hydrogen-rich water ameliorates total body irradiation...

Research ArticleHydrogen-Rich Water Ameliorates Total BodyIrradiation-Induced Hematopoietic Stem Cell Injury byReducing Hydroxyl Radical

Junling Zhang Xiaolei Xue Xiaodan Han Yuan Li Lu Lu Deguan Li and Saijun Fan

Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine Institute of Radiation MedicinePeking Union Medical College and Chinese Academy of Medical Science Tianjin 300192 China

Correspondence should be addressed to Saijun Fan fansaijunirm-camsaccn

Received 4 August 2016 Revised 8 November 2016 Accepted 26 December 2016 Published 22 January 2017

Academic Editor Andreas Daiber

Copyright copy 2017 Junling Zhang 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 examined whether consumption of hydrogen-rich water (HW) could ameliorate hematopoietic stem cell (HSC) injury in micewith total body irradiation (TBI) The results indicated that HW alleviated TBI-induced HSC injury with respect to cell numberalteration and to the self-renewal and differentiation of HSCs HW specifically decreased hydroxyl radical (∙OH) levels in the c-kit+cells of 4Gy irradiated mice Proliferative bone marrow cells (BMCs) increased and apoptotic c-kit+ cells decreased in irradiatedmice uptaken with HW In addition the mean fluorescence intensity (MFI) of 120574-H2AX and percentage of 8-oxoguanine positivecells significantly decreased in HW-treated c-kit+ cells indicating that HW can alleviate TBI-induced DNA damage and oxidativeDNA damage in c-kit+ cells Finally the cell cycle (P21) cell apoptosis (BCL-XL and BAK) and oxidative stress (NRF2 HO-1NQO1 SOD and GPX1) proteins were significantly altered by HW in irradiated mouse c-kit+ cells Collectively the present resultssuggest that HW protects against TBI-induced HSC injury

1 Introduction

Total body irradiation (TBI) may induce injury in manytissues and organs Direct action refers to the effects of TBI onbioactive macromolecules such as proteins and nucleic acidsIonization excitation chemical bond rupture and changes inmolecular structure all occur during this process leading toabnormal function and metabolic disorders Indirect actionoccurs via the generation of free radicals by water radiolysis[1] The hydroxyl radical (∙OH) the free radical componentthat is one of the strongest oxidant species and that reactswithnucleic acids lipids and proteins accounts for approximately75 of lethal DNA damage caused by ionizing radiation [2]Therefore high concentrations of ∙OH radical scavengersmay be promising radioprotective agents

It is well known that molecular hydrogen (H2) acts as

an antioxidant by efficiently reducing ∙OH Studies haveshown that H

2plays an important role in animal disease

models such as focal ischemia and reperfusion in rats [3]depressive-like behavior and obstructive jaundice in mice[4] In addition it has been reported that H

2protects

mice from TBI-induced injury in many tissues and organsincluding bone marrow cells [5ndash7] heart [8] testis [6 7]intestine [5] lung [9] and skin [10ndash12] In the present studywe explore the radioprotective effect of HW on the injury ofhematopoietic stem cells (HSCs)

HSCs have the ability to replenish themselves and todifferentiate into blood cells of all hematopoietic lineages Asone of the best-characterized somatic stem cell types amongall tissue stem cells HSCs reside in a specialized redox nichewith low levels of oxygen and limit production of reactiveoxygen species (ROS) [13] C-kit also named CD117 is acytokine receptor expressed on the surface of hematopoieticstem cells C-kit positive (c-kit+) cells are the subset oftotal HSCs and hematopoietic progenitor cells (HPCs) Nomature hematopoietic cells markers such as Ter119 B220

HindawiOxidative Medicine and Cellular LongevityVolume 2017 Article ID 8241678 16 pageshttpsdoiorg10115520178241678

2 Oxidative Medicine and Cellular Longevity

CD11b Gr1 CD4 CD8 and IL3(lineage) are found on thesurface of murine HSCs and HPCs except c-kit and sca1HSCs are identified as lineageminussca1+c-kit+(LSK) and HPCsare identified as lineageminussca1minusc-kit+ respectivelyThus c-kit+cells may represent the total HSCs and HPCs

The hematopoietic system is highly radiosensitive andTBI may induce acute hematopoietic syndrome residualbone marrow injury and hematopoietic stem cell (HSC)senescence [14 15] The potential mechanisms involved inHSCsrsquo injury caused by TBI include the induction of HSCapoptosis differentiation and senescence [1] Thus it isnecessary to identify and characterize radioprotective agentsthat guard against TBI-induced hematopoietic system injury

Our present results show that HW may elevate the sur-vival rate of mice lethally irradiated Moreover the numberof bone marrow cells (BMCs) and the rate of self-renewaland differentiation are all improved in irradiated mice underthe condition of HW consumption HW significantly affectsthe expression of cell cycle- cell apoptosis- and oxidativestress-related proteins in the c-kit+ cells from irradiatedmiceThese results suggest that HW consumption is able to protectagainst TBI-induced HSC injury

2 Materials and Methods

21 Mice Male C57BL6 mice were purchased from theInstitute of Laboratory Animal Sciences Chinese Academyof Medical Sciences amp Peking Union Medical College Micewere bred in the experimental animal center of the Instituteof Radiation Medicine Mice were used at approximately 6ndash8 weeks of age All of the animal experiments in our studywere approved by the Animal Care and Ethics Committee atthe Institute of RadiationMedicineThe study was performedin accordance with the principle of Institutional Animal Careand Ethics Committee guidelines

22 Antibodies The following antibodies were purchasedfrom eBioscience (San Diego CA USA) anti-mouse CD3-APC (clone 145-2C11) anti-mouse CD117 (c-kit) APC(clone 2B8) anti-mouse Sca-1-PE (clone D7) anti-mouseCD34-FITC (clone RAM34) and anti-mouse Ki67-FITC(clone SolA15) The following antibodies were purchasedfrom Biolegend (San Diego CA USA) Percp-conjugatedstreptavidin anti-mouse B220-PE (clone RA3-6B2) anti-mouse CD11b-Percp (clone M170) anti-mouse Gr1-Percp(clone RB6-8C5) anti-mouse CD451-FITC (clone A20)anti-mouse CD452-PE (clone 104) biotin-conjugated anti-mouse Ter119 (clone TER119) anti-mouse B220 (clone RA3-6B2) anti-mouse Gr1 (clone RB6-8C5) anti-mouse CD11b(clone M170) anti-mouse CD4 (clone GK15) and anti-mouse CD8 (clone 53ndash67) The following antibodies werepurchased from BD Bioscience (San Jose CA USA) anti-mouse annexin V-FITC and anti-mouse 120574-H2AX-FITC Thefollowing antibodies were purchased from Cell SignalingTechnology (Danvers MA USA) anti-mouse NRF2 anti-mouse BCL-XL and anti-mouse BAK Anti-mouse p21 andanti-mouse 120573-actin were purchased from Abcam (Cam-bridge England) Anti-mouse 8-oxoguanine was purchasedfromMillipore (Boston MA USA)

23 Preparation ofHW Thepreparation ofHWwas based ona previous study with slight modification H2 gas was gener-ated from a hydrogen gas generator (SHC-300 Saikesaisi HWEnergy Shandong China) and bubbled into 500mL of sterilewater at a rate of 150mLmin for 20minThe concentration ofH2in the water was detected with a dissolved hydrogenmeter

(Trustlex ENH-1000 Japan) HW was freshly prepared eachday to ensure that an H

2concentration of more than 08 ppm

was maintained

24 TBI and HW Administration Mice were divided intofour groups for a 30-day survival experiment the groupsincluded 68Gy TBI + vehicle (normal water) 68 Gy TBI+ HW 72Gy TBI + vehicle and 72Gy TBI + HW For theother experiments mice were also divided into 4 groupsnamely vehicle HW alone 4Gy TBI and TBI + HW Themice with TBI were irradiated with 120574-rays at a dosage rateof 099Gymin Mice received 05mL HW by gavage for10min before TBI and up to the 7 days following TBI Thecontrol mice with vehicle received normal water for the samefrequency and volume as those in the mice with HW Micewere finally euthanized on the 15th day after TBI

25 Peripheral Blood Cell and Bone Marrow Cell CountsBlood was obtained from mice via the orbital sinus andcollected inmicropipettes coatedwith EDTAK

3 white blood

cells (WBCs) the percentages of lymphocytes (LY) andneutrophil granulocytes (NE) were counted and calculatedBone marrow cells were isolated from tibias and femurs aspreviously reported [16] and cell numbers were analyzedwith a hematology analyzer (Nihon Kohden Japan)

26 Flow Cytometry Analysis Bone marrow cells were sus-pended in phosphate-buffered saline (PBS) and cells werefiltered and counted prior to antibody staining To analyzethe B cells T cells and myeloid cells in peripheral blood50 120583L of peripheral blood was harvested and stained withB220 CD3 CD11b and Gr1 antibodies at room temperatureThe red blood cells in the blood samples were removedusing the BD FACS lysing solution For HSC analysis 5 times106 bone marrow cells were first stained with biotin-labeledTer119 B220 Gr1 CD11b CD4 and CD8 antibodies andsubsequently stained with streptavidin c-kit sca1 and CD34antibodies To assess ROS levels 1 times 106 bone marrow cellswere stainedwith anti-c-kit antibody and then incubatedwith27-dichlorodihydrofluorescein diacetate (DCFDA BeyotimeBiotechnology Nanjing China 10120583M) MitoSox (Life Tech-nologies Grand Island NY USA 10 120583M) and dihydroethid-ium (DHE Beyotime Biotechnology Nanjing China 5120583M)in a 37∘Cwater bath For cell cycle analysis bonemarrow cellswere first stained with a c-kit antibody and fixed and per-meabilized with CytofixCytoperm buffer (BD BiosciencesUSA) before being stained with anti-Ki67 antibody and PIFor analysis of 120574-H2AX NRF2 HO-1 NQO1 BCL-XL andBAK bone marrow cells were first processed as describedfor ki67 staining and then staining 120574-H2AX NRF2 HO-1NQO1 BCL-XL and BAK antibodies at room temperatureFor apoptosis analysis bone marrow cells were also stained

Oxidative Medicine and Cellular Longevity 3

with c-kit and annexin V and PI according to the instructionsfor a BD apoptosis kit Data acquisition was conducted on aBDAccuri C6 and analyzed usingBDAccuri C6 software (BDBioscience San Jose CA USA)

27 Colony-Forming Units of Granulocyte Macrophage Cells(CFU-GM) Assay A total of 1 times 104 bone marrow cells in thenonirradiated control mice or 1 times 105 bone marrow cells inthe 4-Gy TBI mice were cultured in M3534 methylcellulosemedium (Stem Cell Technologies Vancouver Canada) for 5days CFU-GMcolonieswithmore than 30 cells were countedaccording to the kit instructions The results were expressedas the number of CFU-GM per 105 bone marrow cells

28 Competitive Transplantation Assay For competitivebone marrow cell transplantation 1 times 106 bone marrow cellsfrom C57BL6(CD452) mice and 1 times 106 competitive cellsfrom C57BL6(CD451452) mice were transplanted intolethally irradiated C57BL6 mice (CD451) The percentageof donor-derived (CD452 positive) cells in the recipientsrsquoperipheral blood was examined 4 months after transplanta-tion

29 Isolation of c-kit+ Cells Bone marrow cells were stainedwith c-kit-APC antibody for 30min on ice washed withPBS and resuspended the pellet with anti-APC microbeads(Miltenyi Biotec Germany) for 15min c-kit+ cells weresorted with a LS column in the magnetic separation filed Foranalysis of ROS level and DNA damage 1 times 106 c-kit positivecells were cultured in 1mL hydrogen-rich Stem Span serum-free expansion medium (SFEM) 10min before irradiation at4Gy The cells were harvested for 120574-H2AX and ROS analysis05 hours and 18 hours after irradiation respectively Foranalysis of ROS level SFEM medium (both hydrogen-richmedium and normal medium) renewed half of the liquidvolume every 3 hours

210 Protein Analyses C-kit+ cells were lysed in ice-coldlysis buffer (BOSTER Wuhan China) containing 1mMphenylmethanesulfonyl fluoride (PMSF) Western blot anal-ysis was performed with anti-p21 (1 1000) and anti-GAPDH(1 5000)

211 Immunofluorescence Staining C-kit positive cells weresorted as described above and fixed in 4 paraformaldehydefor 10mins at room temperature Cells were permeabilizedin 02 Triton-X-100PBS and blocked with 5 BSA for 1 hat room temperature and then incubated with anti-120574-H2AX(1 400) and 8-oxoguanine antibodies (1 300) overnight at4∘C and incubated with FITC-conjugated goat anti-rabbitIgG (1 300) and goat anti-mouse IgG (1 300) antibody for1 h at room temperature

212 Analysis of Hydroxyl Radicals (∙OH) and of SOD andGPX Enzyme Activities Hydroxyl radicals and SOD andGPX enzyme activity were detected using detection kits(Nanjing JianchengBioengineering Institute Nanjing China

and Beyotime Biotechnology Nanjing China) according tothe manufacturerrsquos instructions A Fenton reaction occurredin the process of ∙OH detection following the manufacturerrsquosinstruction and absorbance value was measured after chro-mogenic reaction happened Level of ∙OH was evaluatedand calculated by the formula following the manufacturerrsquosinstruction

Ability of ∙OH production

=OD value (T minus C)OD value (S minus B)

times 8824 times1

sample volume

times dilution ratio

(1)

B is the blank tube S is the standard tube C is the controltube and T is the testing tube

213 Statistical Analysis Data are shown as means plusmn SEMand an unpaired 119905-test (two-tails) was used for the majorityof comparisons along with Welchrsquos correction 119905-test whenthe variances were not equal Comparisons of overall survivalwere performed using log-rank test Statistical analyses wereperformed using GraphPad Prism 5 software A 119875 lt 005represented statistical significance

3 Results

31 HW Improves the Survival of Lethally Irradiated MiceTo test whether HW affected the survival of mice after TBIwe fed mice with 05mL of HW 10min before 68Gy or72Gy TBI and then kept HW consumption daily for 7 daysafter irradiation As shown in Figure 1 all mice irradiated at68Gy or 72Gy died within 27 days or 15 days following TBIHowever approximately 67 of mice exposed to 68Gy and40 of mice exposed to 72Gy were alive 30 days after TBIunder HW consumption These findings suggest that HWsignificantly increases the survival of irradiated mice at least68 Gy and 72Gy

32 HW Alleviates Myelosuppression and Promotes MyeloidSkewing Recovery in Irradiated Mice It has been well estab-lished that TBI can induce myelosuppression a conditionin which bone marrow activity decreased resulting in asignificant decline of peripheral blood cells [17 18]Wang andcolleagues showed that lymphoid-biased HSCs were moresensitive to radiation-induced differentiation than myeloid-biased HSCs resulting in myeloid skewing in irradiatedmice [19] Thus to determine if HW consumption affectedradiation-causedmyelosuppression we analyzed the numberalteration of peripheral blood cells and the percentages of Bcells T cells and myeloid cells As illustrated in Figure 2 theirradiated mice exposed to 4Gy TBI exhibited a significantdecrease of WBCs and lymphocyte percentage (LY) inperipheral blood 15 days following irradiation comparedto the unirradiated controls Moreover the percentages ofB cells and T cells as detected by flow cytometry werealso declined Conversely there was an increase in bothneutrophilic granulocyte percentage (NE) and myeloid


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Figure 1 HW elevates the 30-day survival rate of mice receiving 68Gy and 72Gy TBI Mice received 05mL of vehicle water or HWadministrated intragastrically 10min before TBI and for 7 days after TBI Curve chart shows the 30-day survival rate after exposure to alethal dose of TBI119873 = 15 in 68Gy and 68Gy + HW119873 = 18 in 72Gy and 72Gy + HW

cell number in irradiated mice compared to unirradiatedmice (Figures 2(c) and 2(f)) These findings indicated thatTBI could result in myelosuppression and myeloid skewingIrradiated mice with HW uptaken showed an increase ofWBC counts LY and B cell percentages and a decreaseof NE and myeloid cell percentage in the peripheral blood(Figures 2(c) and 2(f)) No alteration of T cell numberswas found in mice with TBI + HW These results suggestthat HW consumption improves mice recovery from TBI-induced myelosuppression and myeloid skewing

33 HW Increases Number of Bone Marrow Cells (BMCs) ofIrradiated Mice To determine whether HW consumptionaffected BMCs we analyzed number alteration of BMCsper femur and the percentages of c-kit+ cells (Lineageminusc-kit+BMCs) HPCs (Lineageminussca1minusc-kit+BMCs) LSKs(Lineageminussca1+c-kit+BMCs) CD34minusLSK and CD34+LSKcells As shown in Figure 3 4Gy TBI caused a decreasednumber of BMCs a decrease of c-kit+ cells HPCs andLSKs CD34+LSK frequency and an increase of CD34minusLSKpercentage in mice at day 15 after irradiation compared tounirradiated mice However HW consumption reducedor inhibited these effects caused by TBI that is BMCs(Figure 3(a)) c-kit+ cells (Figure 3(b)) HPCs (Figure 3(c))LSKs (Figure 3(d)) CD34minusLSK frequency (Figure 3(e)) andCD34+LSK frequency (Figure 3(f)) in mice with TBI Theseresults suggested that HW could also improve mice recoveryfrom TBI-induced alterations in BMCs

34 HW Increases BMC Self-Renewal Ability in IrradiatedMice TBI causes an injury in self-renewal ability of HSCs[20 21] 4 Gy TBI significantly decreased CFU-GM numbercompared to the unirradiatedmice HWrescued such decline(Figure 4(a)) The competitive bone marrow transplantationassay is a gold standard for evaluating the self-renewaland regeneration capability of HSC [16] Thus we also

transplanted lethally irradiated (CD451+) recipientmicewithbone marrow cells from unirradiated donor mice or fromdonor mice with 4Gy or 4Gy + HW The donor-derivedcells (CD452+) in the peripheral blood of recipient micewere measured by FACS 16 weeks after transplantation Thechimerism of donor cells in irradiated mice (about 4) wassignificantly lower than that in unirradiated mice (about25) while the donor cells in mice with 4Gy + HW showedincreased chimerism (about 15) (Figures 4(b) and 4(c))These results suggested thatHWconsumption could improveBMC self-renewal ability of irradiated mice

35 HW Decreases ∙OH Levels in the c-kit+ Cells of IrradiatedMice To determine whether HW consumption affected ROSlevels in irradiated c-kit+ cells we treated c-kit+ cells with10 120583M DCFDA 10 120583M MitoSox and 5 120583M DHE to detecttotal cellular ROS mitochondria-derived ROS (superoxide)and superoxide free radicals respectively The ∙OH level wasmeasured with a detection kit and evaluated by the abilityto produce ∙OH as described in the Materials and MethodsAll of three ROS types increased in c-kit+ cells of micewith 4Gy TBI in comparison to unirradiated mice whileHW consumption decreased the total ROS (Figures 5(a) and5(e)) and ∙OH levels (Figure 5(b)) and showed no effectson the mitochondria-derived ROS levels (Figures 5(c) and5(f)) or the superoxide free radical levels (Figures 5(d) and5(g)) in irradiated mice These results indicated that HWconsumption decreases ∙OH levels in c-kit+ cells of irradiatedmouse

36 HW Affects Cell Cycle and Cell Apoptosis in c-kit+ Cellsof Irradiated Mice As shown in Figure 6 4Gy TBI caused asignificant arrest at G1-phase cells (about 28) and SG2Mphase (about 27) of c-kit+ cells (about 38) after 15 daysin mice receiving a TBI at 4Gy Apoptosis (early and lateapoptosis) was also observed to be increased in irradiated


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Figure 2 HW alleviates TBI-induced differentiation dysfunction in the hematopoietic system (a)The bar graph shows the number ofWBCsin peripheral blood (b)The bar graph shows the percentage of lymphocytes (LY) in peripheral blood (c)The bar graph shows the percentageof neutrophilic granulocytes (NE) in peripheral blood (d) The bar graph shows the percentage of B cells in peripheral blood as detected byFACS (e) The bar graph shows the percentage of T cells in peripheral blood as detected by FACS (f) The bar graph shows the percentage ofmyeloid cells in peripheral blood as detected by FACS (g) Representative FACS analysis showing the percentage of B cells and T cells (h)Representative FACS analysis showing the percentage of myeloid cells All the data represent the mean plusmn SEM (119899 = 5) 119875 lt 005 versus 0Gycontrol lowast119875 lt 005 versus 4Gy control


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Figure 3 HW attenuates the TBI-induced decrease in BMCs hematopoietic stem cells and progenitor cells (a) The bar graph showsthe number of BMCs per femur (b) The bar graph shows the percentage of c-kit+ cells among lineage-negative cells (c) The bar graphshows the percentage of HPCs (Lineageminussca1minusc-kit+BMCs) among lineage-negative cells (d) The bar graph shows the percentage of LSKs(Lineageminussca1+c-kit+BMCs) among lineage-negative cells (e) The bar graph shows the percentage of CD34minusLSKs among LSK cells (f) Thebar graph shows the percentage of CD34+LSKs among LSK cells The data are presented as the mean plusmn SEM of cell numbers per femur(119899 = 6) (g) Representative FACS analysis of the percentage of LSKs and HPCs (h) Representative analysis of the percentage of CD34minusLSKsand CD34+LSKs All the data represent the mean plusmn SEM (119899 = 5) 119875 lt 005 versus 0Gy control lowast119875 lt 005 versus 4Gy control


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Figure 4 HW attenuates the TBI-induced decrease in self-renewal in HPCs and HSCs (a) The bar graph shows the number of CFU-GMper 105 BMCs (b) The scatter plot shows the percentage of donor-derived cells in peripheral blood cells 4 months after transplantation (c)Representative FACS analysis of the donor cell engraftment All the data represent the mean plusmn SEM (119899 = 6 in panels (a) 119899 = 8 in panel (b))119875 lt 005 versus 0Gy control lowast119875 lt 005 versus 4Gy control

c-kit+ cells after irradiation As expected HW rescued TBI-induced cell cycle arrest and decreased the percentage ofearly apoptosis These results showed that HW consumptionindeed affected TBI-mediated cell cycle progression and earlyapoptosis of c-kit+ cells in irradiated mice

37 HWDecreases DNADamage andOxidative DNADamagein c-kit+ Cells of Irradiated Mice As reported previously TBIcan induce persistent oxidative stress in HSCs which maycause sustained DNA damage and oxidative DNA damage[22] 8-oxoguanine (8-oxoG) is a naturally abundant base andimpactful oxidative DNA lesions with a well-characterizedmutagenic potential [23] it is easily generated in DNA byreactive oxygen species induced by TBI [24] Our results

showed that MFI of 120574-H2AX and percentage of 8-oxoGpositive cells increased significantly in irradiated c-kit+ cellscompared to control group and HW consumption down-regulated expression of 120574-H2AX and 8-oxoG (Figure 7)These results showed that HW consumption decreased DNAdamage and oxidative DNA damage in c-kit+ cells 15 daysafter 4Gy TBI

38 HW Upregulates Expression Antioxidation Proteins Nu-clear factor erythroid 2-related factor 2 (NRF2) is a cellularsensor of oxidative stress [25] In response to oxidative stressNRF2 dissociates from kelch like ECH-associated protein 1(KEAP1) and translocates into the nucleus which in turnsregulates the transcription of heme oxygenase-1(HO-1) and


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Figure 5 HW decreases TBI-induced ROS production in c-kit+ cells (a) The bar graph shows the levels of intracellular ROS as detected byDCF mean fluorescence intensity (MFI) (b) The bar graph shows hydroxyl radical production as detected by the Fenton reaction (c) Thebar graph shows the levels of intracellular ROS as detected by DHE MFI (d) The bar graph shows the intracellular ROS in mitochondriaas detected by the MitoSox MFI (endashg) Representative FACS plots of the ROS levels detected by DCF DHE and MitoSox MFI All the datarepresent the mean plusmn SEM (119899 = 5 in panels (a) (c) and (d) 119899 = 3 in panel (b)) 119875 lt 005 versus 0Gy control lowast119875 lt 005 versus 4Gy control


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101 101

101101

c-kit+ cells

(g)

Figure 6 HW increases proliferation and decreases apoptosis in irradiated c-kit+ cells (a) The bar graph shows the percentage of G0 phase(ki67minusPIminus) cells among BMCs (b)The bar graph shows the percentage of G1 phase (ki67+PIminus) cells among BMCs (c)The bar graph shows thepercentage of SG2M phase (ki67+PI+) cells among BMCs (d)The bar graph shows the percentage of early apoptotic cells (annexin V+PIminus)(e)The bar graph shows the percentage of late apoptotic cells (annexin V+PI+) (f) Representative FACS analysis of cell cycle status in BMCs(g) Representative FACS plots of apoptosis in c-kit+ cells All data represent themean plusmn SEM (119899 = 4) 119875 lt 005 versus 0Gy control lowast119875 lt 005versus 4Gy control


120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)

HWControl 4Gy 4Gy + HW

(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)

Figure 7 HW decreases DNA damage and oxidative DNA damage in irradiated c-kit+ cells (a) The bar graph shows MFI of 120574-H2AX inc-kit+ cells as detected by flow cytometry (b) Representative FACS plots of 120574-H2AX (c) Representative images of 120574-H2AX in c-kit+ cellsas detected by immunofluorescence (IF) (d) The bar graph shows the percentage of 8-oxoG positive cells in c-kit+ cells (e) Representativeimages of 8-oxoG in c-kit+ cells as detected by IF All the data represent the mean plusmn SEM (119899 = 5 in panel (a) and 119899 = 3 in panel (d)) 119875 lt 005versus 0Gy control lowast119875 lt 005 versus 4Gy control


0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)

Figure 8 HW upregulates expression of NRF2 targeted proteins and enzyme activity of SOD2 and GPX1 in mice c-kit+ cells 15 days after4Gy TBI (a) Bar graphs show the protein level of NRF2 in c-kit+ cells as detected by FACS (b) Representative FACS plots of NRF2 (c-d)Bar graphs show the protein level HO-1 and NQO1 in c-kit+ cells as detected by FACS (e-f) Bar graphs show the total enzyme activities ofSOD and GPX in c-kit+ cells All the data represent the mean plusmn SEM (119899 = 5 in panels (a) (c) and (d) and 119899 = 3 in panels (e)-(f)) 119875 lt 005versus 0Gy control lowast119875 lt 005 versus 4Gy control


0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)

Figure 9 Protein levels of P21 BCL-XL and BAK in mice c-kit+ cells 15 days after 4Gy TBI (a) Bar graph shows the relative expressionof P21 protein level (b) Representative western blotting analysis of the P21 in c-kit+ cells (c) Bar graph shows the protein levels of BCL-XLas detected by FACS (d) Representative FACS plots of BCL-XL protein levels (e) Bar graph shows the protein levels of BAK as detected byFACS (f) Representative FACS plots of BAK protein levels Values represent the mean plusmn SEM (119899 = 3 in panel (a) and 119899 = 5 in panels (c) and(e)) 119875 lt 005 versus 0Gy control lowast119875 lt 005 versus 4Gy control


ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)

Control HW 4Gy 4Gy + HW

(c)

Figure 10 HW decreases ROS level and DNA damage in c-kit+ cells exposed to 4Gy IR in vitro (a) Bar graph shows the level of intracellularROS 18 hours after 4Gy IR in vitro (b) Bar graph shows the number of 120574-H2AX foci per cell 05 hours after 4Gy IR in vitro (c) Representativeimages of 120574-H2AX in c-kit+ cells as detected by IF All the data represent the mean plusmn SEM (119899 = 3) 119875 lt 005 versus 0Gy control lowast119875 lt 005versus 4Gy control

NAD (P)H quinine oxidoreductase 1 (NQO1) [26 27]More-over NRF2 is found to promote the survival of irradiatedcells including BM cells as a result of ROS scavenging[28] To explore whetherHWconsumption affects expressionantioxidation proteins we detected expression of NRF2 HO-1 and NQO1 by FACS and SOD GPX1 enzyme activity Asshown in Figure 8 HW consumption upregulated expressionof NRF2 targeted proteins and enzyme activity of SOD andGPX1 These results illustrated that HW decreases ROS levelthrough upregulating expression antioxidation proteins 15days after 4Gy TBI

39 HWAffects the Expression of Proteins Related to Prolifera-tion and Apoptosis As shown in Figure 9 a downregulationof P21 (also known as cdkn1a or Cip1) a cell cycle inhibitor isalso involved in cell apoptosis and transcriptional regulationafterDNAdamage [29ndash31] (Figures 9(a) and 9(b)) andBAK aproapoptotic protein were observed at protein levels in HW-treated irradiated mice c-kit+ cells (Figures 9(e) and 9(f)) Incontrast the protein levels of the antiapoptotic protein BCL-XLwere upregulated in c-kit+ cells ofmice bearing TBI +HW(Figures 9(c) and 9(d)) These results revealed the alterationof these proteins expressions may be potential mechanismsinvolved in HW radioprotection in c-kit cells

310 HW Decreases ROS Level and DNA Damage in c-kit+Cells with 4Gy IR In Vitro To explore the direct radioprotec-tive effect of HW c-kit+ cells were cultured with hydrogen-rich SFEM medium 10min before 4Gy IR and then wedetected ROS level and DNA damage 18 hours and 05 hoursafter IR respectively As shown in Figure 10 when culturedwith hydrogen-rich medium DCF MFI and 120574-H2AX fociin irradiated c-kit positive cells decreased significantly com-pared to cells cultured with normal medium These resultssuggested that HW alleviated ROS production and DNAdamage in vitro

4 Discussion

It has been reported that H2protects mice from TBI-

induced bone marrow injury however existing research inthe literature has focused on cultured hematopoietic cellsor WBCs in peripheral blood colony-forming units in thespleen or the number of bone marrow cells in mice Theeffect of H

2on TBI-induced HSC injury and its underlying

mechanisms are poorly understoodGuo et al [32] and Chuai et al [6] reported that mice

withHWconsumption 5min before TBI alleviated radiation-induced hematopoietic system injury Liu and colleagues


reported that the hydrogen concentrations in the blood andtissues reached a maximum at 5 minutes after the oraladministration of HW and then decreased slowly within30mins [33] It has been widely accepted that with oral intakedrugs or any other agents drugs were absorbed into bloodthrough gastrointestinal venous and then transported to allthe tissues and organs in the body In the publication of Liuet al with oral administration of HW with concentrationof 125 ppm H2 blood levels did not increase up to anadministered dose of 25 ppm however H2 levels in liverkidney heart spleen pancreas intestine muscle and brainincreased significantly when rats were treated with 125 ppmSo it is probably that H2 increase in bone marrow cells aftermice were treated with 125 ppm HW (concentration of H2in our experiment is about 1 ppm) Based on those previousstudies we administrated HW to mice 10min (exactly 5ndash10min each mice per group) before TBI and 7 days afterTBI To confirm whether H2 reached bone marrow 10minsafter oral administration of HW we measured ROS levelin bone marrow cells after 4Gy TBI (data not shown) Inbrief mice were divided into 4 groups control 4 Gy 4Gy+ amifostine and 4Gy + HW 4 mice per group and twoindependent experimentswere executed Amifostine is a ROSscavenger and radioprotective drug that has been approvedby the US Food and Drug Administration (FDA) and micereceived 200mgkg amifostine 30mins before TBI in 4Gy +amifostine group and 05mL HW 10mins before TBI in 4Gy+ HW group Mice were sacrificed immediately after 4GyTBI and bonemarrow cells were harvested thenwemeasuredROS level with flow cytometryWhen treating irradiatedmicewith amifostine or HW ROS decreased significantly whichshow indirect evidence thatH2 reach to bonemarrow 10minsafter HW consumption

Our results show that the number of WBCs in peripheralblood and the number of BMCs per femur 15 days afterirradiation are increased when irradiated mice are suppliedwith HW These results are consistent with other studies [6]Our present findings demonstrate that HW consumption cancounteract imbalances in myeloid-lymphoid differentiationincrease the percentages of c-kit+ cells LSKs HPCs andCD34+LSKs and decrease the CD34minusLSKs frequency inTBI mice Notably self-renewal and reconstitution weresignificantly improved by HW

To explore the underlying mechanisms we measuredthe ROS levels cell proliferation cell apoptosis DNA dam-age oxidative DNA damage and the expression of relatedproteins HW consumption decreases total ROS level andselectively reduces the ∙OH level in irradiated c-kit+ cellsIn contrast no significant alteration ofmitochondria-derivedROS (superoxide) and superoxide free radicals were observedbetween c-kit+ cells with TBI and c-kit+ cells with TBI + HWThese data confirm that H

2selectively reduces ∙OH level

this result is consistent with the results obtained by otherresearchers [3 34] In addition HW results in an increasedpercentage of proliferative cells a decreased proportion ofearly apoptotic cells and the frequency of DNA damageand oxidative DNA damage in irradiated c-kit+ cells byregulating related proteins Consistent with the experimentsin vivo when culturing c-kit+ cells with hydrogen-rich SFEM

medium in vitro ROS level and 120574-H2AX foci decreasedsignificantly compared to these cells cultured with normalSFEM medium The variation in cell proliferation cell apop-tosis and DNA damage may contribute to the increased cellnumbers and frequencies observed in HW-treated BMCs

It has been reported that ROS in bone marrow cellsincreased significantly at 7 9 15 22 30 and 56 days aftermice received TBI [22 35ndash39] However it is a very shortperiod that ROS persist after TBI how should these radicalsbe generated 15 days after TBI In our experiments there arealmost no LSKs and HPCs 4 h after TBI then survival HSCsreplenish themselves and differentiate into blood cells of allhematopoietic lineages LSKs andHPCs appear about 10 daysafter TBI with high level ROS production and the reasonwhyROS is produced in newly generated hematopoietic stem cellsdeserved to be further explored According to the publishedresearch NRF2 and targeted genes and proteins play animportant role in ROS production in irradiated hematopoi-etic stem cell [28] Our unpublished works focus on the effectof the systemic (circulatory) environment on TBI-inducedbonemarrow cells injury indicating that components in irra-diatedmice circulatory environmentmay regulate expressionof NRF2 and targeted genes and proteins So next we detectedexpression of antioxidative proteins in c-kit+ cells

H2has been reported to elevate the production of SOD

and GSH in the thymus [40] and testis [41] Consistent withthese studies we demonstrate that H

2enhances the activity

of SOD and GPX enzyme A number of in vitro and invivo experiments have found that H

2as a potent antioxidant

activates NRF2 and its downstream signaling molecules [42ndash49] Our results show that H

2elevates the expression of NRF2

and targeted protein (HO-1 and NQO1) in c-kit+ cells of TBImice However the mechanism by which H

2activates NRF2

and its related signaling pathways is unknown and warrantsfurther exploration In addition our results show that H

2

upregulates the expression of the antiapoptotic protein BCL-XL consistently with prior studies [9] H

2downregulates the

expression of the cell cycle inhibitor P21 and the proapoptoticprotein BAK in irradiated c-kit+ cells These findings suggestthat H

2may regulate many other signaling pathways to

protect BMCs from TBI-induced injuryTaken together our present study preliminarily reveals

the protective effect of H2by HW consumption on TBI-

induced HSC injury with alteration of the related proteinsFuture studies are carried on to further study the moleculemechanism by which H

2mediated radioprotection of HSCs

H2 as a low-toxicity agent shows promising radioprotective

effects on TBI-induced injury in many organs and tissuesFuture studies are needed to determine how to counteractissues related to solubility and stability

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Junling Zhang and Saijun Fan initiated the research anddeveloped the concept of the paper Junling Zhang and


Saijun Fan designed the research Junling Zhang Xiaolei XueXiaodan Han and Yuan Li performed the experiments withthe help from Lu Lu Deguan Li Junling Zhang and XiaoleiXue analyzed and interpreted the data and Junling Zhangwrote the manuscript with contributions from Saijun FanJunling Zhang and Xiaolei Xue contributed equally to thiswork

Acknowledgments

This study was supported by the grants from the NationalNatural Science Foundation of China (81172127 81572969)the Technology and Development and Research Projectsfor Research Institutes Ministry of Science and Technology(2014EG150134) and the Tianjin Science amp Technology PillarProgram (14ZCZDSY00001) to Saijun Fan National NaturalScience Foundation of China (81402633) Natural ScienceFoundation of Tianjin (16JCQNJC13600) and the IRM-CAMS Research Fund (1524 1544) to Junling Zhang

References

[1] L Shao Y Luo and D Zhou ldquoHematopoietic stem cell injuryinduced by ionizing radiationrdquoAntioxidants amp Redox Signalingvol 20 no 9 pp 1447ndash1462 2014

[2] P Maier FWenz and C Herskind ldquoRadioprotection of normaltissue cellsrdquo Strahlentherapie und Onkologie vol 190 no 8 pp745ndash752 2014

[3] I Ohsawa M Ishikawa K Takahashi et al ldquoHydrogen actsas a therapeutic antioxidant by selectively reducing cytotoxicoxygen radicalsrdquo Nature Medicine vol 13 no 6 pp 688ndash6942007

[4] Y Zhang W Su Y Chen et al ldquoEffects of hydrogen-rich wateron depressive-like behavior in micerdquo Scientific Reports vol 6article 23742 2016

[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] Y Chuai J Shen L Qian et al ldquoHydrogen-rich saline pro-tects spermatogenesis and hematopoiesis in irradiated BALBcmicerdquo Medical Science Monitor vol 18 no 3 pp BR89ndashBR942012

[7] Z Jiang B Xu M Yang Z Li Y Zhang and D JiangldquoProtection by hydrogen against gamma ray-induced testiculardamage in ratsrdquo Basic amp Clinical Pharmacology amp Toxicologyvol 112 no 3 pp 186ndash191 2013

[8] L Qian F Cao J Cui et al ldquoThe potential cardioprotectiveeffects of hydrogen in irradiated micerdquo Journal of RadiationResearch vol 51 no 6 pp 741ndash747 2010

[9] Y Terasaki I Ohsawa M Terasaki et al ldquoHydrogen therapyattenuates irradiation-induced lung damage by reducing oxida-tive stressrdquo American Journal of PhysiologymdashLung Cellular andMolecular Physiology vol 301 no 4 pp L415ndashL426 2011

[10] K Mei S Zhao L Qian B Li J Ni and J Cai ldquoHydrogenprotects rats from dermatitis caused by local radiationrdquo Journalof Dermatological Treatment vol 25 no 2 pp 182ndash188 2014

[11] Z Guo B Zhou W Li X Sun and D Luo ldquoHydrogen-richsaline protects against ultraviolet B radiation injury in ratsrdquoJournal of Biomedical Research vol 26 no 5 pp 365ndash371 2012

[12] M H Shin R Park H Nojima H-C Kim Y K Kim and JH Chung ldquoAtomic hydrogen surrounded by water moleculesH(H2O)m modulates basal and UV-induced gene expressionsin human skin in vivordquo PLoS ONE vol 8 no 4 Article IDe61696 2013

[13] J Zhang R Yang D Zhou K L Rudolph A Meng and ZJu ldquoExonuclease 1 is essential for maintaining genomic stabilityand the proliferative capacity of neural but not hematopoieticstem cellsrdquo Stem Cell Research vol 12 no 1 pp 250ndash259 2014

[14] R Van Os S Robinson T Sheridan J M K Mislow DDawes and P M Mauch ldquoGranulocyte colony-stimulatingfactor enhances bone marrow stem cell damage caused byrepeated administration of cytotoxic agentsrdquo Blood vol 92 no6 pp 1950ndash1956 1998

[15] P Mauch M Rosenblatt and S Hellmann ldquoPermanent loss instem cell self renewal capacity following stress to the marrowrdquoBlood vol 72 no 4 pp 1193ndash1196 1988

[16] H Ema Y Morita S Yamazaki et al ldquoAdult mouse hematopoi-etic stem cells purification and single-cell assaysrdquo NatureProtocols vol 1 no 6 pp 2979ndash2987 2007

[17] P L Doan J L Russell H A Himburg et al ldquoTie2+ bonemarrow endothelial cells regulate hematopoietic stem cellregeneration following radiation injuryrdquo STEM CELLS vol 31no 2 pp 327ndash337 2013

[18] J S Kim W S Jang S Lee Y Son S Park and S S Lee ldquoAstudy of the effect of sequential injection of 5-androstenediolon irradiation-induced myelosuppression in micerdquo Archives ofPharmacal Research vol 38 no 6 pp 1213ndash1222 2015

[19] J Wang Q Sun Y Morita et al ldquoA differentiation checkpointlimits hematopoietic stem cell self-renewal in response to DNAdamagerdquo Cell vol 148 no 5 pp 1001ndash1014 2012

[20] S Wen M Dooner Y Cheng et al ldquoMesenchymal stromalcell-derived extracellular vesicles rescue radiation damage tomurine marrow hematopoietic cellsrdquo Leukemia vol 30 no 11pp 2221ndash2231 2016

[21] R L Porter M A Georger O Bromberg et al ldquoProstaglandinE2 increases hematopoietic stem cell survival and accelerateshematopoietic recovery after radiation injuryrdquo STEM CELLSvol 31 no 2 pp 372ndash383 2013

[22] YWang L Liu S K PazhanisamyH Li AMeng andD ZhouldquoTotal body irradiation causes residual bone marrow injury byinduction of persistent oxidative stress inmurine hematopoieticstem cellsrdquo Free Radical Biology and Medicine vol 48 no 2 pp348ndash356 2010

[23] H Kamiya ldquoMutagenic potentials of damaged nucleic acidsproduced by reactive oxygennitrogen species approachesusing synthetic oligonucleotides and nucleotidesrdquoNucleic AcidsResearch vol 31 no 2 pp 517ndash531 2003

[24] J J Hoppins D R Gruber H L Miears et al ldquo8-Oxoguanineaffects DNA backbone conformation in the ecori recognitionsite and inhibits its cleavage by the enzymerdquo PLOS ONE vol 11no 10 Article ID e0164424 2016

[25] H Pan D Guan X Liu et al ldquoSIRT6 safeguards humanmesenchymal stem cells from oxidative stress by coactivatingNRF2rdquo Cell Research vol 26 no 2 pp 190ndash205 2016

[26] A M Byrne A M Ruiz-Lopez S L Roche J N MoloneyA C Wyse-Jackson and T G Cotter ldquoThe synthetic progestinnorgestrel modulates Nrf2 signaling and acts as an antioxidantin a model of retinal degenerationrdquo Redox Biology vol 10 pp128ndash139 2016


[27] R ZhangM Xu YWang F Xie G Zhang andXQin ldquoNrf2mdasha promising therapeutic target for defensing against oxidativestress in strokerdquoMolecular Neurobiology 2016

[28] K R Sekhar and M L Freeman ldquoNrf2 promotes survivalfollowing exposure to ionizing radiationrdquo Free Radical Biologyand Medicine vol 88 pp 268ndash274 2015

[29] M A Marques-Torrejon E Porlan A Banito et al ldquoCyclin-dependent kinase inhibitor p21 controls adult neural stem cellexpansion by regulating Sox2 gene expressionrdquo Cell Stem Cellvol 12 no 1 pp 88ndash100 2013

[30] A R Choudhury Z Ju M W Djojosubroto et al ldquoCdkn1adeletion improves stem cell function and lifespan of mice withdysfunctional telomeres without accelerating cancer forma-tionrdquo Nature Genetics vol 39 no 1 pp 99ndash105 2007

[31] A Karimian Y Ahmadi and B Yousefi ldquoMultiple functions ofp21 in cell cycle apoptosis and transcriptional regulation afterDNA damagerdquo DNA Repair vol 42 pp 63ndash71 2016

[32] J Guo D Zhao X Lei et al ldquoProtective effects of hydrogenagainst low-dose long-term radiation-induced damage to thebehavioral performances hematopoietic system genital sys-tem and splenic lymphocytes in micerdquo Oxidative Medicine andCellular Longevity vol 2016 Article ID 1947819 15 pages 2016

[33] C Liu R Kurokawa M Fujino S Hirano B Sato and X-K LildquoEstimation of the hydrogen concentration in rat tissue usingan airtight tube following the administration of hydrogen viavarious routesrdquo Scientific Reports vol 4 article 5485 2014

[34] Y Yang B Li C Liu et al ldquoHydrogen-rich saline protectsimmunocytes from radiation-induced apoptosisrdquo Medical Sci-ence Monitor vol 18 no 4 pp BR144ndashBR148 2012

[35] D Li L Lu J Zhang et al ldquoMitigating the effects of Xuebijinginjection on hematopoietic cell injury induced by total bodyirradiation with 120574 rays by decreasing reactive oxygen specieslevelsrdquo International Journal of Molecular Sciences vol 15 no6 pp 10541ndash10553 2014

[36] L Lu J Dong D Li J Zhang and S Fan ldquo331015840-Diindolyl-methane mitigates total body irradiation-induced hematopoi-etic injury in micerdquo Free Radical Biology and Medicine vol 99pp 463ndash471 2016

[37] G Xu H Wu J Zhang et al ldquoMetformin ameliorates ionizingirradiation-induced long-term hematopoietic stem cell injuryin micerdquo Free Radical Biology and Medicine vol 87 pp 15ndash252015

[38] J Zhang H Li L Lu et al ldquoThe Yiqi and Yangyin Formulaameliorates injury to the hematopoietic system induced by totalbody irradiationrdquo Journal of Radiation Research 2016

[39] J Zhang X Xue X Han et al ldquoVam3 ameliorates totalbody irradiation-inducedhematopoietic system injury partly byregulating the expression of Nrf2-targeted genesrdquo Free RadicalBiology and Medicine vol 101 pp 455ndash464 2016

[40] L Zhao C Zhou J Zhang et al ldquoHydrogen protects micefrom radiation induced thymic lymphoma in BALBc micerdquoInternational Journal of Biological Sciences vol 7 no 3 pp 297ndash300 2011

[41] Y Chuai F Gao B Li et al ldquoHydrogen-rich saline attenuatesradiation-inducedmale germ cell loss inmice through reducinghydroxyl radicalsrdquo Biochemical Journal vol 442 no 1 pp 49ndash56 2012

[42] D-Z Li Q-X Zhang X-X Dong H-D Li and X Ma ldquoTreat-ment with hydrogen molecules prevents RANKL-inducedosteoclast differentiation associated with inhibition of ROSformation and inactivation of MAPK AKT and NF-kappa B

pathways in murine RAW2647 cellsrdquo Journal of Bone andMineral Metabolism vol 32 no 5 pp 494ndash504 2014

[43] G Song C Zong Z Zhang et al ldquoMolecular hydrogen stabi-lizes atherosclerotic plaque in low-density lipoprotein receptor-knockout micerdquo Free Radical Biology and Medicine vol 87 pp58ndash68 2015

[44] Y Li Q Li H Chen et al ldquoHydrogen gas alleviates theintestinal injury caused by severe sepsis in mice by increasingthe expression of heme oxygenase-1rdquo Shock vol 44 no 1 pp90ndash98 2015

[45] T Kawamura N Wakabayashi N Shigemura et al ldquoHydrogengas reduces hyperoxic lung injury via the Nrf2 pathway in vivordquoAmerican Journal of PhysiologymdashLung Cellular and MolecularPhysiology vol 304 no 10 pp L646ndashL656 2013

[46] S Spulber K Edoff L Hong S Morisawa S Shirahata and SCeccatelli ldquoMolecular hydrogen reduces LPS-induced neuroin-flammation and promotes recovery from sickness behaviour inmicerdquo PLoS ONE vol 7 no 7 Article ID e42078 2012

[47] X Zhai X Chen J Shi et al ldquoLactulose ameliorates cerebralischemiandashreperfusion injury in ratsby inducing hydrogen byactivating Nrf2 expressionrdquo Free Radical Biology and Medicinevol 65 pp 731ndash741 2013

[48] Y Li K Xie H Chen G Wang and Y Yu ldquoHydrogen gasinhibits high-mobility group box 1 release in septic mice byupregulation of heme oxygenase 1rdquo Journal of Surgical Researchvol 196 no 1 pp 136ndash148 2015

[49] Q Xie X-X Li P Zhang et al ldquoHydrogen gas protects againstserum and glucose deprivation-induced myocardial injury inH9c2 cells through activation of the NF-E2-related factor2heme oxygenase 1 signaling pathwayrdquo Molecular MedicineReports vol 10 no 2 pp 1143ndash1149 2014

Submit your manuscripts athttpswwwhindawicom

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

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


CD11b Gr1 CD4 CD8 and IL3(lineage) are found on thesurface of murine HSCs and HPCs except c-kit and sca1HSCs are identified as lineageminussca1+c-kit+(LSK) and HPCsare identified as lineageminussca1minusc-kit+ respectivelyThus c-kit+cells may represent the total HSCs and HPCs

The hematopoietic system is highly radiosensitive andTBI may induce acute hematopoietic syndrome residualbone marrow injury and hematopoietic stem cell (HSC)senescence [14 15] The potential mechanisms involved inHSCsrsquo injury caused by TBI include the induction of HSCapoptosis differentiation and senescence [1] Thus it isnecessary to identify and characterize radioprotective agentsthat guard against TBI-induced hematopoietic system injury

Our present results show that HW may elevate the sur-vival rate of mice lethally irradiated Moreover the numberof bone marrow cells (BMCs) and the rate of self-renewaland differentiation are all improved in irradiated mice underthe condition of HW consumption HW significantly affectsthe expression of cell cycle- cell apoptosis- and oxidativestress-related proteins in the c-kit+ cells from irradiatedmiceThese results suggest that HW consumption is able to protectagainst TBI-induced HSC injury

2 Materials and Methods

21 Mice Male C57BL6 mice were purchased from theInstitute of Laboratory Animal Sciences Chinese Academyof Medical Sciences amp Peking Union Medical College Micewere bred in the experimental animal center of the Instituteof Radiation Medicine Mice were used at approximately 6ndash8 weeks of age All of the animal experiments in our studywere approved by the Animal Care and Ethics Committee atthe Institute of RadiationMedicineThe study was performedin accordance with the principle of Institutional Animal Careand Ethics Committee guidelines

22 Antibodies The following antibodies were purchasedfrom eBioscience (San Diego CA USA) anti-mouse CD3-APC (clone 145-2C11) anti-mouse CD117 (c-kit) APC(clone 2B8) anti-mouse Sca-1-PE (clone D7) anti-mouseCD34-FITC (clone RAM34) and anti-mouse Ki67-FITC(clone SolA15) The following antibodies were purchasedfrom Biolegend (San Diego CA USA) Percp-conjugatedstreptavidin anti-mouse B220-PE (clone RA3-6B2) anti-mouse CD11b-Percp (clone M170) anti-mouse Gr1-Percp(clone RB6-8C5) anti-mouse CD451-FITC (clone A20)anti-mouse CD452-PE (clone 104) biotin-conjugated anti-mouse Ter119 (clone TER119) anti-mouse B220 (clone RA3-6B2) anti-mouse Gr1 (clone RB6-8C5) anti-mouse CD11b(clone M170) anti-mouse CD4 (clone GK15) and anti-mouse CD8 (clone 53ndash67) The following antibodies werepurchased from BD Bioscience (San Jose CA USA) anti-mouse annexin V-FITC and anti-mouse 120574-H2AX-FITC Thefollowing antibodies were purchased from Cell SignalingTechnology (Danvers MA USA) anti-mouse NRF2 anti-mouse BCL-XL and anti-mouse BAK Anti-mouse p21 andanti-mouse 120573-actin were purchased from Abcam (Cam-bridge England) Anti-mouse 8-oxoguanine was purchasedfromMillipore (Boston MA USA)

23 Preparation ofHW Thepreparation ofHWwas based ona previous study with slight modification H2 gas was gener-ated from a hydrogen gas generator (SHC-300 Saikesaisi HWEnergy Shandong China) and bubbled into 500mL of sterilewater at a rate of 150mLmin for 20minThe concentration ofH2in the water was detected with a dissolved hydrogenmeter

(Trustlex ENH-1000 Japan) HW was freshly prepared eachday to ensure that an H

2concentration of more than 08 ppm

was maintained

24 TBI and HW Administration Mice were divided intofour groups for a 30-day survival experiment the groupsincluded 68Gy TBI + vehicle (normal water) 68 Gy TBI+ HW 72Gy TBI + vehicle and 72Gy TBI + HW For theother experiments mice were also divided into 4 groupsnamely vehicle HW alone 4Gy TBI and TBI + HW Themice with TBI were irradiated with 120574-rays at a dosage rateof 099Gymin Mice received 05mL HW by gavage for10min before TBI and up to the 7 days following TBI Thecontrol mice with vehicle received normal water for the samefrequency and volume as those in the mice with HW Micewere finally euthanized on the 15th day after TBI

25 Peripheral Blood Cell and Bone Marrow Cell CountsBlood was obtained from mice via the orbital sinus andcollected inmicropipettes coatedwith EDTAK

3 white blood

cells (WBCs) the percentages of lymphocytes (LY) andneutrophil granulocytes (NE) were counted and calculatedBone marrow cells were isolated from tibias and femurs aspreviously reported [16] and cell numbers were analyzedwith a hematology analyzer (Nihon Kohden Japan)

26 Flow Cytometry Analysis Bone marrow cells were sus-pended in phosphate-buffered saline (PBS) and cells werefiltered and counted prior to antibody staining To analyzethe B cells T cells and myeloid cells in peripheral blood50 120583L of peripheral blood was harvested and stained withB220 CD3 CD11b and Gr1 antibodies at room temperatureThe red blood cells in the blood samples were removedusing the BD FACS lysing solution For HSC analysis 5 times106 bone marrow cells were first stained with biotin-labeledTer119 B220 Gr1 CD11b CD4 and CD8 antibodies andsubsequently stained with streptavidin c-kit sca1 and CD34antibodies To assess ROS levels 1 times 106 bone marrow cellswere stainedwith anti-c-kit antibody and then incubatedwith27-dichlorodihydrofluorescein diacetate (DCFDA BeyotimeBiotechnology Nanjing China 10120583M) MitoSox (Life Tech-nologies Grand Island NY USA 10 120583M) and dihydroethid-ium (DHE Beyotime Biotechnology Nanjing China 5120583M)in a 37∘Cwater bath For cell cycle analysis bonemarrow cellswere first stained with a c-kit antibody and fixed and per-meabilized with CytofixCytoperm buffer (BD BiosciencesUSA) before being stained with anti-Ki67 antibody and PIFor analysis of 120574-H2AX NRF2 HO-1 NQO1 BCL-XL andBAK bone marrow cells were first processed as describedfor ki67 staining and then staining 120574-H2AX NRF2 HO-1NQO1 BCL-XL and BAK antibodies at room temperatureFor apoptosis analysis bone marrow cells were also stained












times 8824 times1

sample volume


(1)



3 Results




0

20

40

60

80

100

Perc

ent s

urvi

val


P = 00001

P = 00003

68Gy68Gy + HW

72Gy72Gy + HW









ControlHW

0

2

4

6

8

10

WBC

(times10

3 120583

L)

lowast

0Gy 4Gy

(a)

ControlHW

0

20

40

60

80

100

LY

lowast

0Gy 4Gy

(b)

ControlHW

0

20

40

60

80

NE lowast

0Gy 4Gy

(c)

B cell

ControlHW

0

20

40

60

Perc

enta

ge

lowast

0Gy 4Gy

(d)

T cell

ControlHW

0

10

20

30Pe

rcen

tage

0Gy 4Gy

(e)

Myeloid cell

ControlHW

0

10

20

30

40

50

Perc

enta

ge

lowast

0Gy 4Gy

(f)

B cell

T cell

B220

CD3Control HW

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

101101

101

101 101101101

101

(g)

Myeloid cell

CD3

CD11

bG

r1

Control HW

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 106 1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

101

101101

101101

101

101101

0G

y4

Gy

(h)



0Gy 4Gy

BMCs

(times10

6)

0

10

20

30

40

lowast

ControlHW

(a)

0

5

10

15

20

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

c-kit+ cells

(b)

HPC

0

5

10

15

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(c)

LSK

0

1

2

3

4

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(d)

0

5

10

15

20

25

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34minusLSK

(e)

0

20

40

60

80

100

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34+ LSK

(f)

Control HW

c-ki

t

Sca1

HPC LSK

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

0G

y4

Gy

101 101

101101

101 101

101101

(g)Control HW

c-ki

t

CD34

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072


101

101101

101

101101101

101

(h)



400500600700800

020406080

100

0Gy 4Gy

lowast

ControlHW

CFU

-GM

105

BMCs

(a)

Chimerism

0

10

20

30

40

Perc

enta

ge

0Gy 4Gy

lowast

(b)

Control HW

CD45

1

CD452


103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 1061072

101101

101

101

101

101101

101

(c)







0

200000

400000

600000

ROS

(DCF

MFI

)

ControlHW

0Gy 4Gy

lowast

(a)

Abili

ty o

f hyd

roxy

l rad

ical

0

10

20

30

40

prod

uctio

n

ControlHW

0Gy 4Gy

lowast

(b)

0

10000

20000

30000

40000

Mito

Sox

(MFI

)

ControlHW

0Gy 4Gy

(c)

0

20000

40000

60000

80000

DH

E (M

FI)

ControlHW

0Gy 4Gy

(d)

ROS

HWControl

Cou

nt

102 103 104 105 106 1072

4Gy

0

50

100

150

200

4Gy + HW

101

(e)

MitoSox

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(f)

DHE

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(g)



0

20

40

60

80Pe

rcen

tage

G0 phase

0Gy 4GyControlHW

lowast

(a)

G1 phase

0

10

20

30

40

Perc

enta

ge

0Gy 4GyControlHW

lowast

(b)

SG2M phase

0

10

20

30

Perc

enta

ge

0Gy 4GyControlHW

lowast

(c)

Early apoptosis

0

5

10

15

20

25

0Gy 4Gy

ControlHW

lowast

Perc

enta

ge

(d)

Late apoptosis

0

2

4

6

8

0Gy 4Gy

ControlHW

Perc

enta

ge

(e)

Ki67

PIControl HW

0G

y4

Gy

1027

104

104

105

1058

1027

104

105

1058

1027

104

105

1058

1027

104

105

10581033 105 1058 1041033 105 1058

1041033 105 1058 1041033 105 1058

(f)

PI

Ann

exin

V

Control HW

102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102

103

104

105

106

1072

102 103 104 105 1061072

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

101 101

101101

101101

101 101

101101

c-kit+ cells

(g)



120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)


(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)



0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























times 8824 times1

sample volume


(1)



3 Results




0

20

40

60

80

100

Perc

ent s

urvi

val


P = 00001

P = 00003

68Gy68Gy + HW

72Gy72Gy + HW









ControlHW

0

2

4

6

8

10

WBC

(times10

3 120583

L)

lowast

0Gy 4Gy

(a)

ControlHW

0

20

40

60

80

100

LY

lowast

0Gy 4Gy

(b)

ControlHW

0

20

40

60

80

NE lowast

0Gy 4Gy

(c)

B cell

ControlHW

0

20

40

60

Perc

enta

ge

lowast

0Gy 4Gy

(d)

T cell

ControlHW

0

10

20

30Pe

rcen

tage

0Gy 4Gy

(e)

Myeloid cell

ControlHW

0

10

20

30

40

50

Perc

enta

ge

lowast

0Gy 4Gy

(f)

B cell

T cell

B220

CD3Control HW

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

101101

101

101 101101101

101

(g)

Myeloid cell

CD3

CD11

bG

r1

Control HW

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 106 1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

101

101101

101101

101

101101

0G

y4

Gy

(h)



0Gy 4Gy

BMCs

(times10

6)

0

10

20

30

40

lowast

ControlHW

(a)

0

5

10

15

20

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

c-kit+ cells

(b)

HPC

0

5

10

15

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(c)

LSK

0

1

2

3

4

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(d)

0

5

10

15

20

25

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34minusLSK

(e)

0

20

40

60

80

100

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34+ LSK

(f)

Control HW

c-ki

t

Sca1

HPC LSK

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

0G

y4

Gy

101 101

101101

101 101

101101

(g)Control HW

c-ki

t

CD34

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072


101

101101

101

101101101

101

(h)



400500600700800

020406080

100

0Gy 4Gy

lowast

ControlHW

CFU

-GM

105

BMCs

(a)

Chimerism

0

10

20

30

40

Perc

enta

ge

0Gy 4Gy

lowast

(b)

Control HW

CD45

1

CD452


103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 1061072

101101

101

101

101

101101

101

(c)







0

200000

400000

600000

ROS

(DCF

MFI

)

ControlHW

0Gy 4Gy

lowast

(a)

Abili

ty o

f hyd

roxy

l rad

ical

0

10

20

30

40

prod

uctio

n

ControlHW

0Gy 4Gy

lowast

(b)

0

10000

20000

30000

40000

Mito

Sox

(MFI

)

ControlHW

0Gy 4Gy

(c)

0

20000

40000

60000

80000

DH

E (M

FI)

ControlHW

0Gy 4Gy

(d)

ROS

HWControl

Cou

nt

102 103 104 105 106 1072

4Gy

0

50

100

150

200

4Gy + HW

101

(e)

MitoSox

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(f)

DHE

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(g)



0

20

40

60

80Pe

rcen

tage

G0 phase

0Gy 4GyControlHW

lowast

(a)

G1 phase

0

10

20

30

40

Perc

enta

ge

0Gy 4GyControlHW

lowast

(b)

SG2M phase

0

10

20

30

Perc

enta

ge

0Gy 4GyControlHW

lowast

(c)

Early apoptosis

0

5

10

15

20

25

0Gy 4Gy

ControlHW

lowast

Perc

enta

ge

(d)

Late apoptosis

0

2

4

6

8

0Gy 4Gy

ControlHW

Perc

enta

ge

(e)

Ki67

PIControl HW

0G

y4

Gy

1027

104

104

105

1058

1027

104

105

1058

1027

104

105

1058

1027

104

105

10581033 105 1058 1041033 105 1058

1041033 105 1058 1041033 105 1058

(f)

PI

Ann

exin

V

Control HW

102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102

103

104

105

106

1072

102 103 104 105 1061072

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

101 101

101101

101101

101 101

101101

c-kit+ cells

(g)



120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)


(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)



0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

Perc

ent s

urvi

val


P = 00001

P = 00003

68Gy68Gy + HW

72Gy72Gy + HW









ControlHW

0

2

4

6

8

10

WBC

(times10

3 120583

L)

lowast

0Gy 4Gy

(a)

ControlHW

0

20

40

60

80

100

LY

lowast

0Gy 4Gy

(b)

ControlHW

0

20

40

60

80

NE lowast

0Gy 4Gy

(c)

B cell

ControlHW

0

20

40

60

Perc

enta

ge

lowast

0Gy 4Gy

(d)

T cell

ControlHW

0

10

20

30Pe

rcen

tage

0Gy 4Gy

(e)

Myeloid cell

ControlHW

0

10

20

30

40

50

Perc

enta

ge

lowast

0Gy 4Gy

(f)

B cell

T cell

B220

CD3Control HW

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

101101

101

101 101101101

101

(g)

Myeloid cell

CD3

CD11

bG

r1

Control HW

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 106 1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

101

101101

101101

101

101101

0G

y4

Gy

(h)



0Gy 4Gy

BMCs

(times10

6)

0

10

20

30

40

lowast

ControlHW

(a)

0

5

10

15

20

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

c-kit+ cells

(b)

HPC

0

5

10

15

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(c)

LSK

0

1

2

3

4

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(d)

0

5

10

15

20

25

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34minusLSK

(e)

0

20

40

60

80

100

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34+ LSK

(f)

Control HW

c-ki

t

Sca1

HPC LSK

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

0G

y4

Gy

101 101

101101

101 101

101101

(g)Control HW

c-ki

t

CD34

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072


101

101101

101

101101101

101

(h)



400500600700800

020406080

100

0Gy 4Gy

lowast

ControlHW

CFU

-GM

105

BMCs

(a)

Chimerism

0

10

20

30

40

Perc

enta

ge

0Gy 4Gy

lowast

(b)

Control HW

CD45

1

CD452


103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 1061072

101101

101

101

101

101101

101

(c)







0

200000

400000

600000

ROS

(DCF

MFI

)

ControlHW

0Gy 4Gy

lowast

(a)

Abili

ty o

f hyd

roxy

l rad

ical

0

10

20

30

40

prod

uctio

n

ControlHW

0Gy 4Gy

lowast

(b)

0

10000

20000

30000

40000

Mito

Sox

(MFI

)

ControlHW

0Gy 4Gy

(c)

0

20000

40000

60000

80000

DH

E (M

FI)

ControlHW

0Gy 4Gy

(d)

ROS

HWControl

Cou

nt

102 103 104 105 106 1072

4Gy

0

50

100

150

200

4Gy + HW

101

(e)

MitoSox

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(f)

DHE

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(g)



0

20

40

60

80Pe

rcen

tage

G0 phase

0Gy 4GyControlHW

lowast

(a)

G1 phase

0

10

20

30

40

Perc

enta

ge

0Gy 4GyControlHW

lowast

(b)

SG2M phase

0

10

20

30

Perc

enta

ge

0Gy 4GyControlHW

lowast

(c)

Early apoptosis

0

5

10

15

20

25

0Gy 4Gy

ControlHW

lowast

Perc

enta

ge

(d)

Late apoptosis

0

2

4

6

8

0Gy 4Gy

ControlHW

Perc

enta

ge

(e)

Ki67

PIControl HW

0G

y4

Gy

1027

104

104

105

1058

1027

104

105

1058

1027

104

105

1058

1027

104

105

10581033 105 1058 1041033 105 1058

1041033 105 1058 1041033 105 1058

(f)

PI

Ann

exin

V

Control HW

102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102

103

104

105

106

1072

102 103 104 105 1061072

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

101 101

101101

101101

101 101

101101

c-kit+ cells

(g)



120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)


(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)



0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ControlHW

0

2

4

6

8

10

WBC

(times10

3 120583

L)

lowast

0Gy 4Gy

(a)

ControlHW

0

20

40

60

80

100

LY

lowast

0Gy 4Gy

(b)

ControlHW

0

20

40

60

80

NE lowast

0Gy 4Gy

(c)

B cell

ControlHW

0

20

40

60

Perc

enta

ge

lowast

0Gy 4Gy

(d)

T cell

ControlHW

0

10

20

30Pe

rcen

tage

0Gy 4Gy

(e)

Myeloid cell

ControlHW

0

10

20

30

40

50

Perc

enta

ge

lowast

0Gy 4Gy

(f)

B cell

T cell

B220

CD3Control HW

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

101101

101

101 101101101

101

(g)

Myeloid cell

CD3

CD11

bG

r1

Control HW

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 106 1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

101

101101

101101

101

101101

0G

y4

Gy

(h)



0Gy 4Gy

BMCs

(times10

6)

0

10

20

30

40

lowast

ControlHW

(a)

0

5

10

15

20

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

c-kit+ cells

(b)

HPC

0

5

10

15

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(c)

LSK

0

1

2

3

4

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(d)

0

5

10

15

20

25

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34minusLSK

(e)

0

20

40

60

80

100

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34+ LSK

(f)

Control HW

c-ki

t

Sca1

HPC LSK

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

0G

y4

Gy

101 101

101101

101 101

101101

(g)Control HW

c-ki

t

CD34

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072


101

101101

101

101101101

101

(h)



400500600700800

020406080

100

0Gy 4Gy

lowast

ControlHW

CFU

-GM

105

BMCs

(a)

Chimerism

0

10

20

30

40

Perc

enta

ge

0Gy 4Gy

lowast

(b)

Control HW

CD45

1

CD452


103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 1061072

101101

101

101

101

101101

101

(c)







0

200000

400000

600000

ROS

(DCF

MFI

)

ControlHW

0Gy 4Gy

lowast

(a)

Abili

ty o

f hyd

roxy

l rad

ical

0

10

20

30

40

prod

uctio

n

ControlHW

0Gy 4Gy

lowast

(b)

0

10000

20000

30000

40000

Mito

Sox

(MFI

)

ControlHW

0Gy 4Gy

(c)

0

20000

40000

60000

80000

DH

E (M

FI)

ControlHW

0Gy 4Gy

(d)

ROS

HWControl

Cou

nt

102 103 104 105 106 1072

4Gy

0

50

100

150

200

4Gy + HW

101

(e)

MitoSox

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(f)

DHE

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(g)



0

20

40

60

80Pe

rcen

tage

G0 phase

0Gy 4GyControlHW

lowast

(a)

G1 phase

0

10

20

30

40

Perc

enta

ge

0Gy 4GyControlHW

lowast

(b)

SG2M phase

0

10

20

30

Perc

enta

ge

0Gy 4GyControlHW

lowast

(c)

Early apoptosis

0

5

10

15

20

25

0Gy 4Gy

ControlHW

lowast

Perc

enta

ge

(d)

Late apoptosis

0

2

4

6

8

0Gy 4Gy

ControlHW

Perc

enta

ge

(e)

Ki67

PIControl HW

0G

y4

Gy

1027

104

104

105

1058

1027

104

105

1058

1027

104

105

1058

1027

104

105

10581033 105 1058 1041033 105 1058

1041033 105 1058 1041033 105 1058

(f)

PI

Ann

exin

V

Control HW

102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102

103

104

105

106

1072

102 103 104 105 1061072

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

101 101

101101

101101

101 101

101101

c-kit+ cells

(g)



120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)


(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)



0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0Gy 4Gy

BMCs

(times10

6)

0

10

20

30

40

lowast

ControlHW

(a)

0

5

10

15

20

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

c-kit+ cells

(b)

HPC

0

5

10

15

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(c)

LSK

0

1

2

3

4

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

(d)

0

5

10

15

20

25

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34minusLSK

(e)

0

20

40

60

80

100

Perc

enta

ge

0Gy 4Gy

lowast

ControlHW

CD34+ LSK

(f)

Control HW

c-ki

t

Sca1

HPC LSK

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

0G

y4

Gy

101 101

101101

101 101

101101

(g)Control HW

c-ki

t

CD34

102 103 104 105 1061072 102 103 104 105 1061072

102 103 104 105 1061072102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072


101

101101

101

101101101

101

(h)



400500600700800

020406080

100

0Gy 4Gy

lowast

ControlHW

CFU

-GM

105

BMCs

(a)

Chimerism

0

10

20

30

40

Perc

enta

ge

0Gy 4Gy

lowast

(b)

Control HW

CD45

1

CD452


103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 1061072

101101

101

101

101

101101

101

(c)







0

200000

400000

600000

ROS

(DCF

MFI

)

ControlHW

0Gy 4Gy

lowast

(a)

Abili

ty o

f hyd

roxy

l rad

ical

0

10

20

30

40

prod

uctio

n

ControlHW

0Gy 4Gy

lowast

(b)

0

10000

20000

30000

40000

Mito

Sox

(MFI

)

ControlHW

0Gy 4Gy

(c)

0

20000

40000

60000

80000

DH

E (M

FI)

ControlHW

0Gy 4Gy

(d)

ROS

HWControl

Cou

nt

102 103 104 105 106 1072

4Gy

0

50

100

150

200

4Gy + HW

101

(e)

MitoSox

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(f)

DHE

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(g)



0

20

40

60

80Pe

rcen

tage

G0 phase

0Gy 4GyControlHW

lowast

(a)

G1 phase

0

10

20

30

40

Perc

enta

ge

0Gy 4GyControlHW

lowast

(b)

SG2M phase

0

10

20

30

Perc

enta

ge

0Gy 4GyControlHW

lowast

(c)

Early apoptosis

0

5

10

15

20

25

0Gy 4Gy

ControlHW

lowast

Perc

enta

ge

(d)

Late apoptosis

0

2

4

6

8

0Gy 4Gy

ControlHW

Perc

enta

ge

(e)

Ki67

PIControl HW

0G

y4

Gy

1027

104

104

105

1058

1027

104

105

1058

1027

104

105

1058

1027

104

105

10581033 105 1058 1041033 105 1058

1041033 105 1058 1041033 105 1058

(f)

PI

Ann

exin

V

Control HW

102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102

103

104

105

106

1072

102 103 104 105 1061072

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

101 101

101101

101101

101 101

101101

c-kit+ cells

(g)



120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)


(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)



0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















400500600700800

020406080

100

0Gy 4Gy

lowast

ControlHW

CFU

-GM

105

BMCs

(a)

Chimerism

0

10

20

30

40

Perc

enta

ge

0Gy 4Gy

lowast

(b)

Control HW

CD45

1

CD452


103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

102

103

104

105

106

1072

0G

y4

Gy

102 103 104 105 106 1072 102 103 104 105 106 1072

102 103 104 105 106 1072102 103 104 105 1061072

101101

101

101

101

101101

101

(c)







0

200000

400000

600000

ROS

(DCF

MFI

)

ControlHW

0Gy 4Gy

lowast

(a)

Abili

ty o

f hyd

roxy

l rad

ical

0

10

20

30

40

prod

uctio

n

ControlHW

0Gy 4Gy

lowast

(b)

0

10000

20000

30000

40000

Mito

Sox

(MFI

)

ControlHW

0Gy 4Gy

(c)

0

20000

40000

60000

80000

DH

E (M

FI)

ControlHW

0Gy 4Gy

(d)

ROS

HWControl

Cou

nt

102 103 104 105 106 1072

4Gy

0

50

100

150

200

4Gy + HW

101

(e)

MitoSox

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(f)

DHE

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(g)



0

20

40

60

80Pe

rcen

tage

G0 phase

0Gy 4GyControlHW

lowast

(a)

G1 phase

0

10

20

30

40

Perc

enta

ge

0Gy 4GyControlHW

lowast

(b)

SG2M phase

0

10

20

30

Perc

enta

ge

0Gy 4GyControlHW

lowast

(c)

Early apoptosis

0

5

10

15

20

25

0Gy 4Gy

ControlHW

lowast

Perc

enta

ge

(d)

Late apoptosis

0

2

4

6

8

0Gy 4Gy

ControlHW

Perc

enta

ge

(e)

Ki67

PIControl HW

0G

y4

Gy

1027

104

104

105

1058

1027

104

105

1058

1027

104

105

1058

1027

104

105

10581033 105 1058 1041033 105 1058

1041033 105 1058 1041033 105 1058

(f)

PI

Ann

exin

V

Control HW

102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102

103

104

105

106

1072

102 103 104 105 1061072

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

101 101

101101

101101

101 101

101101

c-kit+ cells

(g)



120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)


(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)



0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

200000

400000

600000

ROS

(DCF

MFI

)

ControlHW

0Gy 4Gy

lowast

(a)

Abili

ty o

f hyd

roxy

l rad

ical

0

10

20

30

40

prod

uctio

n

ControlHW

0Gy 4Gy

lowast

(b)

0

10000

20000

30000

40000

Mito

Sox

(MFI

)

ControlHW

0Gy 4Gy

(c)

0

20000

40000

60000

80000

DH

E (M

FI)

ControlHW

0Gy 4Gy

(d)

ROS

HWControl

Cou

nt

102 103 104 105 106 1072

4Gy

0

50

100

150

200

4Gy + HW

101

(e)

MitoSox

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(f)

DHE

Cou

nt

102 103 104 105 106 1072

HWControl 4Gy

4Gy + HW

0

50

100

150

200

101

(g)



0

20

40

60

80Pe

rcen

tage

G0 phase

0Gy 4GyControlHW

lowast

(a)

G1 phase

0

10

20

30

40

Perc

enta

ge

0Gy 4GyControlHW

lowast

(b)

SG2M phase

0

10

20

30

Perc

enta

ge

0Gy 4GyControlHW

lowast

(c)

Early apoptosis

0

5

10

15

20

25

0Gy 4Gy

ControlHW

lowast

Perc

enta

ge

(d)

Late apoptosis

0

2

4

6

8

0Gy 4Gy

ControlHW

Perc

enta

ge

(e)

Ki67

PIControl HW

0G

y4

Gy

1027

104

104

105

1058

1027

104

105

1058

1027

104

105

1058

1027

104

105

10581033 105 1058 1041033 105 1058

1041033 105 1058 1041033 105 1058

(f)

PI

Ann

exin

V

Control HW

102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102

103

104

105

106

1072

102 103 104 105 1061072

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

101 101

101101

101101

101 101

101101

c-kit+ cells

(g)



120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)


(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)



0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80Pe

rcen

tage

G0 phase

0Gy 4GyControlHW

lowast

(a)

G1 phase

0

10

20

30

40

Perc

enta

ge

0Gy 4GyControlHW

lowast

(b)

SG2M phase

0

10

20

30

Perc

enta

ge

0Gy 4GyControlHW

lowast

(c)

Early apoptosis

0

5

10

15

20

25

0Gy 4Gy

ControlHW

lowast

Perc

enta

ge

(d)

Late apoptosis

0

2

4

6

8

0Gy 4Gy

ControlHW

Perc

enta

ge

(e)

Ki67

PIControl HW

0G

y4

Gy

1027

104

104

105

1058

1027

104

105

1058

1027

104

105

1058

1027

104

105

10581033 105 1058 1041033 105 1058

1041033 105 1058 1041033 105 1058

(f)

PI

Ann

exin

V

Control HW

102 103 104 105 1061072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102103104105106

1072

102

103

104

105

106

1072

102 103 104 105 1061072

102 103 104 105 106 1072

102 103 104 105 106 1072 102 103 104 105 106 1072

101 101

101101

101101

101 101

101101

c-kit+ cells

(g)



120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)


(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)



0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120574-H

2AX

(MFI

)

0

50000

100000

150000

lowast

0Gy 4Gy

ControlHW

(a)

120574-H2AX

Cou

nt

HWControl 4Gy

4Gy + HW

0

200

400

600

800

1018 103 104 105

(b)


(c)

0

10

20

30

40

Perc

enta

ge o

f 8-o

xoG

posit

ive c

ells

0Gy 4Gy

ControlHW

lowast

(d)

Control HW

0Gy

4Gy

(e)



0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5000

10000

15000

20000N

RF2

(MFI

)

0Gy 4Gy

ControlHW

lowast

(a)

Cou

nt

NRF21021 103 104 1049

HWControl 4Gy

4Gy + HW

0

100

200

300

400

(b)

0

50000

100000

150000

200000

250000

HO

-1 (M

FI)

0Gy 4Gy

ControlHW

(c)

0

20000

40000

60000

80000N

QO

1 (M

FI)

0Gy 4Gy

ControlHW

lowast

(d)

0

10

20

30

SOD

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(e)

0

10

20

30

40

50

GPX

enzy

me a

ctiv

ity

0Gy 4Gy

ControlHW

lowast

(f)



0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

P21

120573-a

ctin

0Gy 4Gy

ControlHW

lowast

(a)

P21

120573-actin

TBI + +

HW + +

minus minus

minusminus

(b)

0

10000

20000

30000

BCL-

XL (M

FI)

0Gy 4Gy

ControlHW

lowast

(c)

Cou

nt

BCL-XL

HWControl 4Gy

4Gy + HW

103 104 105 10540

50

100

150

200

(d)

0

20000

40000

60000

80000

BAK

(MFI

)

0Gy 4GyControlHW

lowast

(e)

Cou

nt

BAK

HWControl 4Gy

4Gy + HW

1041 105 10570

50

100

150

200

(f)



ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ControlHW

0

1000000

2000000

3000000

4000000

ROS

(DCF

MFI

)

lowast

0Gy 4Gy

(a)

0

50

100

150

200

250

120574-H

2AX

foci

per

cell

lowast

0Gy 4Gy

ControlHW

(b)


(c)





4 Discussion






















2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































2activates NRF2


2


2downregulates the









Competing Interests






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















hydrogen-rich water ameliorates total body irradiation...

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