fentonsystem [4] In the current study we demonstrated thatGSPs protected AHH-1 cells and mice from IR-induced

injury which may result from their radical scavengingeffect

Membrane lipids are themajor targets of ROS and the freeradical chain reaction [14] The increase of lipid peroxidationproducts such as malondialdehyde is the index of lipiddamage [6] Also DNA is one of the major targets of ROS[15] One of the consequences of DNA damage is apoptosis ofthe cells [9] In our study we observed a significant increasein the levels of plasma MDA and severe DNA strand breakswhen exposed to ionizing radiation However pretreatmentof GSPs prior to radiation exposure decreased the levels ofMDA and mitigated DNA strand breaks thus attenuatingapoptosis of the cells

Weight is one of the important indices to measure thequality of life Ionizing radiation leads to disorders suchas dyspepsia and thus results in weight loss [16] In thecurrent study we observed that the GSPs-treated group had asignificant increase in body weight compared to the radiationalone group indicating that GSPs improved the digestion


0 5 10 15 20 25 30

Time (d)

0

20

40

60

80

100Su

rviv

al (

)

GSPs+IR+GSPsGSPs+IR+GSPs

ControlIR

(200mgkg)(400mgkg)

(a)

00

02

04

06

08

10

Frac

tion

surv

ival

5 10 15 20 25 300Time (d)

ControlIR GSPs+IR+GSPsGSPs+IR

IR+GSPs

(b)

Figure 4 Survival rates of mice (a) Different dosages of GSPs were given by gavage about 1 h before radiation and maintained throughdrinking until 1 week after radiation (b) Different effects of the three strategies of GSPs (400mgkg of the body weight) delivery includingtherapy (after radiation survival 40) prevention (before radiation survival 50) and the combination of therapy and preventionmedication (after radiation and before radiation survival 80) Representative results from one of three independent experiments are shownlowast119875 lt 005 control 119899 = 10 others 119899 = 20

Wei

ght (

g)

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0Time (d)

12

13

14

15

16

17

18

19

20

lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

IR Control GSPs+IR+GSPs

(a)

0 642 8 10 282624222018161412 30Time (d)

lowastlowastlowast

lowast

lowastlowastlowastlowastlowast

lowast

lowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast

lowast0

5

10

15

20

25

ControlIR

GSPs+IR+GSPs

WBC

coun

t (lowast10

9L

)

(b)

Figure 5 Effects of GSPs on (a) body weight and (b) WBC count of mice after radiation (5Gy) Representative results from one of threeindependent experiments are shown lowast119875 lt 005 for each group 119899 = 10

and absorption function of radiated mice thereby increasingbody weight of mice

The lowest level of WBC after radiation plays the decisiverole in the survival of radiated mice IR-induced WBCdecrease can result in life-threatening infections [17] In thisstudy we observed that GSPs significantly increase the lowestlevel of WBC after radiation indicating that GSPs could pro-tect hematopoietic system from IR-induced damage whichmay account for the significant increased survival rate in theGSPs-treated group

In order to find a radioprotectant of real worth weshould consider the following features such as bioavailabilitysafety and toxicity distribution and free radical scavengingability It is much to be regretted that WR-2721 is the onlyradioprotectant registered in use for humans However ithas relatively high toxicity limiting its clinical use [18] Andcytokines and immunomodulators are effective to be usedonly when suffering from low radiation doses [19]

GSPs widely available in grapes have been reportedto possess greater free radical scavenging ability and better


0

1

2

3

4

5

MD

A (n

mol

mg

prot

ein)



lowast

Figure 6 GSPs-rich saline significantly decreased levels of plasmaMDA a marker of oxidative stress Representative results from oneof three independent experiments are shown lowast119875 lt 005 for eachgroup 119899 = 3

protection against IR-induced lipid peroxidation and DNAdamage than vitamins C and E singly and in combination[20] Previously we have assessed the dose-dependent antiox-idant ability of GSPs by ESR In addition it is important tonote that GSPs can remain in the plasma and tissues for upto 7ndash10 days and exert antioxidant properties with no toxicity[21 22] Given these properties GSPs have great potential asa clinical therapeutic agent

In this study we demonstrated that GSPs had a radio-protective effect in vitro and in vivo The radical scavengingability plays an important role in the radioprotective effect ofGSPs However the exact mechanism remains to be studied

Competing Interests

The authors have no competing interests to disclose

Authorsrsquo Contributions

Yijuan Huang Hainan Zhao and Kun Cao contributedequally to this work

Acknowledgments

This work was supported in part by grants from NationalNatural Science Foundation of China (Grants nos 81472911and 31270900) and Natural Science Foundation of Shanghai(no 14ZR1449200)

References

[1] I Szumiel ldquoIonizing radiation-induced cell deathrdquo Interna-tional Journal of Radiation Biology vol 66 no 4 pp 329ndash3411994

[2] T K Gosselin and B Mautner ldquoAmifostine as a radioprotec-tantrdquo Clinical Journal of Oncology Nursing vol 6 no 3 pp 175ndash180 2002

[3] G Facorro M M Sarrasague H Torti et al ldquoOxidative studyof patients with total body irradiation effects of amifostinetreatmentrdquo Bone Marrow Transplantation vol 33 no 8 pp793ndash798 2004

[4] Y Huang W Liu H Liu et al ldquoGrape seed pro-anthocyanidinsameliorates radiation-induced lung injuryrdquo Journal of Cellularand Molecular Medicine vol 18 no 7 pp 1267ndash1277 2014

[5] L Qian F Cao J Cui et al ldquoRadioprotective effect of hydrogenin cultured cells and micerdquo Free Radical Research vol 44 no 3pp 275ndash282 2010

[6] D Dubner P Gisone I Jaitovich and M Perez ldquoFree radicalsproduction and estimation of oxidative stress related to 120574irradiationrdquo Biological Trace Element Research vol 47 no 1ndash3pp 265ndash270 1995

[7] S Zhao KMei L Qian et al ldquoTherapeutic effects of hydrogen-rich solution on aplastic anemia in vivordquoCellular Physiology andBiochemistry vol 32 no 3 pp 549ndash560 2013

[8] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[9] W Zhao and M E C Robbins ldquoInflammation and chronicoxidative stress in radiation-induced late normal tissue injurytherapeutic implicationsrdquo Current Medicinal Chemistry vol 16no 2 pp 130ndash143 2009

[10] K Fleckenstein L Zgonjanin L Chen et al ldquoTemporal onsetof hypoxia and oxidative stress after pulmonary irradiationrdquoInternational Journal of Radiation Oncology Biology Physicsvol 68 no 1 pp 196ndash204 2007

[11] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007

[12] A Shirazi G Ghobadi and M Ghazi-Khansari ldquoA radiobio-logical review on melatonin a novel radioprotectorrdquo Journal ofRadiation Research vol 48 no 4 pp 263ndash272 2007

[13] S-X Yan X-Y Hong Y Hu and K-H Liao ldquoTempol oneof nitroxides is a novel ultraviolet-A1 radiation protector forhuman dermal fibroblastsrdquo Journal of Dermatological Sciencevol 37 no 3 pp 137ndash143 2005

[14] M-A Barbacanne J-P Souchard B Darblade et al ldquoDetectionof superoxide anion released extracellularly by endothelial cellsusing cytochrome c reduction ESR fluorescence and lucigenin-enhanced chemiluminescence techniquesrdquo Free Radical Biologyand Medicine vol 29 no 5 pp 388ndash396 2000

[15] H Kasai ldquoAnalysis of a form of oxidative DNA damage 8-hydroxy-21015840-deoxyguanosine as a marker of cellular oxidativestress during carcinogenesisrdquo Mutation ResearchReviews inMutation Research vol 387 no 3 pp 147ndash163 1997

[16] A C Couto-Silva C Trivin H Esperou J Michon A Fischerand R Brauner ldquoChanges in height weight and plasma leptinafter bone marrow transplantationrdquo Bone Marrow Transplanta-tion vol 26 no 11 pp 1205ndash1210 2000

[17] B P Alter andP S Rosenberg ldquoGranulocyte colony-stimulatingfactor and severe aplastic anemiardquo Blood vol 109 no 10 article4589 2007

[18] S J Hosseinimehr ldquoTrends in the development of radioprotec-tive agentsrdquo Drug Discovery Today vol 12 no 19-20 pp 794ndash805 2007


[19] F Herodin and M Drouet ldquoCytokine-based treatment of acci-dentally irradiated victims and new approachesrdquo ExperimentalHematology vol 33 no 10 pp 1071ndash1080 2005

[20] D Bagchi M Bagchi S J Stohs et al ldquoFree radicals and grapeseed proanthocyanidin extract importance in human healthand disease preventionrdquo Toxicology vol 148 no 2-3 pp 187ndash197 2000

[21] P C Hollman and M B Katan ldquoBioavailability and healtheffects of dietary flavonols in manrdquo Archives of ToxicologySupplement vol 20 pp 237ndash248 1998

[22] J H M de Vries P C H Hollman S Meyboom et alldquoPlasma concentrations and urinary excretion of the antiox-idant flavonols quercetin and kaempferol as biomarkers fordietary intakerdquo American Journal of Clinical Nutrition vol 68no 1 pp 60ndash65 1998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


5mins before radiation and further cultured for 24 hrs afterradiation Cell viability was determined by Cell Counting Kit(Dojindo Laboratories Kumamoto Japan)

24 Apoptosis Assays for Cultured Cells The level of theapoptosis was determined by Apoptosis Detection Kit (BipecBiopharma Massachusetts MA) Treated cells were analysedby flow cytometry Alternatively apoptosis was determinedby Hoechst 33258 fluorescein diacetate (FDA) and PI stain-ing as described previously [5]

25 Comet Assay TheDNA strand breaks (DSBs) were mea-sured by using single-cell gel electrophoresis (comet assay)based on the method of Dubner et al [6]

26 Animals andGSPs Treatment All the protocols of animalexperiment were consistent with the Guide for Care andUse of Laboratory Animals published by the US NIH Six-week-old male BALBc mice (Chinese Academy of SciencesChina) were used in the experiments The animal housewas kept with unwavering temperature (24∘C) and the micewere fed ad libitum About 1 h before radiation grape seedproanthocyanidins were administered to mice by gavagewhich were provided by Pure-one Bio Technology Co Ltd(Shanghai China) GSPs were maintained through drinkingfor 1 week after radiation

27 Survival Assays Survival rates of the mice were recordeddaily for 30 days after radiation with 8Gy at a dose rate of1 Gymin

28 Body Weight All mice were weighed by an electronicscale (GH-202 AND Japan) at the same time of the day

29 White Blood Cell Caudal vein WBC was counted in cellcount plate at the same time of the day as described previously[7]

210 MDA Assay Arterial blood samples (06mL) of themice which were collected 12 hrs after radiation were takenfor MDA assay with the method defined by Ohkawa et al[8]

211 Statistical Analysis Statistics were expressed as meansplusmn SEM for each experiment and analysed by using One-WayAnalysis of Variance Between groups a two-tailed Studentrsquos119905-test was used A 119875 value less than 005 was considered to bestatistically significant

3 Results

31 GSPs-Rich PBS Increases Cell Viability of Radiated AHH-1Cells Figure 1(a) showed that GSPs inhibited cellular prolif-eration and diminished the viability of AHH-1 cells when theconcentration of GSPs was more than 80 120583gmL In additionwe demonstrated that pretreatment of AHH-1 cells with5ndash40 120583gmL GSPs-rich PBS before radiation significantly

increased cell survival rates as compared to cells treated withradiation alone at all examined doses (up to 10Gy) and theradioprotective effect of GSPs was dose-dependent (Figures1(b) and 1(c))

32 GSPs-Rich PBS Attenuated Apoptosis in Radiated AHH-1 Cells The early apoptotic cells decreased when AHH-1cells were pretreated with 40 120583gmL GSPs-rich PBS as com-pared to cells pretreated with PBS alone in flow cytometryassay (Figure 2(a) 151 versus 342 resp) These findingswere corroborated by the morphology of dying cells usingHoechst 33258 fluorescein diacetate and propidium iodidestaining Radiated AHH-1 cells pretreated with GSPs-richPBS had a reduced apoptosis of 284 as compared to 573in PBS pretreated radiated cells (Figure 2(b)) These datasuggest that GSPs attenuate apoptosis in radiated AHH-1cells

33 Comet Assay The radiated mice resulted in an increasein the levels of all comet parameters (comet tail lengthand tail DNA) whereas pretreatment of GSPs before radi-ation inhibited the increase of these parameters significantlyindicating the protective effect of GSPs on IR-induced DNAdamage (Figure 3)

34 Survival Rates of Mice 70 of radiated mice withoutGSPs treatment died by the 15th day after radiation while50 of the mice survived when treated with 200mgkgof the body weight GSPs and 80 survived when treatedwith 400mgkg of the body weight GSPs (Figure 4(a))Figure 4(b) showed the different effects of the three strategiesof GSPs (400mgkg of the body weight) delivery includingtherapy (after radiation survival 40) prevention (beforeradiation survival 50) and the combination of therapy andprevention medication (after and before radiation survival80) We found that the combined medication improved thesurvival rate of the mice most

35 Effect of GSPs on Body Weight of Mice after Radiation(5 Gy) Weight of mice fell sharply within 4 days after radi-ation and GSPs cannot slow down its decrease (Figure 5(a))However in the subsequent time period weight of micebegan to increase and GSPs significantly speeded up theweight back

36 Effect of GSPs on WBC Count after Radiation (5 Gy)GSPs slowed down the decrease ofWBC count after radiationand speeded up the recovery of WBC count from the 4th dayafter radiation while WBC count remained at lower levelsuntil the 11th day after radiation in the radiation alone group(Figure 5(b))WBC count was back up to the peak at the 24thday after radiation and then fell to a stable level HoweverWBC count remained at higher levels in the GSPs-treatedgroup than in the radiation alone group at the end of theexperiment

37 Changes in the Levels of Plasma MDA The plasma MDAconcentrations were measured at 12 hrs after 5Gy radiation


00

02

04

06

08

10

12C

ell s

urvi

val f

ract

ion


lowast

(a)

00

02

04

06

08

Cell

surv

ival

frac

tion

(8G

y)


lowast

lowast

lowast

(b)

00

02

04

06

08

10

12

Cel

l sur

viva

l fra

ctio

n





Ann

exin

V-p

ositi

ve ce

lls (

of t

otal

)

0

10

20

30

40



lowast

(a)

Apop

totic

cells

( o

f tot

al)

0

20

40

60

80


lowast




(a)

0

20

40

60

80

100

Com

et (

)


lowast


(b)

0

20

40

60

80

Tail

leng

th (120583

m)


lowast


(c)

0

10

20

30

Tail

DN

A (

)



lowast

(d)



4 Discussion






0 5 10 15 20 25 30

Time (d)

0

20

40

60

80

100Su

rviv

al (

)


ControlIR

(200mgkg)(400mgkg)

(a)

00

02

04

06

08

10

Frac

tion

surv

ival

5 10 15 20 25 300Time (d)


IR+GSPs

(b)


Wei

ght (

g)

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0Time (d)

12

13

14

15

16

17

18

19

20


lowastlowast

lowastlowast


(a)

0 642 8 10 282624222018161412 30Time (d)

lowastlowastlowast

lowast


lowast

lowast



lowast

lowast0

5

10

15

20

25

ControlIR

GSPs+IR+GSPs

WBC

coun

t (lowast10

9L

)

(b)







0

1

2

3

4

5

MD

A (n

mol

mg

prot

ein)



lowast




Competing Interests




Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

02

04

06

08

10

12C

ell s

urvi

val f

ract

ion


lowast

(a)

00

02

04

06

08

Cell

surv

ival

frac

tion

(8G

y)


lowast

lowast

lowast

(b)

00

02

04

06

08

10

12

Cel

l sur

viva

l fra

ctio

n





Ann

exin

V-p

ositi

ve ce

lls (

of t

otal

)

0

10

20

30

40



lowast

(a)

Apop

totic

cells

( o

f tot

al)

0

20

40

60

80


lowast




(a)

0

20

40

60

80

100

Com

et (

)


lowast


(b)

0

20

40

60

80

Tail

leng

th (120583

m)


lowast


(c)

0

10

20

30

Tail

DN

A (

)



lowast

(d)



4 Discussion






0 5 10 15 20 25 30

Time (d)

0

20

40

60

80

100Su

rviv

al (

)


ControlIR

(200mgkg)(400mgkg)

(a)

00

02

04

06

08

10

Frac

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5 10 15 20 25 300Time (d)


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2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0Time (d)

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13

14

15

16

17

18

19

20


lowastlowast

lowastlowast


(a)

0 642 8 10 282624222018161412 30Time (d)

lowastlowastlowast

lowast


lowast

lowast



lowast

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5

10

15

20

25

ControlIR

GSPs+IR+GSPs

WBC

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t (lowast10

9L

)

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1

2

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4

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A (n

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Competing Interests




Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

0

20

40

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4 Discussion






0 5 10 15 20 25 30

Time (d)

0

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)


ControlIR

(200mgkg)(400mgkg)

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00

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5 10 15 20 25 300Time (d)


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2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0Time (d)

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(a)

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GSPs+IR+GSPs

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Competing Interests




Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 5 10 15 20 25 30

Time (d)

0

20

40

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5 10 15 20 25 300Time (d)


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2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0Time (d)

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17

18

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(a)

0 642 8 10 282624222018161412 30Time (d)

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Competing Interests




Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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2

3

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MD

A (n

mol

mg

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Competing Interests




Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article radioprotective effect of grape seed ...week-old male balb/c mice (chinese academy...

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