Tocotrienol-Rich Fraction Ameliorates Antioxidant Defense Mechanisms...

Research ArticleTocotrienol-Rich Fraction Ameliorates Antioxidant DefenseMechanisms and Improves Replicative Senescence-AssociatedOxidative Stress in Human Myoblasts

Shy Cian Khor Wan Zurinah Wan Ngah Yasmin Anum Mohd YusofNorwahidah Abdul Karim and Suzana Makpol

Department of Biochemistry Faculty of Medicine Level 17 Preclinical Building Universiti Kebangsaan Malaysia Medical Centre(UKMMC) Jalan Yaacob Latif Bandar Tun Razak 56000 Cheras Kuala Lumpur Malaysia

Correspondence should be addressed to Suzana Makpol suzanamakpolppukmukmedumy

Received 18 October 2016 Revised 22 November 2016 Accepted 18 December 2016 Published 24 January 2017

Academic Editor Andrea Vasconsuelo

Copyright copy 2017 Shy Cian Khor 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

During aging oxidative stress affects the normal function of satellite cells with consequent regeneration defects that lead tosarcopenia This study aimed to evaluate tocotrienol-rich fraction (TRF) modulation in reestablishing the oxidative status ofmyoblasts during replicative senescence and to compare the effects of TRF with other antioxidants (120572-tocopherol (ATF) and N-acetyl-cysteine (NAC)) Primary human myoblasts were cultured to young presenescent and senescent phases The cells weretreated with antioxidants for 24 h followed by the assessment of free radical generation lipid peroxidation antioxidant enzymemRNA expression and activities and the ratio of reduced to oxidized glutathione Our data showed that replicative senescenceincreased reactive oxygen species (ROS) generation and lipid peroxidation in myoblasts Treatment with TRF significantlydiminished ROS production and decreased lipid peroxidation in senescent myoblasts Moreover the gene expression of superoxidedismutase (SOD2) catalase (CAT) and glutathione peroxidase (GPX1)was modulated by TRF treatment with increased activity ofsuperoxide dismutase and catalase and reduced glutathione peroxidase in senescent myoblasts In comparison to ATF and NACTRF was more efficient in heightening the antioxidant capacity and reducing free radical insults These results suggested that TRFis able to ameliorate antioxidant defense mechanisms and improves replicative senescence-associated oxidative stress in myoblasts

1 Introduction

Adult skeletal muscle contains a subpopulation of cellsthat readily proliferate and differentiate when required tomaintain the structure and function of skeletal muscle [1]These cells were first identified by Mauro in 1961 as quiescentcells located between the basal lamina and sarcolemma ofmyofibers known as satellite cells [2] Human satellite cellscan be isolated and cultured in vitro with a limited prolif-erative capacity depending on the donor age Proliferatingsatellite cells are known as myoblasts [3] The proliferativelifespan of myoblasts remains stable during adulthood butdecreases from infants to adolescents and the cells ultimatelyreach replicative senescent [4]

During aging a progressive loss of muscle mass andstrength is observed and this phenomenon is known as

sarcopenia Although the underlying mechanism is still un-certain sarcopenia is believed to be the result of certainintrinsic or extrinsic factors such as immobilization chronicdiseases changes in hormone and proinflammatory factorsas well as nutritional status in older adults [5] Additionallythe accumulation of reactive oxygen species (ROS) has beensuggested to play a vital role in this age-related muscle atro-phy [6] Redox imbalance observed in senescent satellite cellscan be attributed to elevated ROS production or an impairedendogenous antioxidant defense system leading to oxidativedamage [7 8] The vulnerability of proliferating myoblasts tooxidative damage will affect muscle regeneration and con-tributes to the development of sarcopenia suggesting thatoxidative stress satellite cells and sarcopenia are interrelated[6 7]

HindawiOxidative Medicine and Cellular LongevityVolume 2017 Article ID 3868305 17 pageshttpsdoiorg10115520173868305

2 Oxidative Medicine and Cellular Longevity

Oxidative stress in aged skeletal muscle can cause oxida-tive damage in cells manifested as damaged DNA lipid per-oxidation and protein carbonylation [9 10] In muscle fibersfree radicals can be produced intrinsically by mitochondriaand regulate fundamental signaling pathways in skeletalmuscle The presence of reactive oxygen species (ROS) orreactive nitrogen species (RNS) can be counteracted bythe antioxidant defense system which includes antioxidantenzymes vitamins and glutathione resulting in sustainedredox balance [9] If the antioxidant defense is overwhelmedby excess ROS or RNS oxidative stress occurs which leads tomuscle injury [8 10]

In addition to the existing oxidative stress during aginginsufficient antioxidant intake among the elderly can con-tribute to the occurrence of sarcopenia [11] Low antioxidantlevels in older individuals were associated with poor musclestrength and low physical performance and can cause frailtyin the elderly [12 13] An in vivo study demonstratedthat vitamin E deficiency caused poor muscle performanceand accelerated aging development [14] Hence introducingantioxidants such as vitamin E could be a relevant strategyto delay sarcopenia progression however more studies areneeded [15]

Vitamin E is a lipid-soluble vitamin with two subclassestocopherols and tocotrienols [16] A previous study reportedthat 120572-tocopherol was able to repair laser-induced disruptedmyoblast membranes indicating a therapeutic effect forvitamin E in the muscle [17] However the less-exploredsubtype of vitamin E is the class of tocotrienols Similar to120572-tocopherol a potential therapeutic effect of tocotrienol-rich fraction (TRF) was proposed owing to its reversal effecton stress-induced presenescence (SIPS) model of myoblasts[18] In our laboratory we also found that TRF was supe-rior to 120572-tocopherol in ameliorating replicative senescence-related aberration and promoting myogenic differentiation[19] Thus it would be of interest to elucidate the effects oftocotrienols on the dynamics of oxidative status in senes-cent myoblasts Therefore the aims of this study were toinvestigate the effects of tocotrienol-rich fraction (TRF) inreestablishing oxidative status during replicative senescenceof myoblasts and to compare these effects with other antiox-idants such as 120572-tocopherol (ATF) and N-acetyl-cysteine(NAC) in young presenescent and senescent myoblastsfollowed by the measurement of cell viability and apoptosisas the final outcomes of antioxidant treatment

2 Materials and Methods

21 Cell Culture HumanSkeletalMuscleMyoblasts (HSMM)were purchased from Lonza (Walkersville MD USA)Briefly myoblasts were cultured in Skeletal Muscle BasalMedium (SkBM) that was supplemented with human epi-dermal growth factor fetal bovine serum dexamethasoneL-glutamine and gentamicin sulfateamphotericin B (LonzaWalkersville MD USA) Cells were cultivated at 37∘C in ahumid atmosphere containing 5 CO

2 The myoblasts then

underwent serial passaging The number of divisions wascalculated for each passage using the formula ln(119873119899) ln 2where N is the number of cells at the harvest stage and

n is the number of cells at the seeding stage [20] Whencells reached replicative senescence they were unable toproliferate within 10 days in culture Myoblasts were dividedinto 3 different stages young (lt15 cell divisions) presenescent(18-19 cell divisions) and senescent (gt20 cell divisions)based on their decreasing proliferative capacity which wasrepresented by hyperbolic proliferative lifespan curve anddiminishing percentage of BrdU incorporationThe presenceof senescent cells was confirmed by SA-120573-gal staining [19]

22 Antioxidants Tocotrienol-rich fraction (TRF) was pur-chased from Sime Darby Sdn Bhd Selangor Malaysia (TRFGold TRI E 70) while alpha-tocopherol (ATF)was a gift fromthe Malaysian Palm Oil Board (MPOB) (Selangor Malaysia)Both vitamin E subclasses are palm oil-derived TRF consistsof 120572-tocotrienol (ATT 2689) 120573-tocotrienol (BTT 364)120574-tocotrienol (GTT 3166) 120575-tocotrienol (DTT 1366)and 120572-tocopherol (ATF 2415) Briefly stock solutions ofTRF were freshly prepared in 100 ethanol (1 1) and keptat minus20∘C for no longer than one month A similar processwas performed for ATF TRF and ATF were then incubatedovernight with fetal bovine serum at 37∘C before use Thenboth TRF and ATF were diluted in culture medium and usedat a final concentration of 50 120583gmL [19] N-Acetyl-cysteine(NAC) was purchased from Sigma-Aldrich (St Louis USA)NAC was freshly prepared in culture medium to the desiredfinal concentration A dosage of 10mgmLNACwas used forsubsequent experiments which was determined using a cellviability assay (Supplemental 1A in Supplementary Materialavailable online at httpsdoiorg10115520173868305) Adosage of 25 120583gmL of TRF or ATF with 10mgmL NACwas used for combined treatment of TRF and NAC (TRF+ NAC) and combination of ATF and NAC (ATF + NAC)(Supplemental 1B and C)

23 Cell Viability The optimal concentrations of NAC thecombination of TRF and NAC (TRF + NAC) and the combi-nation of ATF andNAC (ATF+NAC)were determined usinga cell viability assay (Supplemental 1)The effects of H

2O2and

antioxidants were also determined using cell viability assayA CellTiter 96 Aqueous Nonradioactive Cell ProliferationAssay (MTS Promega Madison USA) was used accordingto the manufacturerrsquos instructions Cells were incubated withseveral concentrations of antioxidants for 24 h and 45min forH2O2 After that the treatments were replaced by MTS for

another 2 h of incubation The absorbance of MTS formazanwas measured at 490 nm with a microtiter plate reader(VersaMax Molecular Devices USA) The optimum dosageof treatments was used for subsequent experiments

24 Analysis of Cell Morphology In brief cells were platedand fixed with cold ethanol in 120583-Slide 8 wells (ibidi Martin-sried Germany) followed by incubation with an anti-Desminantibody (dilution 1 50) (D33 Dako Produktionsvej Den-mark) and Alexa Fluor 488 goat anti-mouse (dilution 1 500)(Molecular Probes Eugene OR USA) Nuclei were visual-ized using Hoechst 33342 (Molecular Probes Eugene ORUSA) Slides were then observed under a Leica TCS SP5 II

Oxidative Medicine and Cellular Longevity 3

Confocal Laser Scanning Microscope (Leica MicrosystemsWetzlar Germany)

25 Senescence-Associated 120573-Galactosidase (SA 120573-gal) Stain-ing SA 120573-gal was evaluated using a Senescent Cell Histo-chemical Staining Kit (Sigma-Aldrich St Louis MissouriUSA) Cells were stained according to the manufacturerrsquosinstructions After the cells were incubated in staining solu-tion for 8 h at 37∘C in the absence of CO

2 at least 100 cells

were observed under the microscope and the percentage ofblue stained cells was calculated

26 Assessment of the Intracellular Production of ReactiveOxygen Species (ROS) ROS generation in myoblasts wasdetermined using two dyes dihydroethidium (DHE) and 5-(and-6)-carboxy-2101584071015840-dichlorodihydrofluorescein diacetate(carboxy-H

2DCFDA) (Molecular Probes EugeneORUSA)

The fluorescence of DHE indicated oxidation by superoxideanions while carboxy-H

2DCFDA is oxidized by hydro-

gen peroxide (H2O2) peroxynitrite or hydroxyl radicals

Superoxide anions may contribute to carboxy-H2DCFDA

oxidation albeit to a lesser degree Briefly myoblasts wereincubated in 20120583MDHE and 40 120583Mcarboxy-H

2DCFDA for

45min After that cells were washed with PBS and recoveredinmedium for 30minThen wemeasured the intensity of thefluorescence using a flow cytometer (CytoFLEX BeckmanCoulter Pasadena CA USA) with the 58542 bandpassand 52540 bandpass channels The percentage of cells thatwas positively labeled with a particular dye was reportedindependently For observation the same staining protocolwas applied followed by visualization of the nuclei usingHoechst 33342 (Molecular Probes Eugene OR USA) Cellswere then observed under a fluorescence microscope (EVOSFL digital inverted microscope Thermo Fisher ScientificUSA)

27 Assessment of Lipid Peroxidation Lipid peroxidation inmyoblasts was measured using a dye C11-BODIPY (581591) (Molecular Probes Eugene OR USA) which is alipid peroxidation sensor that can shift from red to greenfluorescence emission upon oxidation of the polyunsaturatedbutadienyl segment of the fluorophore Briefly myoblastswere incubated in 10 120583MC11-BODIPY for 30min After thatcells were washed with PBS trypsinized and reconstitutedin PBS and the oxidized BODIPY was quantitated usinga flow cytometer (CytoFLEX Beckman Coulter PasadenaCA USA) with the 52540 bandpass channel while reducedBODIPY was quantitated with 58542 bandpass channelThe percentage of cells that was positively labeled witheither oxidized or reduced BODIPY was obtained and theratio of these percentages (oxidizedreduced BODIPY) wasreported For observation the same staining protocol wasapplied followed by visualization of the nuclei using Hoechst33342 (Molecular Probes Eugene OR USA) Cells were thenobserved under a fluorescent microscope (EVOS FL digitalinverted microscope Thermo Fisher Scientific USA)

28 Cellular Uptake of Vitamin E Vitamin E extractionwas performed according to the protocol by Mazlan et al[22] After trypsinizing and counting the cells 50mgmLbutylated hydroxytoluene (BHT) was added to stop autooxi-dation The hexane layer of supernatant was then collectedvacuum-dried and stored at minus80∘C before analysis withHPLC A total of 100 120583L hexane was added before furtherdilution with hexane for HPLC analysis The uptake ofvitamin E was analyzed using an HPLC fluorescence detec-tor (ExEm 294 nm330 nm) (RF-10A Shimadzu Japan)A TRF standard was used and the concentrations of 120572-tocopherol (ATF)120572-tocotrienol (ATT)120573-tocotrienol (BTT)120574-tocotrienol (GTT) and 120575-tocotrienol (DTT) uptake in cellswere calculated in 120583gmL per million cells

29 Determination of Antioxidant Enzymes at the Transcrip-tional Level Total RNA was extracted using TRI reagentand polyacryl carrier (Molecular Research Center Inc OhioUSA) For gene expression determination quantitative real-time RT-PCR (qRT-PCR) was usedThe expression of SOD1SOD2 CAT and GPX1 mRNA was quantitatively analyzedusing KAPA SYBR FAST One-Step qPCR kit (Kapa Biosys-tems Boston Massachusetts USA) For RT-PCR 400 nMof each primer was used and the primer sequences areshown in Table 1 [21] The master mix was prepared andPCR reactions were carried out in a Bio-Rad iQ5 Cycler(Hercules CA USA)The program included cDNA synthesisfor 5min at 42∘C predenaturation for 4min at 95∘C andPCR amplification for 40 cycles of 3 sec at 95∘C and 20 sec at60∘C These reactions were followed by a melt curve analysisof each targeted gene The melt curve analysis of each pair ofprimers and agarose gel electrophoresis that was performedon the PCR products were used to determine the primerspecificity (Supplemental 2) The expression level of eachtargeted gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) The relative expressionvalue (REV) was calculated using the 2minusΔΔCt method ofrelative quantification and the following equation

REV = 2Ct value of GAPDHminusCt value of the gene of interest (1)

210 Activities of Antioxidant Enzymes Theactivities of threeantioxidant enzymes superoxide dismutase (Sod) catalase(Cat) and glutathione peroxidase (Gpx) were determinedThese enzymes were extracted in PBS by sonication followingthe 24-hour treatments

A Sod assay was performed according to Beyer Jr andFridovich [23] In brief substrate solution was freshly pre-pared bymixing L-methionine nitro blue tetrazolium (NBT)1 Triton-X and PBS pH 78 (Sigma St Louis USA) Then20120583L of enzyme extract and 10 120583L of 4mg100mL riboflavin(Sigma St Louis USA) were added before incubation under20-W Sylvania GroLux lamps in a cupboard for 7minAbsorbance was measured using a UVVIS spectrophotome-ter (Shimadzu Kyoto Japan) at 560 nm Sod specific activitywas expressed in mUmg of protein

A Cat assay was carried out using the method describedby Aebi [24] Enzyme extract was added to a quartz cuvetteThe reaction was started by adding 30mM H

2O2(Merck


Table 1 The primer sequences of SOD1 SOD2 CAT and GPX1 [21]

Gene Forward Reverse Product sizeSOD1 GAAGGTGTGGGGAAGCATTA ACATTGCCCAAGTCTCCAAC 174SOD2 CGTCACCGAGGAGAAGTACC CTGATTTGGACAAGCAGCAA 313CAT CGTGCTGAATGAGGAACAGA AGTCAGGGTGGACCTCAGTG 119GPX1 CCAAGCTCATCACCTGGTCT TCGATGTCAATGGTCTGGAA 127GAPDH TCCCTGAGCTGAACGGGAAG GGAGGAGTGGGTGTCGCTGT 218

Darmstadt German) and absorbance was measured kinet-ically for 30 seconds using a UVVIS spectrophotometer(Shimadzu Kyoto Japan) at 240 nm Cat-specific activity wasexpressed in mUmg of protein

A Gpx assay was carried out according to Paglia andValentine [25] Substrate solution was freshly prepared bymixing reduced glutathione PBS at pH 70 sodium azidethe reduced form of nicotinamide adenine dinucleotidephosphate (NADPH) and glutathione reductase (1 UmL)(Sigma St Louis USA) Enzyme extract was added to thesubstrate solution and the reaction was started by addingH2O2(Merck Darmstadt German) The conversion of

NADPH toNADP+ wasmeasured kinetically using aUVVISspectrophotometer (Shimadzu Kyoto Japan) at 340 nm for5min Gpx-specific activity was expressed in mUmg ofprotein

The total protein was extracted using lysis buffer andits concentration was determined using a Bio-Rad ProteinAssay (Hercules CA USA) at 595 nm with a microtiter platereader (VersaMax Molecular Devices USA) The proteinconcentration was used to normalize the enzyme activity

211 Measurement of Reduced to Oxidized Glutathione (GSHGSSG) Ratio The GSHGSSG ratio was determined usinga GSHGSSG-Glo Assay Kit (Promega Madison WI)which is based on the firefly luciferase reaction Based onthe manufacturerrsquos instructions both the GSH and GSSGluminescent reaction schemes were performed andmeasuredusing a microplate reader with an integration time of 1 swell(Infinite 200 Tecan USA)The ratio was calculated directlyfrom Net RLU as stated in the technical manual

212 Assessment of Apoptotic Events Apoptosis profiles weredetermined using an Annexin V-FITC Apoptosis DetectionKit II (BD Pharmingen CA USA) according to the man-ufacturerrsquos instructions Two dyes were used Annexin V-FITC which was detected by FL1 and propidium iodide (PI)which was detected by FL3 The cells were analyzed with aFACS Calibur flow cytometer (Becton Dickinson CA USA)The percentage of cells that were negatively stained withboth dyes (FITCminusvePIminusve) was reported as viable cells whilepercentage of cells that were positively stained with AnnexinV-FITC only (FITC+vePIminusve) or both Annexin V-FITC andPI (FITC+vePI+ve) was reported as early and late apoptoticcells respectively

213 Statistical Analysis Statistical analyses were performedusing SPSS 220 software (IBM NY USA) All of the data

are reported as the means plusmn standard deviation (SD) fromat least three replicates For all of the tests 119901 lt 005 wasconsidered statistically significant To determine the signif-icance between two treatment groups comparisons weremade using an independent 119905-test while ANOVAwas used toanalyze multiple groups followed by a post hoc Tukeyrsquos HSDor LSD (if equal variance was assumed) and Dunnettrsquos T3 (ifequal variance was not assumed) tests

3 Results

31 Effects of Antioxidants on Senescent Myoblasts To deter-mine whether antioxidant properties alone are sufficientto ameliorate senescent myoblasts in our study we testeda synthetic antioxidant N-acetyl-cysteine (NAC) and itscombination with TRF and ATF besides treatment with TRFor ATF alone Previously the beneficial effects of TRF or ATFon senescentmyoblasts were reported where a concentrationof 50120583gmL TRF or ATF alone was able to increase cellviability improve cellular morphology (more spindle-shapedcells were observed) and decrease the total of SA-120573-galstaining positive cells during replicative senescence [19] Inthe present study we found that NACwas not toxic to cells upto the highest concentration used with a 24-hour incubation(Supplemental 1A) Thus 10mgmL of NAC was used in thesubsequent experiments Different concentrations of TRF orATF in combination with NACwere tested using cell viabilityassay (Supplemental 1B and 1C) in which a concentrationof 25 120583gmL TRF or ATF was used with 10mgmL NAC inthe subsequent experiment NAC alone and in combinationwith TRF (TRF + NAC) or ATF (ATF + NAC) significantlyimproved the cellular morphology of senescent myoblasts(cells became spindle-shaped) and reduced the percentage ofcells positive for a senescence biomarker (SA-120573-gal staining)(Figures 1(a) and 1(b))

32 Effects of Antioxidants on ROS Generation during Replica-tive Senescence To elucidate the effects of aging on theoxidative status of myoblasts we expanded cells in cultureuntil replicative senescence was achieved The generationof ROS was observed at all stages of cell culture usingcarboxy-H

2DCFDA (in green H

2O2 peroxynitrite hydroxyl

radicals etc) and DHE (in orange superoxide anion) (Fig-ures 2(a)ndash2(c)) The presence of carboxy-H

2DCFDA grad-

ually increased in untreated control of young to senescentmyoblasts (Figures 2(a)ndash2(c)) However similar observa-tions were not observed with DHE Quantitative analysisshowed that the amount of intracellular ROSwas significantly


TRF + NAC ATF + NAC

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minus100

10203040506070

(b)

Figure 1 Protective effects of NAC alone and in combination with TRF and ATF on senescent myoblasts (a) More spindle-shaped cellsobserved in senescent myoblasts after NAC TRF + NAC and ATF + NAC treatment which were similar to the effect of TRF and ATFtreatment alone on senescent myoblasts Scale bar 100120583m (b) The percentage of SA-120573-gal positive cells decreased in all treatment groupsaDenoting 119901 lt 005 significantly different compared to the young control b119901 lt 005 significantly different compared to the presenescentcontrol and c119901 lt 005 significantly different compared to the senescent control Data are presented as the mean plusmn SD 119899 = 3

increased in presenescent and senescentmyoblasts comparedto young myoblasts (119901 lt 005) and senescent myoblastshad the highest levels of ROS suggesting that senescent cellsexperience more severe oxidant insults compared to youngand presenescent cells (Figure 2(d))

Both TRF and ATF treatments reduced the amountof intracellular ROS in senescent myoblasts which wasindicated by the decline in fluorescence intensity as well asthe total number of positively stained cells (Figure 2(c))The treatments were likely to diminish ROS in young andpresenescent cells as well (Figures 2(a) and 2(b)) Quanti-tative analysis showed that both TRF and ATF significantlydiminished intracellular H

2O2generation in presenescent

and senescent myoblasts (119901 lt 005) (Figure 2(d)) Howeveronly ATF-treated senescentmyoblasts had significantly lowerintracellular superoxide anion levels compared to untreated

senescent control cells (119901 lt 005) (Figure 2(d)) Presenescentcells treated with TRF + NAC and ATF + NAC exhibitedsignificantly lower ROS generation (119901 lt 005) (Figure 2(d))In senescent myoblasts only TRF + NAC treatment causeda significant decrease in ROS generation (119901 lt 005)(Figure 2(d)) A similar reduction in free radical generationwas not observed in cells treated with NAC alone indicatingthat the antioxidant effects observed were the result of TRFand ATF actions The presence of ROS in all treatmentgroups in young presenescent and senescent myoblasts canbe visualized in Figures 2(a) 2(b) and 2(c) respectively

To confirm the effects of ROS on normal myoblasts wemeasured the cell viability immediately after a short-term ofH2O2insult Our results showed decreased cell viability at all

stages of aging after 45min of incubation with 1mM 15mM2mM and 25mM H

2O2 119901 lt 005 (Figure 2(e)) The steady


Hoe

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Control TRF ATF NAC TRF + NAC ATF + NAC

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Figure 2 Continued


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ell v

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lowast

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H2O2 concentration (mM)

(e)

Figure 2 Effects of antioxidants treatment on the presence of ROS inmyoblast cells (a) Young (b) presenescent and (c) senescent myoblastslabeled with 20 120583MDHE and 40 120583M carboxy-H

2DCFDA to detect the presence of ROS which were observed under fluorescence microscope

(magnification 200x scale bar 200 120583m) TRF and ATF diminished the presence of ROS in senescent cells (d) Quantitative analysis of ROSgeneration in myoblasts The percentage of positively stained cells was significantly increased in presenescent and senescent myoblastshowever the percentage was decreased in TRF- ATF- and TRF + NAC-treated senescent myoblasts (e) A gradual decrease in cell viabilitywith increasing concentrations of H

2O2was observed indicating the vital role of redox balance maintenance in cells aDenoting 119901 lt 005

significantly different compared to young control b119901 lt 005 significantly different compared to presenescent myoblasts c119901 lt 005significantly different compared to senescent myoblasts and lowastyen119901 lt 005 significantly different compared to 0mM Data are presentedas the mean plusmn SD 119899 = 3

decline in cell viability with increasing H2O2concentration

exposure may indicate the vital role of a maintained redoxbalance in muscle cells

33 Effects of Antioxidants on Lipid Peroxidation during Repli-cative Senescence To determine the oxidative damage inmyoblasts lipid peroxidation levels were measured with C11-BODIPY (581591) a sensitive fluorescent reporter for lipidperoxidationThe total number of cells that underwent a shiftfrom red to green was gradually augmented from young tosenescent cells (Figures 3(a)ndash3(c)) In senescent myoblaststhe amount of cells with lipid peroxidation which wasindicated by the percentage of cells with oxidized BODIPY(in green) was increased as represented in the right-shiftedfluorescence intensity histogram (Figure 3(d)) Quantitativeanalysis showed that the ratio of cells with oxidized BODIPYto reduced BODIPY (in red) (oxidizedreduced BODIPYratio) was significantly increased in presenescent and senes-cent myoblasts compared to young myoblasts (119901 lt 005)(Figure 3(e))

Evaluation of lipid peroxidation levels which was per-formed after antioxidant treatment showed that both TRFandATF reduced the level of lipid peroxidation in young pre-senescent and senescent myoblasts (Figures 3(a) 3(b) 3(c)and 3(e))The number of oxidized BODIPY-positive cells wasdecreased in both TRF- andATF-treated senescentmyoblasts(Figure 3(c)) Statistical analysis of the oxidizedreducedBODIPY ratio revealed that both TRF and ATF significantlyreduced lipid peroxidation in presenescent and senescentmyoblasts (119901 lt 005) (Figure 3(e)) Surprisingly NACincreased oxidizedreduced BODIPY ratio in presenescentmyoblasts (119901 lt 005) (Figure 3(e)) However senescent

cells treated with ATF + NAC and TRF + NAC showedsignificantly decreased lipid peroxidation (oxidizedreducedBODIPY ratio) compared to untreated control senescentmyoblasts (119901 lt 005) (Figure 3(e))

34 Cellular Uptake of Vitamin E To validate the cellularuptake of vitamin E we determined the concentration ofvitamin E isomers (ATF ATT BTT GTT and DTT) in allgroups of cells (Figures 4(a) and 4(b)) Young presenescentand senescent TRF-treatedmyoblasts showed the presence of5 vitamin E isomers while a significantly inferior numberof isomers were found in control cells (Figure 4(a)) Asignificantly higher concentration of ATF was observed inyoung presenescent and senescent ATF-treated myoblastscompared to untreated cells (119901 lt 005) (Figure 4(a)) TheNAC-treated cells contained the lowest concentrations ofvitamin E isomers compared to its combination with TRF orATF group (Figure 4(b)) TRF + NAC-treated cells containedhigh levels of all 5 isomers of vitamin E while ATF + NACcells only contained a high ATF concentration (Figure 4(b))

35 TRFTreatmentModulatesAntioxidantCapacity Themod-ulation of antioxidant capacity by TRF ATF NAC TRF +NAC and ATF + NAC during the replicative senescenceof myoblasts was investigated by determining the mRNAexpression of antioxidant enzymes (SOD1 SOD2 CAT andGPX1) and activities of antioxidant enzymes (Sod Catand Gpx) as well as the ratio of GSHGSSG in youngpresenescent and senescent myoblasts (Figures 5 and 6)Overall TRF treatment was more effective in modulatingantioxidant enzymes expression especially at transcriptionallevel compared to other antioxidants in senescent myoblasts


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Figure 3 Continued



1519 2832 3228

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(e)

Figure 3 Effects of antioxidants treatment on lipid peroxidation levels of myoblasts (a) Young (b) presenescent and (c) senescent myoblastslabeled with C11-BODIPY (581591) to detect lipid peroxidation which were observed under fluorescence microscope (magnification 200xscale bar 200 120583m) TRF and ATF treatment effectively reduced lipid peroxidation (d) Representative histogram of myoblasts based on theoxidized BODIPY (in green 52540 bandpass channel) (e) Ratio of oxidizedreduced BODIPY virtually representing lipid peroxidationin myoblast cells aDenoting 119901 lt 005 significantly different compared to young control b119901 lt 005 significantly different compared topresenescent myoblasts c119901 lt 005 significantly different compared to senescent myoblasts Data are presented as the mean plusmn SD 119899 = 3

There was no significant change in SOD1 mRNA expres-sion levelswith aging and antioxidant treatment (Figure 5(a))However SOD2 mRNA expression in the presenescent con-trol was significantly increased compared to the youngcontrol (119901 lt 005) (Figure 5(b)) TRF treatment upregulatedSOD2 mRNA expression in young presenescent and senes-cent myoblasts compared to their corresponding untreatedcontrols (119901 lt 005) (Figure 5(b)) Furthermore our resultsshowed that only TRF + NAC modulated the expressionof SOD2 mRNA while NAC and ATF + NAC did notmodulate any antioxidant enzymes at the transcriptional level(Figure 5(b)) suggesting that TRF exerted a modulatoryeffect at the transcriptional level Moreover Sod activitywas increased in TRF-treated presenescent and senescentmyoblasts compared to their untreated controls (119901 lt 005)(Figure 6(a)) In senescent myoblasts treatment with TRF +NAC significantly increased the activity of Sod compared tothe senescent control (119901 lt 005) However similar increases

in Sod activity were observed in youngmyoblasts treatedwithATF+NACwhile in presenescentmyoblasts treatmentwithNAC TRF + NAC and ATF + NAC decreased Sod activity

In the presenescent control CAT mRNA expression wassignificantly higher than the young control 119901 lt 005 (Fig-ure 5(c)) In contrast Cat activity in the presenescent controlwas lower than in the young control (119901 lt 005) (Figure 6(b))Treatment with either TRF or ATF upregulated CAT mRNAexpression in senescent myoblasts compared to untreatedcontrols (119901 lt 005) (Figure 5(c)) while only TRF increasedCat activity in senescent myoblasts and ATF increasedCat activity in presenescent myoblasts (Figure 6(b)) TRF+ NAC significantly increased CAT mRNA expression inyoung myoblasts in comparison to their untreated controls(Figure 5(c)) Treatment with NAC TRF + NAC and ATF+ NAC increased Cat activity in presenescent myoblasts (119901 lt005) while in youngmyoblasts Cat activitywas significantlyincreased with TRF + NAC and ATF + NAC treatment


01020304050607080

Control TRF ATF Control TRF ATF Control TRF ATFYoung Presenescent Senescent

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)

Figure 4 Cellular uptake of vitamin E isomers bymyoblasts (a)Untreated control TRF- andATF-treated young presenescent and senescentmyoblasts Cellular uptake of 5 vitamin E isomers was significantly higher in TRF-treated myoblasts (b) NAC- TRF + NAC- and ATF +NAC-treated young presenescent and senescent myoblasts Cellular uptake of 5 vitamin E isomers was significantly higher in TRF + NAC-treated myoblasts aDenoting 119901 lt 005 significantly different compared to the young control b119901 lt 005 significantly different compared tothe presenescent control and c119901 lt 005 significantly different compared to the senescent control Data are presented as themean plusmn SD 119899 = 3

(Figure 6(b)) However only ATF + NAC-treated senescentmyoblasts demonstrated increased Cat activity

No significant changes were observed in GPX1 mRNAexpression with aging in myoblasts even though expressionof this gene was modulated by TRF which was evident by theupregulation of its mRNA expression in young presenescentand senescent cells (119901 lt 005) (Figure 5(d)) Howevera similar pattern of expression was not observed for theenzyme activity of Gpx Gpx activity was significantly higherin the senescent control compared to both the young andpresenescent controls (119901 lt 005) (Figure 6(c)) while bothTRF- and ATF-treated senescent myoblasts exhibited lowerenzyme activities compared to the senescent control (119901 lt005) (Figure 6(c)) Gpx activity levels were increased in TRF+ NAC-treated presenescent myoblasts but decreased in ATF+ NAC-treated presenescent myoblasts (Figure 6(c)) NAC-TRF + NAC- and ATF + NAC-treated senescent myoblastsexhibited significantly lower Gpx activity compared to theuntreated senescent control A similar decrease in Gpxactivity was observed in young myoblasts treated with NACalone

The GSHGSSG ratio is always used as an indicator forantioxidant capacity However in our study there was nosignificant change observed in the GSHGSSG ratio betweenyoung presenescent and the senescent control (Figure 6(d))In young and presenescent myoblasts TRF treatmentreduced the ratio significantly compared to untreated con-trols (119901 lt 005) (Figure 6(d)) Determination of the GSHGSSG ratio in myoblasts showed that treatment with NACalone in young presenescent and senescent myoblastsresulted in a significantly increased GSHGSSG ratio (119901 lt005) (Figure 6(d)) A similar increase in theGSHGSSG ratiowas observed in presenescent cells treated with TRF + NAC

36 Effects of Antioxidants on Cell Viability and ApoptosisProfile To evaluate the beneficial effects of TRF ATF NACand their combinations on oxidative status and its final out-come cell viability (Figure 7(a)) and apoptosis profile (Fig-ures 7(b)ndash7(d)) weremeasured Our results showed that TRFATFNAC and their combinations significantly increased thenumber of viable cells in young presenescent and senescent


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)

Figure 5 Effects of antioxidants treatment on antioxidant enzymes mRNA expression in young presenescent and senescent myoblasts (a)SOD1 mRNA (b) SOD2 mRNA (c) CAT mRNA and (d) GPX1 mRNA expression aDenoting 119901 lt 005 significantly different compared tothe young control b119901 lt 005 significantly different compared to the presenescent control c119901 lt 005 significantly different compared to thesenescent control and lowast119901 lt 005 significantly different compared to TRF-treated senescent myoblasts Data are presented as the mean plusmn SD119899 = 3

myoblasts compared to their corresponding untreated con-trols (Figure 7(a))

Apoptotic changes were determined by Annexin V-FITCstaining Figure 7(b) shows the dot plot of FITC-AnnexinVPI double staining as determined by flow cytometry anal-ysis The three quadrants represent different cell conditionsthe upper right quadrant (R1) indicates nonviable and lateapoptotic cells (FITC+vePI+ve) the lower left quadrant (R2)indicates viable cells (FITCminusvePIminusve) and the lower rightquadrant (R3) indicates early apoptotic cells (FITC+vePIminusve)which is demonstrated by Annexin V binding and cytoplas-mic membrane integrity The quantitative data for the viablecells is shown in Figure 7(c) The percentage of viable cellsin the presenescent and senescent controls was significantlylower than in the young control (119901 lt 005) Treatment withTRF ATF TRF + NAC or ATF + NAC was able to increasethe number of viable cells during replicative senescencesignifying the protective role of antioxidant treatment againstcell death A similar increase in cell viability was observed inpresenescent myoblasts treated with NAC TRF + NAC orATF + NAC (119901 lt 005)

Increases in early and late apoptotic events were observedin senescent myoblasts compared to young and presenescentcells (119901 lt 005) (Figure 7(d)) Treatment with TRF ATFNAC TRF + NAC or ATF + NAC significantly reducedthe number of early apoptotic cells in senescent myoblastscompared to untreated controls (119901 lt 005) Only TRF TRF

+ NAC and ATF + NAC treatment significantly decreasedthe number of cells undergoing late apoptotic events (119901 lt005) Early and late apoptotic events were also increased inpresenescent myoblasts and were reduced by TRF + NAC orATF + NAC treatment (119901 lt 005)

4 Discussion

Vitamin E is well known for its free radical-scavengingcapacity which plays an important role in antioxidant de-fense mechanisms The lesser known form of vitamin Etocotrienols has been reported to possess greater antioxidanteffects and better membrane penetration ability comparedto tocopherols [26 27] These features contribute to theirefficient uptake by targeted cells in addition to exhibitinghigher scavenging power due to active recycling [26 28]Thepresent study has demonstrated the effectiveness of TRF abroad mixture of vitamin E in combating oxidative stressand enhancing antioxidant defense mechanisms in senescentmyoblasts resulting in the improvement of senescence-associated oxidative stress as evidenced by decreased pro-grammed cell death and increased cell viability

In brief increased oxidative stress as a result of redoximbalance during aging leads to increased susceptibility ofsatellite cells to apoptosis and affects muscle regeneration[6 8 29 30]The findings of this study showed that ROS gen-eration was significantly increased in senescent myoblasts


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)

Figure 6 Effects of antioxidants treatment on antioxidant enzymes activities and GSHGSSG ratio in young presenescent and senescentmyoblasts (a) Sod enzyme activity (b) Cat enzyme activity (c) Gpx enzyme activity and (d) GSHGSSG ratio aDenoting 119901 lt 005significantly different compared to the young control b119901 lt 005 significantly different compared to the presenescent control and c119901 lt 005significantly different compared to the senescent control Data are presented as the mean plusmn SD 119899 = 3

resulting in higher levels of oxidative damage as indicated byelevated lipid peroxidation Increased oxidative stress duringreplicative senescence has been reported to be similar to theconditions in satellite cells derived from aged individuals [810] Moreover elevated ROS in myoblasts endangers cellularendurance as shown in our study We found that the numberof viable cells decreased with increasing exogenous H

2O2

concentration [31]The presence of free radicals in cells can be counteracted

by antioxidant defense systems However antioxidant capac-ity decreases with advancing age resulting in the accumu-lation of free radicals which threaten cell viability [8] Asreported by Fulle and his coworkers [8] the levels of theantioxidant enzymes Cat and glutathione transferase in satel-lite cells isolated from the elderly were drastically decreasedcompared to cells from young donors SOD1 and GPX1mRNA expression were not significantly different across thecell stages However SOD2 and CAT mRNA expression wereupregulated in presenescent myoblasts which was similar toSod activity in a previous study [32] Additionally a declinein Cat activity was observed in presenescent myoblasts com-pared to young myoblasts Because antioxidant enzymes areregulated in response to oxidative stress [33] we postulatedthat presenescent myoblasts were attempting to compensatefor the decreased Cat levels by upregulating CAT mRNAexpression to overcome the progressive increase in oxidativestress Previous study reported that there is a compensatorymachinery in antioxidant defense systems which maintains

the integrity of muscle [34] However we found that senes-cent cells were less responsive to increased oxidative stressThe levels of SOD2 mRNA in senescent cells were lowercompared to presenescent cells The expression of SOD2mRNA was critical because lack of gene SOD2 can causemitochondrial damage and lifespan shortening inDrosophila[35] On the other hand Gpx activity was increased insenescent myoblasts compared to young and presenescentmyoblasts A previous study also reported enhanced Catand Gpx activity during aging which could be an adaptivemechanism to the elevated H

2O2levels [36] but the response

may be inadequate to counteract the existing ROSOur study also reports on the nonenzymatic antioxidants

in senescent myoblasts glutathione [9] The GSHGSSGratio in our study remained unchanged at all ages whichis similar to data reported previously suggesting that thereis no alteration in GSH membrane transportation duringaging [37] Therefore based on the intracellular ROS levelslipid peroxidation and enzymatic antioxidants our resultsindicated that senescentmyoblasts experience oxidant insultsas a result of a less effective antioxidant defense system whichbarely counteracted the elevated levels of cellular senescence-associated oxidative stress

The vitamin E concentration in cells increased withvitamin E treatment (TRF orATF alone) particularly in TRF-treated myoblasts which contained all five isomers that weretested NAC treatment did not affect vitamin E uptake by thecells as shown by higher levels of vitamin E in myoblasts


Young Presenescent SenescentC

ell v

iabi

lity

()

ControlTRFATF

NACTRF + NACATF + NAC

lowastlowast

lowastlowast lowast lowast lowast

lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)

Presenescent control R1

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)

(early apoptosis)Viable cells100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls

Early apoptosisLate apoptosis

a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)

Figure 7 Effects of antioxidants treatment on cell viability and apoptosis (a) The total number of viable cells after antioxidant treatment ofyoung presenescent and senescent myoblasts (b) Dot plot of Annexin V-FITC and PI double staining Each quadrant represents differentcell conditions which are late apoptotic cells (FITC+vePI+ve right upper quadrant R1) viable cells (FITCminusvePIminusve left lower quadrant R2)and early apoptotic cells (FITC+vePIminusve right lower quadrant R3)The cells shifted from the left lower quadrant to the right lower and upperquadrants when transitioning from young to senescent status (c) The percentage of viable cells (d) The apoptosis profile of myoblasts inresponse to all treatments during early and late apoptosis events lowastDenoting 119901 lt 005 significantly different compared to respective controlgroups a119901 lt 005 significantly different compared to young control b119901 lt 005 significantly different compared to the presenescent controland c119901 lt 005 significantly different compared to the senescent control Data are presented as the mean plusmn SD 119899 = 3


treated with TRF + NAC and ATF + NAC compared tocells treated with NAC alone In brief vitamin E can actas a nonenzymatic antioxidant to combat oxidative stress[9] Thus given its free radical-scavenging power both TRF-treated and ATF-treated senescent myoblasts demonstratedreductions in their intracellular ROS generation and lipidperoxidation levels Findings from a previous animal studyreported a similar reduction in lipid peroxidation levels afterTRF and ATF supplementation [38] revealing the protectiveeffects of vitamin E against oxidative damage TRF has beenreported to improve senescence-associated phenotypes inH2O2-induced myoblasts [18] In another study ATF showed

protective effects inH2O2-inducedmyoblasts [39]Therefore

both TRF and ATF could potentially be used to protect cellsagainst reactive oxidants

Our data showed that TRF regulates the expression ofantioxidant enzymes in young presenescent and senescentmyoblasts SOD2 mRNA expression was upregulated in allTRF-treated cells while Sod enzyme activity was upregulatedin TRF-treated presenescent and senescent myoblasts TheSOD2 mRNA encodes one of the Sod isoforms (MnSod)which is located in mitochondrial matrix [40] Gianni etal [41] suggested that a superoxide related mitochondrialstress is more apparent than cytosolic stress during agingPrevious studies also reported that increased mitochondrialDNA or RNA mutations were correlated with increasingage and abnormality of aged muscle [42 43] Hence age-related oxidative stress is thought to lead to mitochondrialdysfunction which eventually may lead to progressive lossof muscle mass and strength [44] Instead of SOD1 mRNATRF regulated SOD2mRNA thus we hypothesized that TRFmay act on the mitochondria and potentially protect thisorganelle during aging The upregulation of SOD2mRNA byTRF could be a compensatory mechanism to counteract ele-vated oxidative challenges inmitochondria during replicativesenescence and prevent accumulation of oxidative damagethat can trigger cellular aberration Evidence demonstratedthat 120574-tocotrienol potentially protects renal proximal tubularcells from oxidant-induced mitochondria dysfunctional andcellular injury [45] Increased Sod activity with TRF treat-ment further validated the ability of TRF to modulate thedecomposition of superoxide anions to H

2O2[38] However

neither Sod mRNA nor enzyme activity was modulated byATF

In addition to the significant regulation of Sod activityTRF treatment also upregulated CAT and GPX1 mRNA inyoung and senescent myoblasts However at the enzymeactivity level TRF increasedCat activity whereasGpx activitywas reduced in senescent myoblasts Cat and Gpx work inparallel to remove H

2O2in cells [9] Gpx also catalyzes

the elimination of hydroperoxides originated from unsat-urated fatty acids at the expense of reduced glutathione[46] However the protective role of the glutathione redoxcycle is limited at low levels of oxidative stress Duringsevere oxidant insults Cat becomes more substantial [46]Thus increased Cat at the mRNA and enzyme activityrevealed the effectiveness of TRF in enhancing the antiox-idant defense system in senescent myoblasts Because Gpxis involved in hydroperoxide degradation decreased Gpx

in TRF-treated senescent myoblasts may be attributable todecreased lipid peroxidation in cells as reported in thisstudy This explanation can also be applied to ATF-treatedmyoblasts which exhibited decreased Gpx enzyme activityin both the presenescent and senescent stages In additionATF treatment upregulated CAT mRNA in senescent cellsbut at levels that were significantly lower than TRF-treatedcells These findings indicate that TRF potentially improvesthe antioxidant defense system in senescent myoblasts andthis effect is better than that of ATF

NAC is a precursor of cysteine that can sustain theproduction of glutathione an important antioxidant in cells[47] In this study NAC treatment successfully increased theGSHGSSG ratio in all myoblasts NAC can scavenge ROSdirectly [47] In a previous study NAC showed protectiveeffects against dystrophic muscle damage in the mdx mouse[48] and attenuates fatigue during prolonged exercise [49]Our results showed that NAC ameliorated myoblast mor-phology and SA-120573-gal staining during senescence which issimilar to the effects of TRF andATF [19] Similar effects werealso observed in cells treated with combinations of TRF +NAC and ATF + NAC However intracellular ROS and lipidperoxidation levels were not modulated by NAC treatmentalone during senescence The rate constants of NAC inreaction with superoxide anion H

2O2 and peroxynitrite

were comparatively low [47] consequently the influenceof NAC on the dyes used in this study would be limitedIn our study lipid peroxidation levels were not improvedby NAC as supported by a previous study that measuredmalondialdehyde (MDA) as a product of lipid peroxidation inthe mdx mouse [48] As expected both combined treatmentwith TRF +NAC and ATF +NAC lowered lipid peroxidationlevels On the other hand intracellular ROS levels were onlyameliorated byTRF+NAC treatment in senescentmyoblastssuggesting that TRF is better than ATF in scavenging ROSeven at lower concentrations compared to the concentrationused for TRF treatment alone

The effects of NAC on antioxidant enzymes were onlyobserved for Gpx activity in senescent myoblasts althougha previous study showed that NAC was able to increaseantioxidant enzymes in cocaine-induced hepatocytes [50]We found that NAC modulated the antioxidant enzymeactivity in presenescent myoblasts as shown by decreasedSod activity and increased Cat activity with NAC treatmentTreatment of senescent myoblasts with the combinationof TRF + NAC resulted in a stronger antioxidant defenseresponse which involves the upregulation of SOD2 mRNAexpression and the increment in Sod activity as well as adecrease in Gpx activity compared to treatment with NACalone These results were similar to those observed for TRF-treated senescentmyoblasts RegardingATF the combinationof ATF +NAC exerted greater effects on antioxidant enzymescompared to treatment with ATF alone Both Sod activityand Gpx activity decreased whereas Cat activity increasedin presenescent myoblasts with ATF + NAC treatmentIncreased Cat activity and decreased Gpx activity were alsoobserved in ATF + NAC-treated senescent myoblasts Fromthe results both TRF + NAC and ATF + NAC treatmentwere more effective than NAC alone in ameliorating the


antioxidant defense systemwhich is similar to previous studythat showed combination of NAC and vitamin E has bettereffect on gentamycin-induced nephrotoxicity compared toNAC alone [51] Therefore the improved antioxidant defensemechanisms observed in this studymay be attributable to themodulatory effects of vitamin E treatment

Satellite cells play very important roles in regeneratinginjured muscle fibers [1] Despite the availability of satellitecells which decline with increasing age [52 53] they maynot be the sole reason underlying the impaired regenerativeresponse to injury during aging Decreased satellite cell pro-liferative capacity was reported with increased susceptibilityto apoptosis signifying the fact that programmed cell deathmay also play a part in age-related muscle regenerationimpairment [29] Under stressful stimuli more satellite cellsin old animals underwent apoptosis thereby distorting skele-tal muscle regeneration [29] However in a previous studyvitamin E was reported to be able to protect against cell deathinduced by low doses of oxidants [39] In another study itwas reported that antioxidant levels were interrelated withthe regenerative capacity of muscle stem cells [30] In shortviable senescent myoblasts were preserved and the amountof cell death was diminished with TRF treatment whichmay be attributed to the improvement of the oxidative statusin senescent cells This may indicate that TRF amelioratesregenerative capacity during aging in human myoblasts [19]

Adequate supply of vitamin E is essential for musclehealth Conversely vitamin E deficiency can lead to poormuscle performance [12 14] A study showed that the antiox-idant properties alone are insufficient to repair the injuredmyoblasts [17] Lipid-soluble vitamin E can easily diffuseinto the hydrophobic membrane and act as a ldquostabilizerrdquo forlipid membranes to facilitate ROS scavenging activity [17]Accordingly the findings of our study showed that both TRFand ATF produced greater effects than NAC in oxidativedamage prevention However ATF which is a well-knownrepresentative of vitamin E was not as effective as TRF in pro-tecting against replicative senescence in myoblasts AlthoughATF improved the oxidative damage to a similar degreeas TRF TRF is superior in enhancing antioxidant defensemechanism Previous findings showed that TRF-treated ratsexhibited better physical performance and oxidative statusthan ATF-treated rats [38] Unlike ATF TRF is a broad mix-ture of vitamin E that contains all four isomers of tocotrienoland ATF thus it should be more potent in scavenging freeradicalsThedistinctive antioxidant properties of each isomerhave been attributed mainly to their chemical structure[54] For instance the unsaturated isoprenoid side chain oftocotrienols accounts for a higher peroxyl radical-scavengingpotential compared to tocopherols [54] As a result even ata lower concentration TRF is able to improve antioxidantdefense mechanism and ameliorate the oxidative status ofsenescent myoblasts Previous report suggested that at lowconcentrations 120574-tocotrienol can protect cells against H

2O2-

induced apoptosis while a higher concentration is requiredfor ATF to produce the similar outcome [22] Our resultsshowed the modulatory effect of TRF is distinguishablecompared to other treatments Previous findings reportedthat tocotrienols can target a broad range of molecules that

might play a role in aging or degenerative diseases [55]Hence we suggest that TRF is better than ATF inmodulatingantioxidant enzymes particularly at the transcriptional levelto reestablish redox balance in senescent myoblasts

In summary our study highlights the effects of TRF onoxidative status in myoblasts during replicative senescenceThe results of our study showed increased oxidative stress insenescent myoblasts with reduced antioxidant capacity andincreased susceptibility towards programmed cell death orapoptosis which were distinguishable from youngmyoblastsTreatment with TRF in senescent myoblasts resulted indiminished ROS and lipid peroxidation in addition to rein-forcing the antioxidant defense systemby augmenting antiox-idant enzymes levels in senescentmyoblasts which ultimatelymaintained the number of myoblast cells In conclusion TRFis a useful antioxidant that can counteract oxidative stressand improve cellular survival during replicative senescenceof myoblasts and thereby it can potentially be used toameliorate muscle regeneration such as sarcopenia althoughfurther experiments should be carried out

Competing Interests

The authors declare no conflict of interests

Authorsrsquo Contributions

Shy Cian Khor performed the experiments analyzed thedata and drafted the manuscript Wan Zurinah Wan Ngahand Norwahidah Abdul Karim participated in designing thestudy and provided a critical analysis of the manuscriptYasmin AnumMohd Yusof provided a critical analysis of themanuscript Suzana Makpol designed the study interpretedthe data and revised the manuscript All of the authors readand approved the final manuscript

Acknowledgments

This research study was financially supported by UniversitiKebangsaan Malaysia Grants AP-2012-012 and UKM-FF-2013-259 The authors would like to express gratitude toall researchers and staff of the Biochemistry DepartmentFaculty ofMedicine Universiti KebangssanMalaysiaMedicalCentre

References

[1] S B P Charge and M A Rudnicki ldquoCellular and molecularregulation of muscle regenerationrdquo Physiological Reviews vol84 no 1 pp 209ndash238 2004

[2] A Mauro ldquoSatellite cell of skeletal muscle fibersrdquoThe Journal ofBiophysical and Biochemical Cytology vol 9 no 2 pp 493ndash4951961

[3] H Yin F Price and M A Rudnicki ldquoSatellite cells and themuscle stem cell nicherdquo Physiological Reviews vol 93 no 1 pp23ndash67 2013

[4] VMouly AAamiri A Bigot et al ldquoThemitotic clock in skeletalmuscle regeneration disease and cell mediated gene therapyrdquoActa Physiologica Scandinavica vol 184 no 1 pp 3ndash15 2005


[5] A J Cruz-Jentoft J P Baeyens J M Bauer et al ldquoSarcopeniaEuropean consensus on definition and diagnosis report of theEuropean Working Group on Sarcopenia in Older Peoplerdquo Ageand Ageing vol 39 no 4 pp 412ndash423 2010

[6] A Vasilaki and M J Jackson ldquoRole of reactive oxygen speciesin the defective regeneration seen in agingmusclerdquo Free RadicalBiology and Medicine vol 65 pp 317ndash323 2013

[7] B Szczesny G Olah D KWalker et al ldquoDeficiency in repair ofthe mitochondrial genome sensitizes proliferating myoblasts tooxidative damagerdquo PLOS ONE vol 8 no 9 Article ID e752012013

[8] S Fulle S Di Donna C Puglielli et al ldquoAge-dependentimbalance of the antioxidative system in human satellite cellsrdquoExperimental Gerontology vol 40 no 3 pp 189ndash197 2005

[9] E Doria D Buonocore A Focarelli and F Marzatico ldquoRela-tionship between human aging muscle and oxidative systempathwayrdquo Oxidative Medicine and Cellular Longevity vol 2012Article ID 830257 13 pages 2012

[10] G Fano P Mecocci J Vecchiet et al ldquoAge and sex influenceon oxidative damage and functional status in human skeletalmusclerdquo Journal of Muscle Research and Cell Motility vol 22no 4 pp 345ndash351 2001

[11] J P Chaput C Lord M Cloutier et al ldquoRelationship betweenantioxidant intakes and class I sarcopenia in elderly men andwomenrdquo Journal of Nutrition Health and Aging vol 11 no 4pp 363ndash369 2007

[12] M Cesari M Pahor B Bartali et al ldquoAntioxidants and physicalperformance in elderly persons the invecchiare in Chianti(InCHIANTI) studyrdquo American Journal of Clinical Nutritionvol 79 no 2 pp 289ndash294 2004

[13] A Ble A Cherubini S Volpato et al ldquoLower plasma vitamin Elevels are associated with the frailty syndrome the InCHIANTIstudyrdquo Journals of GerontologymdashSeries A Biological Sciences andMedical Sciences vol 61 no 3 pp 278ndash283 2006

[14] R Rafique A H V Schapira and J M Cooper ldquoMitochondrialrespiratory chain dysfunction in ageing influence of vitaminE deficiencyrdquo Free Radical Research vol 38 no 2 pp 157ndash1652004

[15] S C Khor N Abdul Karim W Z W Ngah Y A M Yusofand S Makpol ldquoVitamin E in sarcopenia current evidences onits role in prevention and treatmentrdquo Oxidative Medicine andCellular Longevity vol 2014 Article ID 914853 16 pages 2014

[16] C K Sen S Khanna and S Roy ldquoTocotrienols vitamin Ebeyond tocopherolsrdquo Life Sciences vol 78 no 18 pp 2088ndash2098 2006

[17] A C Howard A K McNeil and P L McNeil ldquoPromotion ofplasma membrane repair by vitamin Erdquo Nature Communica-tions vol 2 article no 597 2011

[18] J J Lim W Z Wan Ngah V Mouly and N Abdul KarimldquoReversal of myoblast aging by tocotrienol rich fraction post-treatmentrdquoOxidativeMedicine andCellular Longevity vol 2013Article ID 978101 11 pages 2013

[19] S C Khor A M Razak W ZWan Ngah Y A Mohd Yusof NAbdul Karim and S Makpol ldquoThe tocotrienol-rich fraction issuperior to tocopherol in promoting myogenic differentiationin the prevention of replicative senescence of myoblastsrdquo PLoSONE vol 11 no 2 Article ID e0149265 2016

[20] A Bigot V Jacquemin F Debacq-Chainiaux et al ldquoReplica-tive aging down-regulates the myogenic regulatory factors inhuman myoblastsrdquo Biology of the Cell vol 100 no 3 pp 189ndash199 2008

[21] T A F T Ahmad Z Jubri N F Rajab K A Rahim Y A MYusof and S Makpol ldquoGelam honey protects against gamma-irradiation damage to antioxidant enzymes in human diploidfibroblastsrdquoMolecules vol 18 no 2 pp 2200ndash2211 2013

[22] M Mazlan T Sue Mian G Mat Top and W Zurinah WanNgah ldquoComparative effects of 120572-tocopherol and 120574-tocotrienolagainst hydrogen peroxide induced apoptosis on primary-cultured astrocytesrdquo Journal of the Neurological Sciences vol243 no 1-2 pp 5ndash12 2006

[23] W F Beyer Jr and I Fridovich ldquoAssaying for superoxidedismutase activity some large consequences of minor changesin conditionsrdquo Analytical Biochemistry vol 161 no 2 pp 559ndash566 1987

[24] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[25] D E Paglia and W N Valentine ldquoStudies on the quantitativeand qualitative characterization of erythrocyte glutathione per-oxidaserdquo The Journal of Laboratory and Clinical Medicine vol70 no 1 pp 158ndash169 1967

[26] E Serbinova V Kagan D Han and L Packer ldquoFree radicalrecycling and intramembrane mobility in the antioxidant prop-erties of alpha-tocopherol and alpha-tocotrienolrdquo Free RadicalBiology and Medicine vol 10 no 5 pp 263ndash275 1991

[27] Y J Suzuki M Tsuchiya S R Wassall et al ldquoStructural anddynamic membrane properties of 120572-tocopherol and 120572-tocot-rienol iimplication to themolecularmechanismof their antiox-idant potencyrdquo Biochemistry vol 32 no 40 pp 10692ndash106991993

[28] Y Saito Y Yoshida K Nishio M Hayakawa and E NikildquoCharacterization of cellular uptake and distribution of vitaminErdquo Annals of the New York Academy of Sciences vol 1031 pp368ndash375 2004

[29] S S Jejurikar E A Henkelman P S Cederna C L MarceloM G Urbanchek and W M Kuzon Jr ldquoAging increasesthe susceptibility of skeletal muscle derived satellite cells toapoptosisrdquoExperimental Gerontology vol 41 no 9 pp 828ndash8362006

[30] K L Urish J B Vella M Okada et al ldquoAntioxidant levelsrepresent a major determinant in the regenerative capacity ofmuscle stem cellsrdquo Molecular Biology of the Cell vol 20 no 1pp 509ndash520 2009

[31] V Renault L-E Thornell G Butler-Browne and V MoulyldquoHuman skeletal muscle satellite cells aging oxidative stressand themitotic clockrdquo Experimental Gerontology vol 37 no 10-11 pp 1229ndash1236 2002

[32] S Makpol T W Yeoh F A C Ruslam K T Arifin andY A M Yusof ldquoComparative effect of Piper betle Chlorellavulgaris and tocotrienol-rich fraction on antioxidant enzymesactivity in cellular ageing of human diploid fibroblastsrdquo BMCComplementary and Alternative Medicine vol 13 article 2102013

[33] A A Franco R S Odom and T A Rando ldquoRegulation ofantioxidant enzyme gene expression in response to oxidativestress and during differentiation of mouse skeletal musclerdquo FreeRadical Biology and Medicine vol 27 no 9-10 pp 1122ndash11321999

[34] M J Sullivan-Gunn and P A Lewandowski ldquoElevated hydro-gen peroxide and decreased catalase and glutathione peroxidaseprotection are associated with aging sarcopeniardquo BMC Geri-atrics vol 13 no 1 article 104 2013

[35] I Martin M A Jones D Rhodenizer et al ldquoSod2 knock-down in the musculature has whole-organism consequences in


Drosophilardquo Free Radical Biology andMedicine vol 47 no 6 pp803ndash813 2009

[36] M E Inal G Kanbak and E Sunal ldquoAntioxidant enzymeactivities and malondialdehyde levels related to agingrdquo ClinicaChimica Acta vol 305 no 1-2 pp 75ndash80 2001

[37] B Marzani O Pansarasa and F Marzatico ldquolsquoOxidative stressrsquoand muscle aging influence of age sex fiber composition andfunctionrdquo Basic and Applied Myology vol 14 pp 37ndash44 2004

[38] S-P Lee G-Y Mar and L-T Ng ldquoEffects of tocotrienol-richfraction on exercise endurance capacity and oxidative stress inforced swimming ratsrdquo European Journal of Applied Physiologyvol 107 no 5 pp 587ndash595 2009

[39] V ANunes A J Gozzo I Cruz-Silva et al ldquoVitamin E preventscell death induced by mild oxidative stress in chicken skeletalmuscle cellsrdquo Comparative Biochemistry and Physiology Part CToxicology amp Pharmacology vol 141 no 3 pp 225ndash240 2005

[40] I N Zelko T J Mariani and R J Folz ldquoSuperoxide dismutasemultigene family a comparison of the CuZn-SOD (SOD1)Mn-SOD (SOD2) and EC-SOD (SOD3) gene structures evolutionand expressionrdquo Free Radical Biology and Medicine vol 33 no3 pp 337ndash349 2002

[41] P Gianni K J Jan M J Douglas P M Stuart and M ATarnopolsky ldquoOxidative stress and the mitochondrial theory ofaging in human skeletal musclerdquo Experimental Gerontology vol39 no 9 pp 1391ndash1400 2004

[42] A Cormio F Milella J Vecchiet G Felzani M N Gadaletaand P Cantatore ldquoMitochondrial DNA mutations in RRF ofhealthy subjects of different agerdquoNeurobiology of Aging vol 26no 5 pp 655ndash664 2005

[43] V Pesce A Cormio F Fracasso et al ldquoAge-related mitochon-drial genotypic and phenotypic alterations in human skeletalmusclerdquo Free Radical Biology and Medicine vol 30 no 11 pp1223ndash1233 2001

[44] C M Peterson D L Johannsen and E Ravussin ldquoSkeletalmuscle mitochondria and aging a reviewrdquo Journal of AgingResearch vol 2012 Article ID 194821 20 pages 2012

[45] G Nowak D Bakajsova C Hayes M Hauer-Jensen and CM Compadre ldquo120574-tocotrienol protects against mitochondrialdysfunction and renal cell deathrdquo Journal of Pharmacology andExperimental Therapeutics vol 340 no 2 pp 330ndash338 2012

[46] J M Mates ldquoEffects of antioxidant enzymes in the molecularcontrol of reactive oxygen species toxicologyrdquo Toxicology vol153 no 1ndash3 pp 83ndash104 2000

[47] Y Samuni S Goldstein O M Dean and M Berk ldquoThe chem-istry and biological activities of N-acetylcysteinerdquo Biochimica etBiophysica Acta (BBA)mdashGeneral Subjects vol 1830 no 8 pp4117ndash4129 2013

[48] J R Terrill H G Radley-Crabb M D Grounds and P GArthur ldquoN-Acetylcysteine treatment of dystrophic mdx miceresults in protein thiol modifications and inhibition of exerciseinduced myofibre necrosisrdquo Neuromuscular Disorders vol 22no 5 pp 427ndash434 2012

[49] I Medved M J Brown A R Bjorksten et al ldquoN-acetylcysteineenhancesmuscle cysteine and glutathione availability and atten-uates fatigue during prolonged exercise in endurance-trainedindividualsrdquo Journal of Applied Physiology vol 97 no 4 pp1477ndash1485 2004

[50] A Zaragoza C Dıez-Fernandez A M Alvarez D Andresand M Cascales ldquoEffect of N-acetylcysteine and deferoxamineon endogenous antioxidant defense system gene expression ina rat hepatocyte model of cocaine cytotoxicityrdquo Biochimica et

Biophysica ActamdashMolecular Cell Research vol 1496 no 2-3 pp183ndash195 2000

[51] H M A El-Fattah and N M El-Sheikh ldquoEvaluation ofchemoprotective role of N-acetylcysteine and vitamin E ongentamicininduced nephrotoxicityrdquo Australian Journal of Basicand Applied Sciences vol 6 no 3 pp 263ndash270 2012

[52] V Renault L-EThorne P-O Eriksson G Butler-Browne andV Mouly ldquoRegenerative potential of human skeletal muscleduring agingrdquo Aging Cell vol 1 no 2 pp 132ndash139 2002

[53] C A Collins P S Zammit A Perez Ruiz J E Morgan and TA Partridge ldquoA population of myogenic stem cells that survivesskeletal muscle agingrdquo Stem Cells vol 25 no 4 pp 885ndash8942007

[54] L Muller K Theile and V Bohm ldquoIn vitro antioxidant activityof tocopherols and tocotrienols and comparison of vitamin Econcentration and lipophilic antioxidant capacity in humanplasmardquoMolecular Nutrition amp Food Research vol 54 no 5 pp731ndash742 2010

[55] B B Aggarwal C Sundaram S Prasad and R KannappanldquoTocotrienols the vitamin E of the 21st century its potentialagainst cancer and other chronic diseasesrdquo Biochemical Phar-macology vol 80 no 11 pp 1613ndash1631 2010

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


Oxidative stress in aged skeletal muscle can cause oxida-tive damage in cells manifested as damaged DNA lipid per-oxidation and protein carbonylation [9 10] In muscle fibersfree radicals can be produced intrinsically by mitochondriaand regulate fundamental signaling pathways in skeletalmuscle The presence of reactive oxygen species (ROS) orreactive nitrogen species (RNS) can be counteracted bythe antioxidant defense system which includes antioxidantenzymes vitamins and glutathione resulting in sustainedredox balance [9] If the antioxidant defense is overwhelmedby excess ROS or RNS oxidative stress occurs which leads tomuscle injury [8 10]

In addition to the existing oxidative stress during aginginsufficient antioxidant intake among the elderly can con-tribute to the occurrence of sarcopenia [11] Low antioxidantlevels in older individuals were associated with poor musclestrength and low physical performance and can cause frailtyin the elderly [12 13] An in vivo study demonstratedthat vitamin E deficiency caused poor muscle performanceand accelerated aging development [14] Hence introducingantioxidants such as vitamin E could be a relevant strategyto delay sarcopenia progression however more studies areneeded [15]

Vitamin E is a lipid-soluble vitamin with two subclassestocopherols and tocotrienols [16] A previous study reportedthat 120572-tocopherol was able to repair laser-induced disruptedmyoblast membranes indicating a therapeutic effect forvitamin E in the muscle [17] However the less-exploredsubtype of vitamin E is the class of tocotrienols Similar to120572-tocopherol a potential therapeutic effect of tocotrienol-rich fraction (TRF) was proposed owing to its reversal effecton stress-induced presenescence (SIPS) model of myoblasts[18] In our laboratory we also found that TRF was supe-rior to 120572-tocopherol in ameliorating replicative senescence-related aberration and promoting myogenic differentiation[19] Thus it would be of interest to elucidate the effects oftocotrienols on the dynamics of oxidative status in senes-cent myoblasts Therefore the aims of this study were toinvestigate the effects of tocotrienol-rich fraction (TRF) inreestablishing oxidative status during replicative senescenceof myoblasts and to compare these effects with other antiox-idants such as 120572-tocopherol (ATF) and N-acetyl-cysteine(NAC) in young presenescent and senescent myoblastsfollowed by the measurement of cell viability and apoptosisas the final outcomes of antioxidant treatment

2 Materials and Methods

21 Cell Culture HumanSkeletalMuscleMyoblasts (HSMM)were purchased from Lonza (Walkersville MD USA)Briefly myoblasts were cultured in Skeletal Muscle BasalMedium (SkBM) that was supplemented with human epi-dermal growth factor fetal bovine serum dexamethasoneL-glutamine and gentamicin sulfateamphotericin B (LonzaWalkersville MD USA) Cells were cultivated at 37∘C in ahumid atmosphere containing 5 CO

2 The myoblasts then

underwent serial passaging The number of divisions wascalculated for each passage using the formula ln(119873119899) ln 2where N is the number of cells at the harvest stage and

n is the number of cells at the seeding stage [20] Whencells reached replicative senescence they were unable toproliferate within 10 days in culture Myoblasts were dividedinto 3 different stages young (lt15 cell divisions) presenescent(18-19 cell divisions) and senescent (gt20 cell divisions)based on their decreasing proliferative capacity which wasrepresented by hyperbolic proliferative lifespan curve anddiminishing percentage of BrdU incorporationThe presenceof senescent cells was confirmed by SA-120573-gal staining [19]

22 Antioxidants Tocotrienol-rich fraction (TRF) was pur-chased from Sime Darby Sdn Bhd Selangor Malaysia (TRFGold TRI E 70) while alpha-tocopherol (ATF)was a gift fromthe Malaysian Palm Oil Board (MPOB) (Selangor Malaysia)Both vitamin E subclasses are palm oil-derived TRF consistsof 120572-tocotrienol (ATT 2689) 120573-tocotrienol (BTT 364)120574-tocotrienol (GTT 3166) 120575-tocotrienol (DTT 1366)and 120572-tocopherol (ATF 2415) Briefly stock solutions ofTRF were freshly prepared in 100 ethanol (1 1) and keptat minus20∘C for no longer than one month A similar processwas performed for ATF TRF and ATF were then incubatedovernight with fetal bovine serum at 37∘C before use Thenboth TRF and ATF were diluted in culture medium and usedat a final concentration of 50 120583gmL [19] N-Acetyl-cysteine(NAC) was purchased from Sigma-Aldrich (St Louis USA)NAC was freshly prepared in culture medium to the desiredfinal concentration A dosage of 10mgmLNACwas used forsubsequent experiments which was determined using a cellviability assay (Supplemental 1A in Supplementary Materialavailable online at httpsdoiorg10115520173868305) Adosage of 25 120583gmL of TRF or ATF with 10mgmL NACwas used for combined treatment of TRF and NAC (TRF+ NAC) and combination of ATF and NAC (ATF + NAC)(Supplemental 1B and C)

23 Cell Viability The optimal concentrations of NAC thecombination of TRF and NAC (TRF + NAC) and the combi-nation of ATF andNAC (ATF+NAC)were determined usinga cell viability assay (Supplemental 1)The effects of H

2O2and

antioxidants were also determined using cell viability assayA CellTiter 96 Aqueous Nonradioactive Cell ProliferationAssay (MTS Promega Madison USA) was used accordingto the manufacturerrsquos instructions Cells were incubated withseveral concentrations of antioxidants for 24 h and 45min forH2O2 After that the treatments were replaced by MTS for

another 2 h of incubation The absorbance of MTS formazanwas measured at 490 nm with a microtiter plate reader(VersaMax Molecular Devices USA) The optimum dosageof treatments was used for subsequent experiments

24 Analysis of Cell Morphology In brief cells were platedand fixed with cold ethanol in 120583-Slide 8 wells (ibidi Martin-sried Germany) followed by incubation with an anti-Desminantibody (dilution 1 50) (D33 Dako Produktionsvej Den-mark) and Alexa Fluor 488 goat anti-mouse (dilution 1 500)(Molecular Probes Eugene OR USA) Nuclei were visual-ized using Hoechst 33342 (Molecular Probes Eugene ORUSA) Slides were then observed under a Leica TCS SP5 II













2DCFDA for









2O2(Merck












3 Results



2DCFDA (in green H



2DCFDA grad-



TRF + NAC ATF + NAC

Sene

scen

t myo

blas

ts

Control TRF

NACATF


(a)

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC


S

A-120573

-gal

+ve

cells

a

a b

cc c

minus100

10203040506070

(b)











Hoe

chst

DH

EM

erge


Scale bar

Carb

oxy-

H2D

CFD

A

200 120583m

(a)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(b)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(c)

Figure 2 Continued


0153045607590

105

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f lab

eled

cells

DHE

a

ab

a a bb

c c

c

a a b b

c

Carboxy-H2DCFDA

(d)

40

60

80

100

120

0 05 1 15 2 25Rela

tive c

ell v

iabi

lity

()


lowast

lowastlowast lowast

yen

yenyenyen


(e)














Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(a)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(b)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(c)

Figure 3 Continued



1519 2832 3228

0

50

100

Cou

nt

0

50

Cou

nt0

50

Cou

nt

103 104 105102




(d)

0

01

02

03

04

05

06

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Ratio

of o

xidi

sed

redu

ced

BOD

IPY

aa

b b c c

b

cc

(e)






01020304050607080


Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)







Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























2DCFDA for









2O2(Merck












3 Results



2DCFDA (in green H



2DCFDA grad-



TRF + NAC ATF + NAC

Sene

scen

t myo

blas

ts

Control TRF

NACATF


(a)

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC


S

A-120573

-gal

+ve

cells

a

a b

cc c

minus100

10203040506070

(b)











Hoe

chst

DH

EM

erge


Scale bar

Carb

oxy-

H2D

CFD

A

200 120583m

(a)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(b)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(c)

Figure 2 Continued


0153045607590

105

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f lab

eled

cells

DHE

a

ab

a a bb

c c

c

a a b b

c

Carboxy-H2DCFDA

(d)

40

60

80

100

120

0 05 1 15 2 25Rela

tive c

ell v

iabi

lity

()


lowast

lowastlowast lowast

yen

yenyenyen


(e)














Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(a)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(b)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(c)

Figure 3 Continued



1519 2832 3228

0

50

100

Cou

nt

0

50

Cou

nt0

50

Cou

nt

103 104 105102




(d)

0

01

02

03

04

05

06

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Ratio

of o

xidi

sed

redu

ced

BOD

IPY

aa

b b c c

b

cc

(e)






01020304050607080


Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)







Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results



2DCFDA (in green H



2DCFDA grad-



TRF + NAC ATF + NAC

Sene

scen

t myo

blas

ts

Control TRF

NACATF


(a)

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC


S

A-120573

-gal

+ve

cells

a

a b

cc c

minus100

10203040506070

(b)











Hoe

chst

DH

EM

erge


Scale bar

Carb

oxy-

H2D

CFD

A

200 120583m

(a)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(b)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(c)

Figure 2 Continued


0153045607590

105

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f lab

eled

cells

DHE

a

ab

a a bb

c c

c

a a b b

c

Carboxy-H2DCFDA

(d)

40

60

80

100

120

0 05 1 15 2 25Rela

tive c

ell v

iabi

lity

()


lowast

lowastlowast lowast

yen

yenyenyen


(e)














Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(a)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(b)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(c)

Figure 3 Continued



1519 2832 3228

0

50

100

Cou

nt

0

50

Cou

nt0

50

Cou

nt

103 104 105102




(d)

0

01

02

03

04

05

06

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Ratio

of o

xidi

sed

redu

ced

BOD

IPY

aa

b b c c

b

cc

(e)






01020304050607080


Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)







Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TRF + NAC ATF + NAC

Sene

scen

t myo

blas

ts

Control TRF

NACATF


(a)

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

NAC

TRF

+ N

AC

ATF

+ N

AC


S

A-120573

-gal

+ve

cells

a

a b

cc c

minus100

10203040506070

(b)











Hoe

chst

DH

EM

erge


Scale bar

Carb

oxy-

H2D

CFD

A

200 120583m

(a)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(b)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(c)

Figure 2 Continued


0153045607590

105

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f lab

eled

cells

DHE

a

ab

a a bb

c c

c

a a b b

c

Carboxy-H2DCFDA

(d)

40

60

80

100

120

0 05 1 15 2 25Rela

tive c

ell v

iabi

lity

()


lowast

lowastlowast lowast

yen

yenyenyen


(e)














Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(a)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(b)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(c)

Figure 3 Continued



1519 2832 3228

0

50

100

Cou

nt

0

50

Cou

nt0

50

Cou

nt

103 104 105102




(d)

0

01

02

03

04

05

06

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Ratio

of o

xidi

sed

redu

ced

BOD

IPY

aa

b b c c

b

cc

(e)






01020304050607080


Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)







Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Hoe

chst

DH

EM

erge


Scale bar

Carb

oxy-

H2D

CFD

A

200 120583m

(a)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(b)

Scale bar

Hoe

chst

DH

EM

erge


Carb

oxy-

H2D

CFD

A

200 120583m

(c)

Figure 2 Continued


0153045607590

105

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f lab

eled

cells

DHE

a

ab

a a bb

c c

c

a a b b

c

Carboxy-H2DCFDA

(d)

40

60

80

100

120

0 05 1 15 2 25Rela

tive c

ell v

iabi

lity

()


lowast

lowastlowast lowast

yen

yenyenyen


(e)














Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(a)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(b)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(c)

Figure 3 Continued



1519 2832 3228

0

50

100

Cou

nt

0

50

Cou

nt0

50

Cou

nt

103 104 105102




(d)

0

01

02

03

04

05

06

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Ratio

of o

xidi

sed

redu

ced

BOD

IPY

aa

b b c c

b

cc

(e)






01020304050607080


Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)







Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0153045607590

105

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f lab

eled

cells

DHE

a

ab

a a bb

c c

c

a a b b

c

Carboxy-H2DCFDA

(d)

40

60

80

100

120

0 05 1 15 2 25Rela

tive c

ell v

iabi

lity

()


lowast

lowastlowast lowast

yen

yenyenyen


(e)














Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(a)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(b)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(c)

Figure 3 Continued



1519 2832 3228

0

50

100

Cou

nt

0

50

Cou

nt0

50

Cou

nt

103 104 105102




(d)

0

01

02

03

04

05

06

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Ratio

of o

xidi

sed

redu

ced

BOD

IPY

aa

b b c c

b

cc

(e)






01020304050607080


Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)







Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(a)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(b)

Hoe

chst

BOD

IPY-

red

BOD

IPY-

gree

nM

erge



(c)

Figure 3 Continued



1519 2832 3228

0

50

100

Cou

nt

0

50

Cou

nt0

50

Cou

nt

103 104 105102




(d)

0

01

02

03

04

05

06

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Ratio

of o

xidi

sed

redu

ced

BOD

IPY

aa

b b c c

b

cc

(e)






01020304050607080


Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)







Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















1519 2832 3228

0

50

100

Cou

nt

0

50

Cou

nt0

50

Cou

nt

103 104 105102




(d)

0

01

02

03

04

05

06

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Ratio

of o

xidi

sed

redu

ced

BOD

IPY

aa

b b c c

b

cc

(e)






01020304050607080


Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)







Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















01020304050607080


Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

ATFATTBTT

GTTDTT

aa b b

c c

c

b

ba a

a

b b b

c

c

c

(a)

Con

cent

ratio

n (120583

gm

L pe

r m

illio

n ce

lls)

0102030405060

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC

NAC

TRF

+ N

AC

ATF

+ N

AC


a a bb c

b

c

aa a b bb

cc c

ATFATTBTT

GTTDTT

(b)







Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD1 0

010203040506

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Rela

tive e

xpre

ssio

n va

lue

SOD2

aa

b

b

c

a c000005010015020025030

(b)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


a

a b

c

clowasta

0005101520253035

Relat

ive e

xpre

ssio

n va

lue

CAT

(c)


Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Rela

tive e

xpre

ssio

n va

lue

GPX1

ab

c

00102030405060708

(d)






4 Discussion




Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


Sod

activ

ity(U

mg

prot

ein)

b c

a

b bb

c

0200400600800

10001200

(a)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


ab

caa

b b

b

c

000204060810121416

Cat a

ctiv

ity(m

Um

g pr

otei

n)

(b)

b

ab

c cc

c c

b

b

020406080

100

Gpx

activ

ity(m

Um

g pr

otei

n)

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(c)

ab

ab b

c

0102030405060

GSH

GSS

G ra

tio

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


(d)



2O2










ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















ell v

iabi

lity

()

ControlTRFATF


lowastlowast


lowast lowast

lowastlowast lowast

lowastlowast

lowast

050

100150200250

(a)


R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

R1Senescent control

R2 R3

100

101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

Young controlR1

R2 R3

(late apoptosis)


101

102

103

104

100 101 102 103 104

Prop

idiu

m io

dide

Annexin V-FITC

(b)

8082848688909294

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC

Con

trol

TRF

ATF

NAC

TRF

+ N

ACAT

F +

NAC


o

f via

ble c

ells

a a

a a a bb b

c c c c

(c)

02468

1012

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC

Con

trol

TRF

ATF

NAC

TRF

+ N

AC

ATF

+ N

AC


o

f apo

ptot

ic ce

lls


a a

ab

ab

c cc c c

c c cb b bb ba

aa a

(d)







2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2O2[38] However














2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2O2-




Competing Interests




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Tocotrienol-Rich Fraction Ameliorates Antioxidant Defense Mechanisms...

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Transcript of Tocotrienol-Rich Fraction Ameliorates Antioxidant Defense Mechanisms...