Allium cepa Extract and Quercetin Protect Neuronal Cells ...€¦ · BSO BSO + ACE BSO + QCT CTL 3 10 1 3 10 ACE (mg/mL) VEH 1 QCT (𝜇M) 5 10 15 20 25 TUNEL-positive cells (% of

Research ArticleAllium cepa Extract and Quercetin ProtectNeuronal Cells from Oxidative Stress via PKC-120576InactivationERK12 Activation

Bo Kyung Lee1 and Yi-Sook Jung12

1College of Pharmacy Ajou University Suwon 443-749 Republic of Korea2Research Institute of Pharmaceutical Sciences and Technology Ajou University Suwon 443-749 Republic of Korea

Correspondence should be addressed to Bo Kyung Lee pfifferajouackr and Yi-Sook Jung yisjungajouackr

Received 28 June 2016 Accepted 17 August 2016

Academic Editor Francisco J Romero

Copyright copy 2016 B K Lee and Y-S Jung This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Oxidative stress plays an important role in the pathophysiology of various neurologic disorders Allium cepa extract (ACE) andtheir main flavonoid component quercetin (QCT) possess antioxidant activities and protect neurons from oxidative stress Weinvestigated the underlyingmolecular mechanisms particularly those linked to the antioxidant effects of the ACE Primary corticalneuronal cells derived from mouse embryos were preincubated with ACE or QCT for 30min and exposed to L-buthioninesulfoximine for 4sim24 h We found that ACE and QCT significantly decreased neuronal death and the ROS increase induced byL-buthionine-S R-sulfoximine (BSO) in a concentration-dependent manner Furthermore ACE and QCT activated extracellularsignal-regulated kinase 12 (ERK12) leading to downregulation of protein kinase C-120576 (PKC-120576) in BSO-stimulated neuronal cellsIn addition ACE and QCT decreased the phosphorylated levels of p38 mitogen-activated protein kinase Our results provide newinsight into the protective mechanism of ACE and QCT against oxidative stress in neuronal cells The results suggest that theinactivation of PKC-120576 induced by phosphorylating ERK12 is responsible for the neuroprotective effect of ACE and QCT againstBSO-induced oxidative stress

1 Introduction

Oxidative stress has been implicated in neuronal cell death ina variety of neurologic disorders [1ndash3] Oxidative stress influ-ences the oxidation states of several proteins in neuronal cellsinvolved in intracellular signaling events including mitogen-activated protein kinases (MAPKs) and protein kinaseC (PKC) [4] Particularly MAPKs (extracellular signal-regulated kinase 12 (ERK12) c-Jun NH

2-terminal kinase

(JNK) and p38MAPK) play a pivotal role during oxidativestress of neurons Glutathione (GSH) is a major antioxi-dant in the brain [5] and depletingGSH is quite often accom-panied by increased levels of reactive oxygen species (ROS)[6] We demonstrated previously that the GSH depletingagent L-buthionine-SR-sulfoximine (BSO) causes cell deathby activating PKC-120576 in brain cortical neurons [2] We havedocumented the antioxidant effect of an extract of Allium

cepa (A cepa) which is a bulbous herbaceous biennialmonocot plant [7 8] Furthermore A cepa extracts havea neuroprotective effect during transient cerebral ischemia[9] and have antioxidant activity and lipid peroxidationinhibiting properties in the brain [10] However it is unclearwhether A cepa has protective or antioxidant effects againstneurotoxic conditions in a primary neuronal cell culturesystem Moreover the molecular and cellular mechanismsunderlying the neuroprotective effect of A cepa extractsagainst oxidative stress have not been elucidated Accord-ingly in this study we investigated the protective effect ofan A cepa methanol extract (ACE) and its major flavonoidcomponent quercetin (QCT) against oxidative stress-inducedneuronal cell death We established whether MAPKs andPKC-120576 are involved in the mechanism responsible for theneuroprotective effects of ACE and QCT in neuronal cellsduring oxidative stress

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 2495624 9 pageshttpdxdoiorg10115520162495624

2 Oxidative Medicine and Cellular Longevity

2 Materials and Methods

All experimental procedures were conducted in accordancewith the guidelines on the care and use of laboratory animalsset by the Animal Care Committee of Ajou University

21 Chemicals ACE was obtained from Konkuk UniversityBSO and QCT [2-(34-dihydroxyphenyl)-357-trihydroxy-4H-chromen-4-one] were purchased from Sigma (St LouisMO USA) Trolox [6-hydroxy-2578-tetramethylchroman-2-carboxylic acid] commonly used antioxidant was obtainedfrom Tocris (Ballwin MO USA) Z-DEVD-fmk (cas-pase inhibitor) 120576V1-2-peptide (PKC-120576 inhibitor) U0126[14-diamino-23-dicyano-14-bis(2-aminophenylthio) buta-diene] (ERK inhibitor) and SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] (p38MAPKinhibitor) were purchased from Biomol Research Labs Inc(Plymouth Meeting PA USA) Other reagents were of thehighest grade commercially available

22MouseMixedCorticalNeuronal Cell Culture Mouse neo-cortices were obtained from fetal mouse brains on embryonicdays 14-15 and grown in Eaglersquos minimum essential mediumsupplemented with 21mM glucose 5 fetal bovine serum5 horse serum and 2mM glutamine in 5 CO

2at 37∘C

as described previously [11] At DIV (days in vitro) 7 10 120583Mcytosine arabinofuranoside was added to the cultures to haltovergrowth of glial cells The cells were maintained for 13-14days and then used for experiments

23 Preparation of ACE and Treatment ACE was obtainedusing a method modified slightly from a protocol describedpreviously [12] Briefly after the outer skins or leaves of freshA cepa were removed 50 g of an A cepa bulb was homoge-nized in 70 methanol (100mL) and the homogenate wasfiltered through filter paper The resulting fractions werelyophilized using a vacuum evaporator (N-2N Eyela Tokyo)Lyophilized ACE was dissolved in cell culture medium Thecells were pretreated with ACE (1ndash10mgmL) and QCT (1ndash10 120583M) 30min before and they were not removed from theculture medium during the BSO treatment

24 Lactate Dehydrogenase (LDH) Assay We assayed LDHreleased into the medium after BSO treatment to measureoverall cell injury by spectrophotometric analysis at 340 nmas described previously [2 13] The percentage of LDH wascalculated from the maximum LDH release (100) inducedby lysing cells with 1 Triton X-100

25 Terminal Deoxynucleotidyl Transferase dUTP Nick EndLabeling (TUNEL) Staining Fragmented DNA was labeledin situ using an Apop Taq Plus kit (Millipore Gaithers-burg MD USA) Cells were grown on 24-well plates andfixed in 4 paraformaldehyde in PBS Nucleosome-sizedDNA fragments were tailed with digoxigenin nucleotide andreacted with fluorescein-conjugated antidigoxigenin anti-body as reported previously [14] Percent cell death wascalculated by expressing the number of TUNEL-positive cellsas a percentage of total cell count

26 Intracellular ROS Level We followed a method reportedpreviously to determine ROS levels [15] In brief cells grownon a glass-bottomed dish were loaded with 10 120583M6-carboxy-2101584071015840-dichlorodihydrofluorescein diacetate dicarboxymethylester (DCF-DA) in HCSS buffer containing 120 NaCl 5KCl 16 MgCl

2 23 CaCl

2 15 glucose 20 Hepes and 10

NaOH (mM pH 74) for 20min at 37∘C DCF-DA waspurchased from Molecular Probes (Eugene OR USA) Theexperiments were performed at room temperature on Aconfocal microscope stage and digitized using FLUOVIEWFV300 (Olympus Tokyo)

27 Isolation of ERK12 p38MAPK and JNK fromCell LysatesERK12 p38MAPKs and JNK were isolated as describedpreviously [16] Cells were harvested in RIPA buffer (150NaCl 20 Tris-HCl 1 NP-40 1 Na-deoxycholate 1 EDTAand protease inhibitors at pH 74 [mM]) and homogenizedand nuclei and cell debris were removed by centrifugation at10000timesg for 15min at 4∘C The supernatants were collectedfor immunoblotting Protein contents were determined usingthe BCA protein assay (Pierce Rockford IL USA) Proteinsamples were denatured in Laemmli buffer (4 SDS 20glycerol and 120mM Tris-HCl at pH 68) and total ERK12p38MAPKs and JNK as well as their phosphorylated formswere quantified by immunoblotting using polyclonal andmonoclonal antibodies against the proteins (Cell SignalingTechnology Danvers MA USA)

28 Subcellular Fractionation to Isolate PKC-120576 and Immuno-blotting PKC-120576 was subcellular fractionated as describedpreviously [16] Briefly cells were harvested in homoge-nization buffer (20 Tris-HCl 2 EDTA 5 EGTA 5 DTT 6120573-mercaptoethanol 1 PMSF 002 leupeptin and 10 120583gmLaprotinin pH 74 [mM]) and centrifuged at 100000timesg for 1 hat 4∘CThe supernatant was retained as the cytosolic fractionPellets were resuspended in 1 Triton X-100-containinghomogenization buffer and centrifuged at 10000timesg for10min at 4∘CThe supernatant is referred to as themembranefraction Protein content was determined using the Bradfordprotein assay (Biorad Hercules CA USA)The samples wereresolved on 8 SDS-polyacrylamide gels and transferred topolyvinylidene difluoride membranes (Millipore BedfordMA USA) The blots were incubated in 5 nonfat dry milkfor 1 h at room temperature and then incubated overnightat 4∘C with a polyclonal antibody against PKC-120576 (SantaCruz Biotechnology Santa Cruz CA USA) The blots wererinsed with Tris-buffered saline and incubated with horse-radish peroxidase-conjugated secondary IgG (Cell SignalingTechnology) for 1 h Bound antibody was detected withan enhanced 3D chemiluminescence kit (Intron DaejeonKorea) and the bands were analyzed using a LAS1000 (FujiPhoto Film Tokyo)

29 Statistical Analysis Data are expressed as mean plusmn stan-dard error of at least three separate determinations in eachgroup Numerical data were compared using Studentrsquos 119905-test or one-way ANOVA post hoc test for the unpairedobservations between the two groups A 119901 value lt 005 wasconsidered significant

Oxidative Medicine and Cellular Longevity 3

3 Results

31 Effect of ACE and QCT on BSO-Induced Neuronal CellDeath We measured LDH release after the neuronal cellswere treated with BSO to investigate the effects of ACE andQCT on BSO-induced cell death We pretreated cells for30minwith ACE (1ndash10mgmL) andQCT (1ndash10 120583M) and thenwe add 10mMBSO for 24 hThe concentrations of QCTwereselected because the previous studies for neuroprotectiveeffects of QCT suggest a concentration level of maximum10 120583M [17 18] In the case of ACE we previously per-formed experiments using wide concentration range of theACE (01sim1000mgmL) and found no greater efficacy than10mgmLACE (data not shown) [7] As shown in Figure 1(a)LDH release increased significantly to 521 plusmn 12 in cellsexposed to BSO alone for 24 h versus the untreated controls(138 plusmn 16) This increase in LDH release due to BSOwas concentration-dependently inhibited by ACE or QCTA 1mgmL ACE concentration did not alter BSO-inducedLDH release (478 plusmn 33) but LDH levels decreased slightly(420 plusmn 31) after exposure to 3mgmL ACE and BSO-induced LDH release decreased significantly by 10mgmLACE (295plusmn17) FurthermoreQCThad a partially strongereffect on BSO-induced LDH release from neuronal cellsthan that of ACE QCT at 1 and 3 120583M reduced LDH levelscompared with those of the vehicle (to 424 plusmn 40 and 376 plusmn29 resp) whereas 10 120583MQCT significantly reduced BSO-induced LDH release to 255 plusmn 29 To investigate theantiapoptotic effects of ACE and QCT we analyzed theireffects on BSO-induced cell death by TUNEL staining a well-known indicator of apoptotic cell death [19] As shown inFigure 1(b) TUNELpositivity of vehicle-treated cells exposedto BSO for 24 h was 231 plusmn 15 When cells were pretreatedwith different concentrations (1 3 and 10mgmL) of ACEfor 30min and then exposed to BSO for 24 h the numberof TUNEL-positive cells was significantly decreased at 3 and10mgmL concentrations of ACE (to 102plusmn16 and 65plusmn10resp) indicating that ACE has an antiapoptotic effect oncortical neuronal cells Similarly pretreatment withQCT alsosignificantly reduced the number of TUNEL-positive BSO-treated cells (138 plusmn 21 122 plusmn 18 and 67 plusmn 11 at 1 3 and10 120583M resp)

32 Effects of ACE and QCT on BSO-Induced ROS Accumu-lation We determined whether ACE or QCTmodulated theeffect of BSO onROS Intracellular ROS levels weremeasuredby fluorescence using DCF-DA Treating the cortical cellcultures with 10mM BSO for 8 h increased intracellularROS levels to a peak at 4 h (1748 plusmn 60) Furthermorea 30min ACE pretreatment (10mgmL) after a 4 h BSOtreatment significantly reduced ROS level (1250 plusmn 61 ofcontrol) As observed for ACE a 30min QCT pretreatment(10 120583M) similarly reduced DCF-DA intensity after 4 h of BSOtreatment (1183 plusmn 28 of control) (Figures 1(c) and 1(d))

33 Antioxidant Effects of ACE and QCT Are Mediated byERK12 Phosphorylation and p38MAPK DephosphorylationWe detectedMAPK family phosphorylation such as ERK12JNK12 and p38MAPK in neuronal cells treated with BSO

to identify the mediator of the ACE and QCT antioxidanteffects against BSO-induced cell death The ERK12 phos-phorylation level decreased 60min after BSO treatment anddropped to its lowest level after 120min whereas p38MAPKsincreased In contrast the JNK phosphorylation level wasmaintained (Figure 2(a)) BSO decreased ERK12 phosphory-lation which was abolished by ACE or QCT but p38MAPKinhibition by SB202190 and ERK inhibition by U0126 didnot recover the downregulation of ERK12 In contrast BSOinduced p38MAPK phosphorylation which was preventedby ACE QCT and SB202190 whereas U0126 did not inhibitp38MAPK phosphorylation (Figure 2(b)) Additionally weinvestigated the role of ERK12 and p38MAPK in neuronalcells during BSO-induced ROS accumulation BSO-inducedROS accumulationwas not inhibited byU0126 (1982plusmn48)whereas SB202190 significantly inhibited ROS accumulationafter 4 h (1286plusmn 105) (Figure 2(c))These findings suggestthat p38MAPK phosphorylation is involved in ROS accumu-lation whereas ERK12 phosphorylation was not involved inROS accumulation induced by BSO in neuronal cells

34 The Neuroprotective Effects of ACE and QCT Involve ERKPhosphorylation We conducted LDH release and TUNELassays to investigate the roles of p38MAPK and ERK12phosphorylation during oxidative stress in neuronal cellsAs shown in Figures 3(a) and 3(b) SB202190 blocked theincrease in LDH release (402 plusmn 53) and the number ofTUNEL-positive (214 plusmn 44) neuronal cells induced byBSO (564 plusmn 28 and 333 plusmn 43) but not U0126 (609 plusmn93 and 318 plusmn 26) In addition we examined whetherERK12 phosphorylation regulates the neuroprotective effectsinduced by ACE and QCT in neuronal cells As a result theneuroprotective effects ofACE andQCTagainst neuronal celldeath induced by BSO were partially abolished by U0126 asdetected by LDH release (427 plusmn 62 and 447 plusmn 97) andthe number of TUNEL-positive cells (255 plusmn 38 and 263 plusmn26) respectively suggesting that ACE and QCT protectBSO-treated neuronal cells via ERK12 phosphorylationMoreover the inhibition of ERK12 by U0126 attenuatedERK12 phosphorylation and p38MAPK dephosphorylationinduced by ACE and QCT in BSO-induced neuronal cells(Figure 3(c)) As shown in Figure 3(d) we also investigatedthe effects of ERK12 phosphorylation on oxidative stressU0126 significantly attenuated the antioxidant effects of ACEor QCT at 4 h (1503 plusmn 95 and 1483 plusmn 66) suggesting thatthe inhibiting effects of ACE and QCT on ROS accumulationare mediated by ERK12 phosphorylation in neuronal cells

35TheAntioxidant Effects of ACE andQCT Involve InhibitingPKC-120576 Our previous studies demonstrated that PKC-120576 isthe major PKC isoform involved in pathways triggered byGSH depletion leading to neuronal death in BSO-treatedcortical cells [2] In the present study we investigatedwhetherACE and QCT regulate activation of PKC-120576 in neuronalcells induced by oxidative stress After treating the corticalcells with 10mM BSO PKC-120576 levels in membrane fractionsincreased (sim2 h) whereas the PKC-120576 levels in the cytosolicfractions decreased (Figure 4(a)) ACE or QCT pretreatmentstrongly inhibited PKC-120576 translocation in BSO-induced cells


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Figure 1 Effects of Allium cepa extract (ACE) and quercetin (QCT) on L-buthionine sulfoximine- (BSO-) induced neuronal cell death (a)Cortical cells were pretreated with ACE (1 3 and 10mgmL) and QCT (1 3 and 10120583M) for 30min and then exposed to 10mM BSO for24 h Lactate dehydrogenase (LDH) release was measured after a 24 h BSO treatment (b) Apoptotic cell death was examined by countingthe number of terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL-) positive cells (Top) TUNEL staining photographsarrows indicate TUNEL-positive cells and arrowheads indicate intact cells (Bottom) Cortical cells were pretreated with ACE (1 3 and10mgmL) and QCT (1 3 and 10120583M) for 30min and then exposed to 10mM BSO for 24 h The number of TUNEL-positive () cellswas calculated by dividing the number of TUNEL-stained cells by the total number of cells after a 24 h BSO treatment (c) Fluorescencephotomicrographs (stained with 10120583MDCF-DA) of cells after a 4 h BSO treatment and the control Cortical cells were pretreated with ACE(10mgmL) and QCT (10120583M) for 30min and then exposed to 10mMBSO for 4 h before measuring fluorescence (d) Reactive oxygen species(ROS) generation during BSO treatment was quantified by pretreating the cells with ACE (10mgmL) and QCT (10120583M) simultaneously with10mM BSO ROS levels in the cells were quantified by measuring DCF-DA fluorescence intensity and are represented as the percentage ()of intensity at 0min All data are mean plusmn standard error (119899 ge 5) lowast119901 lt 005 versus control (CTL) 119901 lt 005 versus vehicle (VEH)

(2 h) compared with that by the vehicle Inhibiting PKC-120576 translocation with 120576V1-2 significantly reduced the PKC-120576translocation induced by BSO whereas SB202190 and U0126did not inhibit the translocation of PKC-120576 (Figure 4(b))

Additionally 120576V1-2 significantly inhibited p38MAPK phos-phorylation but did not abolish BSO-induced ERK12 down-regulation (Figure 4(c)) As shown in Figures 4(d) and 4(e)120576V1-2 significantly inhibited LDH release and the number of


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Figure 2 Effects of Allium cepa extract (ACE) and quercetin (QCT) on L-buthionine sulfoximine- (BSO-) induced activation of mitogen-activated protein kinase (MAPK) in cortical cells (a) Representative immunoblots for p-ERK12 p-JNK12 and p-p38MAPK loading werenormalized versus ERK12 JNK12 and p38MAPK in all neuronal cells exposed to 10mM BSO for the indicated treatment periods (0ndash4 h)(b)The cells were treated with 10mMBSO for 2 h in the presence or absence (VEH) of ACE (10mgmL) QCT (10 120583M) SB202190 (SB 10 120583M)or U0126 (10 120583M) (c) Reactive oxygen species (ROS) generation was quantified during BSO treatment after pretreating the cells with ACE(10mgmL) QCT (10120583M) SB (10 120583M) or U0126 (10120583M) simultaneously with 10mM BSO for 4 h ROS levels in cells were quantified bymeasuring DCF-DA fluorescence intensity and are represented as a percentage () of the control (CTL) All data are mean plusmn standard error(119899 = 4) lowast119901 lt 005 versus 0 time 119901 lt 005 versus VEH

TUNEL-positive cells after a 24 h BSO treatment (564 plusmn 28and 333plusmn43 to 308plusmn43 and 162plusmn15 resp)These effectswere similar to those caused by the presence or absence ofSB202190 (327plusmn53 and 160plusmn19 resp) but U0126 did nothave an effect (610plusmn93 and 318plusmn26 resp) Furthermore120576V1-2 partially reduced DCF-DA intensity after a 4 h BSOtreatment (1748 plusmn 60 to 1452 plusmn 58) and no additionaleffects were observed by SB202190 or U0126 (1431 plusmn 84 or1520 plusmn 68) (Figure 4(f)) These results show that ACE andthe major component QCT can reduce neuronal cell deathand intracellular ROS accumulation which ismechanisticallylinked with PKC-120576p38MAPK signaling

4 Discussion and Conclusion

ACE and itsmajor flavonoid component QCT protect againstvarious neurodegenerative disorders and their antioxidantactivities are believed to prevent neuronal death Thereforewe focused on the protective and antioxidant effects of theACE and QCT to identify their molecular mechanismsNatural dietary antioxidants such as A cepa have attractedconsiderable attention Both ACE and QCT have broad-ranging pharmacological effects particularly free radicalscavenging properties that protect against oxidative injurydue to their ability to modulate intracellular signals andpromote cell survival [20] Our results are consistent with


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Figure 3 Role of p38MAPK and ERK12 phosphorylation in neuronal cells during oxidative stress (a) Cortical cells were treated with 10mML-buthionine sulfoximine (BSO) for 24 h in the presence or absence of the Allium cepa extract (ACE) (10mgmL) quercetin (QCT) (10 120583M)SB (10 120583M) or U0126 (10120583M) Lactate dehydrogenase (LDH) release was measured after a 24 h BSO treatment (b) The number of terminaldeoxynucleotidyl transferase dUTP nick end labeling- (TUNEL-) positive () cells was calculated by dividing the number of TUNEL-stainedcells by the total number of cells after a 24 h BSO treatment (c) Representative immunoblots for p-ERK12 and p-p38MAPK loading werenormalized versus ERK12 and p38MAPK respectively Cells were treated with 10mM BSO for 2 h in the presence or absence (VEH) of ACE(10mgmL) QCT (10120583M) or U0126 (10120583M) (d) Reactive oxygen species (ROS) generation was quantified during BSO treatment after cellswere pretreated with ACE (10mgmL) QCT (10120583M) or U0126 (10120583M) simultaneously with 10mMBSO for 4 h All data aremean plusmn standarderror (119899 = 4) lowast119901 lt 005 versus 0 time 119901 lt 005 versus VEH Dagger119901 lt 005 versus ACE or QCT

previous reports that ACE and QCT can scavenge freeradicals thus possibly reducing oxidative stress ACE andQCT has been demonstrated to protect cells from exogenousinsults by activating the endogenous defense system whichinvolves catalase super oxide dismutase and glutathione[21 22] We sought to identify the signaling system involvedin the neuroprotection afforded by ACE and QCT underoxidative stress Accumulating evidence supports that oxida-tive stress including ischemic inflammation apoptosis andother pathological mechanisms is related to regulation ofthe MAPK [23] and PKC [24] signal pathways Our results

shed new light on the beneficial effects of A cepa onoxidative stress by demonstrating a potential mechanismby which oxidative stress is caused by neuronal damageIt is widely acknowledged that MAPKs play a critical roleregulating neuron responses to oxidative stress [25] Recentdata demonstrate that A cepa or QCT activates MAPKpathways in a variety of cell types such as endothelial cells[26 27] However neuronal cells have remained unstudiedPhosphorylation ofMAPKs is critical for producing oxidativestress in neuronal cells Therefore we investigated the effectsof ACE and QCT on phosphorylation-mediated activation of


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Figure 4 Role of protein kinase C (PKC)-120576 in the neuroprotective effects of the Allium cepa extract (ACE) and quercetin (QCT) against L-buthionine sulfoximine- (BSO-) induced cell death in cortical cells (a)Western blot analysis of PKC-120576 in cytosol (C) andmembrane fractions(M) of cortical cells exposed to 10mM BSO for the indicated treatment periods (0ndash4 h) (b) Representative Western blots of PKC-120576 in thecytosolic and membrane fractions after treatment with 10mM BSO for 2 h in the presence or absence of ACE (10mgmL) QCT (10120583M)120576V1-2 (5 120583M) SB (10 120583M) and U0126 (10120583M) (c) Representative Western blots of ERK12 and p38MAPK after treatment with 10mM BSOfor 2 h in the presence or absence of ACE (10mgmL) QCT (10120583M) and 120576V1-2 (5120583M) (d) Cortical cells were treated with 10mM BSO for24 h in the presence or absence of 120576V1-2 (5120583M) SB (10 120583M) or U0126 (10120583M) Lactate dehydrogenase (LDH) release was measured aftera 24 h BSO treatment (e) The number of terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL-) positive () cells wascalculated by dividing the number of TUNEL-stained cells by the total number of cells after a 24 h BSO treatment (f) Reactive oxygen species(ROS) generation was quantified during BSO treatment after cells were pretreated with 10mMBSO for 4 h in the presence or absence of 120576V1-2(5120583M) SB (10 120583M) or U0126 (10120583M) ROS levels in cells were quantified by measuring DCF-DA fluorescence intensity and are representedas a percentage () of the control (CTL) All data are mean plusmn standard error (119899 = 4) lowast119901 lt 005 versus 0 time 119901 lt 005 versus VEH

MAPKs MAPKs are comprised of three major subgroupsthat is ERK12 JNK12 and p38 MAPK which play keyroles transducing various extracellular signals to the nucleusand regulating cell growth differentiation and oxidativestress [28 29] The dynamic balance between the growthfactor-activated ERK12 and stress-activated JNK-p38MAPKpathways is important for determining whether neuronal

cells survive or die [30] Indeed accumulation of phospho-p38MAPK is associated with neurodegenerative diseasesinduced by oxidative stress and p38MAPK pharmacologicalinhibitors have a neuroprotective effect [31] In contrastERK12 promotes survival and enhances differentiation ofnerve cells [32] Additionally ERK12 controls direct or indi-rect antioxidant systems in various cells including neuronal


cells [33] In the present study we observed that p38MAPKwas rapidly activated after BSO exposure whereas ERK12was downregulated (Figure 2) Our results reveal that ACEand QCT may inhibit p38MAPK phosphorylation by acti-vating ERK12 reducing the second increase in ROS whichprotected the neuronal cells Activation of ERK12 wasnoticeable during the antioxidant effect of ACE and QCTand ultimately leads to neuroprotectionThis finding suggestsa beneficial effect of ERK12 against p38MAPK-mediatedoxidative stress Additionally oxidative stress-induced neu-ronal cell death mediated by activating MAPK kinases isattributable to modulating the activities of PKC isozymes[34] PKC-120576 is an importantmember of the PKC family that isactivated in multiple cell types and is believed to function asboth a proapoptotic and an antiapoptotic factor in differentmammalian cells [35] In addition we demonstrated thatBSO induced translocation of PKC-120576 from the cytosol to themembrane followed by increasedROSwhich led to neuronalcell death [2] Our results extended the importance of PKC-120576 in oxidative stress-induced neuronal cell death and weinvestigated a possible link between PKC-120576 and the neu-roprotective effects of ACE and QCT during BSO-inducedneuronal cell death Our results show that BSO increasedintracellular ROS levels which were reducedmarkedly by thePKC-120576 inhibitor 120576V1-2 by preventing activation of p38MAPKOne notable observation was that U0126 completely abol-ished the generation of H

2O2by ACE andQCT (Figure 3(d))

whereas the ACE and QCT-induced neuroprotective effectdisappeared partially after exposure to an ERK12 inhibitor(Figures 3(a) and 3(b)) These findings suggest that BSO-induced neurotoxicitymay be caused bymediators other thanH2O2generation and that these other mediators induced by

BSO may not be blocked by ACE or QCT Our results showfor the first time that ACE and QCT directly interfere withthe activation of PKC-120576 and p38MAPK and ameliorate theharmful effects of oxidative stress caused byBSOby activatingERK12 in neuronal cells Taken together our results providea better understanding of the molecular mechanism of ACEand QCT on protecting neuronal cells and suggest theirpotential therapeutic effects on various neurodegenerativediseases

Competing Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This research was supported by Basic Science ResearchProgram through the National Research Foundation ofKorea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01059515 and NRF-2015R1D1A1A01060069)Republic of Korea

References

[1] M R Jo D Y Choi Y M Lee et al ldquoNeuroprotective effect ofL-theanine on a120573-induced neurotoxicity through anti-oxidative

mechanisms in SK-N-SH and SK-N-MC cellsrdquo Biomolecules ampTherapeutics vol 19 no 3 pp 288ndash295 2011

[2] Y-S Jung B R Ryu B K Lee et al ldquoRole for PKC-120576 in neuronaldeath induced by oxidative stressrdquo Biochemical and BiophysicalResearch Communications vol 320 no 3 pp 789ndash794 2004

[3] A Elmann S Mordechay M Rindner O Larkov M Elkabetzand U Ravid ldquoProtective effects of the essential oil of Salviafruticosa and its constituents on astrocytic susceptibility tohydrogen peroxide-induced cell deathrdquo Journal of Agriculturaland Food Chemistry vol 57 no 15 pp 6636ndash6641 2009

[4] B-H Choi E-M Hur J-H Lee D-J Jun and K-T KimldquoProtein kinase C120575-mediated proteasomal degradation of MAPkinase phosphatase-1 contributes to glutamate-induced neu-ronal cell deathrdquo Journal of Cell Science vol 119 no 7 pp 1329ndash1340 2006

[5] K Aoyama MWatabe and T Nakaki ldquoRegulation of neuronalglutathione synthesisrdquo Journal of Pharmacological Sciences vol108 no 3 pp 227ndash238 2008

[6] Z Liu P Li D Zhao H Tang and J Guo ldquoProtectiveeffect of extract of Cordyceps sinensis in middle cerebral arteryocclusion-induced focal cerebral ischemia in ratsrdquo Behavioraland Brain Functions vol 6 article 61 2010

[7] S ParkM-Y KimDH Lee et al ldquoMethanolic extract of onion(Allium cepa) attenuates ischemiahypoxia-induced apoptosisin cardiomyocytes via antioxidant effectrdquo European Journal ofNutrition vol 48 no 4 pp 235ndash242 2009

[8] S-W Hyun M Jang S W Park E J Kim and Y-S JungldquoOnion (Allium cepa) extract attenuates brain edemardquo Nutri-tion vol 29 no 1 pp 244ndash249 2013

[9] R Shri and K Singh Bora ldquoNeuroprotective effect of methano-lic extracts ofAllium cepa on ischemia and reperfusion-inducedcerebral injuryrdquo Fitoterapia vol 79 no 2 pp 86ndash96 2008

[10] I K Hwang C H Lee K-Y Yoo et al ldquoNeuroprotectiveeffects of onion extract and quercetin against ischemic neuronaldamage in the gerbil hippocampusrdquo Journal of Medicinal Foodvol 12 no 5 pp 990ndash995 2009

[11] B J Gwag D Lobner J Y Koh M B Wie and D W ChoildquoBlockade of glutamate receptors unmasks neuronal apoptosisafter oxygen-glucose deprivation in vitrordquoNeuroscience vol 68no 3 pp 615ndash619 1995

[12] J E Lancaster and K E Kelly ldquoQuantitative analysis of theS-alk(en)yl-L-cysteine sulphoxides in onion (Allium cepa L)rdquoJournal of the Science of Food and Agriculture vol 34 no 11 pp1229ndash1235 1983

[13] W H Choi H D Park S H Baek J P Chu M H Kangand Y J Mi ldquoCannabidiol induces cytotoxicity and cell deathvia apoptotic pathway in cancer cell linesrdquo Biomolecules ampTherapeutics vol 16 no 2 pp 87ndash94 2008

[14] S Lim S-J Lee K-W Nam K H Kim and W Mar ldquoHep-atoprotective effects of reynosin against thioacetamide-inducedapoptosis in primary hepatocytes and mouse liverrdquo Archives ofPharmacal Research vol 36 no 4 pp 485ndash494 2013

[15] G-S Jeong E Byun B Li D-S Lee R-B An and Y-CKim ldquoNeuroprotective effects of constituents of the root barkofDictamnus dasycarpus in mouse hippocampal cellsrdquoArchivesof Pharmacal Research vol 33 no 8 pp 1269ndash1275 2010

[16] B K Lee J S YoonMG Lee andY-S Jung ldquoProtein kinaseC-120573 mediates neuronal activation of Na+H+ exchanger-1 duringglutamate excitotoxicityrdquo Cellular Signalling vol 26 no 4 pp697ndash704 2014


[17] E-J Yang G-S Kim J A Kim and K-S Song ldquoProtectiveeffects of onion-derived quercetin on glutamate-mediated hip-pocampal neuronal cell deathrdquo Pharmacognosy Magazine vol9 no 36 pp 302ndash308 2013

[18] S-K Park D-E Jin C-H Park et al ldquoAntioxidant activity andPC12 cell protective effect of onion flesh and peel(Allium cepaL) fraction on oxidative stressrdquo Journal of Agriculture amp LifeScience vol 49 no 2 pp 83ndash95 2015

[19] M J Kim C-H Moon M-Y Kim et al ldquoKR-32570 a novelNa+H+ exchanger-1 inhibitor attenuates hypoxia-induced celldeath through inhibition of intracellular Ca2+ overload andmitochondrial death pathway in H9c2 cellsrdquo European Journalof Pharmacology vol 525 no 1ndash3 pp 1ndash7 2005

[20] X Dai Y Ding Z Zhang X Cai and Y Li ldquoQuercetin andquercitrin protect against cytokine-induced injuries in RINm5F120573-cells via the mitochondrial pathway and NF-120581B signalingrdquoInternational Journal of Molecular Medicine vol 31 no 1 pp265ndash271 2013

[21] M M Alam D Meerza and I Naseem ldquoProtective effect ofquercetin on hyperglycemia oxidative stress and DNA damagein alloxan induced type 2 diabetic micerdquo Life Sciences vol 109no 1 pp 8ndash14 2014

[22] H Bas S Kalender and D Pandir ldquoIn vitro effects of quercetinon oxidative stress mediated in human erythrocytes by benzoicacid and citric acidrdquo Folia Biologica vol 62 no 1 pp 57ndash642014

[23] L Li X Zhang L Cui et al ldquoUrsolic acid promotes the neuro-protection by activating Nrf2 pathway after cerebral ischemia inmicerdquo Brain Research vol 1497 pp 32ndash39 2013

[24] S Bastianetto W-H Zheng and R Quirion ldquoThe Ginkgobiloba extract (EGb 761) protects and rescues hippocampalcells against nitric oxide-induced toxicity involvement of itsflavonoid constituents and protein kinase Crdquo Journal of Neuro-chemistry vol 74 no 6 pp 2268ndash2277 2000

[25] Y-W Ki J H Park J E Lee I C Shin and H C Koh ldquoJNKand p38 MAPK regulate oxidative stress and the inflammatoryresponse in chlorpyrifos-induced apoptosisrdquo Toxicology Lettersvol 218 no 3 pp 235ndash245 2013

[26] S-M Lee JMoon JHChung Y-J Cha andM-J Shin ldquoEffectof quercetin-rich onion peel extracts on arterial thrombosis inratsrdquo Food and Chemical Toxicology vol 57 pp 99ndash105 2013

[27] S Chuenkitiyanon T Pengsuparp and S Jianmongkol ldquoPro-tective effect of quercetin on hydrogen peroxide-induced tightjunction disruptionrdquo International Journal of Toxicology vol 29no 4 pp 418ndash424 2010

[28] P Cheng I Alberts and X Li ldquoThe role of ERK12 in theregulation of proliferation and differentiation of astrocytesin developing brainrdquo International Journal of DevelopmentalNeuroscience vol 31 no 8 pp 783ndash789 2013

[29] A M Tormos R Talens-Visconti A R Nebreda and J SastreldquoP38 MAPK a dual role in hepatocyte proliferation throughreactive oxygen speciesrdquo Free Radical Research vol 47 no 11pp 905ndash916 2013

[30] Z Xia M Dickens J Raingeaud R J Davis and M EGreenberg ldquoOpposing effects of ERK and JNK-p38 MAPkinases on apoptosisrdquo Science vol 270 no 5240 pp 1326ndash13311995

[31] W-B Song Y-Y Wang F-S Meng et al ldquoCurcumin protectsintestinal mucosal barrier function of rat enteritis via activationof MKP-1 and attenuation of p38 and NF-120581B activationrdquo PLoSONE vol 5 no 9 Article ID e12969 2010

[32] C J Marshall ldquoSpecificity of receptor tyrosine kinase signalingtransient versus sustained extracellular signal-regulated kinaseactivationrdquo Cell vol 80 no 2 pp 179ndash185 1995

[33] D-S Lee K-S Kim W Ko et al ldquoThe cytoprotective effectof sulfuretin against tert-Butyl Hydroperoxide-induced hepa-totoxicity through Nrf2ARE and JNKERK MAPK-mediatedHeme Oxygenase-1 expressionrdquo International Journal of Molec-ular Sciences vol 15 no 5 pp 8863ndash8877 2014

[34] P Maher ldquoHow protein kinase C activation protects nervecells from oxidative stress-induced cell deathrdquo The Journal ofNeuroscience vol 21 no 9 pp 2929ndash2938 2001

[35] T Nakajima ldquoSignaling cascades in radiation-induced apop-tosis roles of protein kinase C in the apoptosis regulationrdquoMedical Science Monitor vol 12 no 10 pp RA220ndashRA2242006


2 Materials and Methods

All experimental procedures were conducted in accordancewith the guidelines on the care and use of laboratory animalsset by the Animal Care Committee of Ajou University

21 Chemicals ACE was obtained from Konkuk UniversityBSO and QCT [2-(34-dihydroxyphenyl)-357-trihydroxy-4H-chromen-4-one] were purchased from Sigma (St LouisMO USA) Trolox [6-hydroxy-2578-tetramethylchroman-2-carboxylic acid] commonly used antioxidant was obtainedfrom Tocris (Ballwin MO USA) Z-DEVD-fmk (cas-pase inhibitor) 120576V1-2-peptide (PKC-120576 inhibitor) U0126[14-diamino-23-dicyano-14-bis(2-aminophenylthio) buta-diene] (ERK inhibitor) and SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] (p38MAPKinhibitor) were purchased from Biomol Research Labs Inc(Plymouth Meeting PA USA) Other reagents were of thehighest grade commercially available

22MouseMixedCorticalNeuronal Cell Culture Mouse neo-cortices were obtained from fetal mouse brains on embryonicdays 14-15 and grown in Eaglersquos minimum essential mediumsupplemented with 21mM glucose 5 fetal bovine serum5 horse serum and 2mM glutamine in 5 CO

2at 37∘C

as described previously [11] At DIV (days in vitro) 7 10 120583Mcytosine arabinofuranoside was added to the cultures to haltovergrowth of glial cells The cells were maintained for 13-14days and then used for experiments

23 Preparation of ACE and Treatment ACE was obtainedusing a method modified slightly from a protocol describedpreviously [12] Briefly after the outer skins or leaves of freshA cepa were removed 50 g of an A cepa bulb was homoge-nized in 70 methanol (100mL) and the homogenate wasfiltered through filter paper The resulting fractions werelyophilized using a vacuum evaporator (N-2N Eyela Tokyo)Lyophilized ACE was dissolved in cell culture medium Thecells were pretreated with ACE (1ndash10mgmL) and QCT (1ndash10 120583M) 30min before and they were not removed from theculture medium during the BSO treatment

24 Lactate Dehydrogenase (LDH) Assay We assayed LDHreleased into the medium after BSO treatment to measureoverall cell injury by spectrophotometric analysis at 340 nmas described previously [2 13] The percentage of LDH wascalculated from the maximum LDH release (100) inducedby lysing cells with 1 Triton X-100

25 Terminal Deoxynucleotidyl Transferase dUTP Nick EndLabeling (TUNEL) Staining Fragmented DNA was labeledin situ using an Apop Taq Plus kit (Millipore Gaithers-burg MD USA) Cells were grown on 24-well plates andfixed in 4 paraformaldehyde in PBS Nucleosome-sizedDNA fragments were tailed with digoxigenin nucleotide andreacted with fluorescein-conjugated antidigoxigenin anti-body as reported previously [14] Percent cell death wascalculated by expressing the number of TUNEL-positive cellsas a percentage of total cell count

26 Intracellular ROS Level We followed a method reportedpreviously to determine ROS levels [15] In brief cells grownon a glass-bottomed dish were loaded with 10 120583M6-carboxy-2101584071015840-dichlorodihydrofluorescein diacetate dicarboxymethylester (DCF-DA) in HCSS buffer containing 120 NaCl 5KCl 16 MgCl

2 23 CaCl

2 15 glucose 20 Hepes and 10

NaOH (mM pH 74) for 20min at 37∘C DCF-DA waspurchased from Molecular Probes (Eugene OR USA) Theexperiments were performed at room temperature on Aconfocal microscope stage and digitized using FLUOVIEWFV300 (Olympus Tokyo)

27 Isolation of ERK12 p38MAPK and JNK fromCell LysatesERK12 p38MAPKs and JNK were isolated as describedpreviously [16] Cells were harvested in RIPA buffer (150NaCl 20 Tris-HCl 1 NP-40 1 Na-deoxycholate 1 EDTAand protease inhibitors at pH 74 [mM]) and homogenizedand nuclei and cell debris were removed by centrifugation at10000timesg for 15min at 4∘C The supernatants were collectedfor immunoblotting Protein contents were determined usingthe BCA protein assay (Pierce Rockford IL USA) Proteinsamples were denatured in Laemmli buffer (4 SDS 20glycerol and 120mM Tris-HCl at pH 68) and total ERK12p38MAPKs and JNK as well as their phosphorylated formswere quantified by immunoblotting using polyclonal andmonoclonal antibodies against the proteins (Cell SignalingTechnology Danvers MA USA)

28 Subcellular Fractionation to Isolate PKC-120576 and Immuno-blotting PKC-120576 was subcellular fractionated as describedpreviously [16] Briefly cells were harvested in homoge-nization buffer (20 Tris-HCl 2 EDTA 5 EGTA 5 DTT 6120573-mercaptoethanol 1 PMSF 002 leupeptin and 10 120583gmLaprotinin pH 74 [mM]) and centrifuged at 100000timesg for 1 hat 4∘CThe supernatant was retained as the cytosolic fractionPellets were resuspended in 1 Triton X-100-containinghomogenization buffer and centrifuged at 10000timesg for10min at 4∘CThe supernatant is referred to as themembranefraction Protein content was determined using the Bradfordprotein assay (Biorad Hercules CA USA)The samples wereresolved on 8 SDS-polyacrylamide gels and transferred topolyvinylidene difluoride membranes (Millipore BedfordMA USA) The blots were incubated in 5 nonfat dry milkfor 1 h at room temperature and then incubated overnightat 4∘C with a polyclonal antibody against PKC-120576 (SantaCruz Biotechnology Santa Cruz CA USA) The blots wererinsed with Tris-buffered saline and incubated with horse-radish peroxidase-conjugated secondary IgG (Cell SignalingTechnology) for 1 h Bound antibody was detected withan enhanced 3D chemiluminescence kit (Intron DaejeonKorea) and the bands were analyzed using a LAS1000 (FujiPhoto Film Tokyo)

29 Statistical Analysis Data are expressed as mean plusmn stan-dard error of at least three separate determinations in eachgroup Numerical data were compared using Studentrsquos 119905-test or one-way ANOVA post hoc test for the unpairedobservations between the two groups A 119901 value lt 005 wasconsidered significant


3 Results









lowast

VEH 10

10

20

30

40

50

60

LDH

rele

ase (

m

ax)

QCT (120583M)BSO (24 h)

(a)

CTL

lowast



VEH 1QCT (120583M)

0

5

10

15

20

25

30

TUN

EL-p

ositi

ve ce

lls (

of t

otal

cells

)

BSO (24h)

(b)


4h

0h

(c)

0 2 41 8Time (h)


lowast

lowast

lowast lowast

80

100

120

140

160

180

DCF

-DA

inte

nsity

(d)





P-ERK

ERK

P-JNK

JNK

P-p38

p38

0 15 30 60 120 240BSO (min)

(a)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

(b)

lowast

lowast

CTL

VEH AC

E

QCT SB

U0126

BSO (4h)

0

50

100

150

200

250

D

CF-D

A in

tens

ity

(c)






DaggerDagger

lowast

0

10

20

30

40

50

60

70LD

H re

leas

e (

of m

ax)





minus + + + + + + + + +BSO

ACEQCT

SBU0126

(a)





minus + + + + + + + + +

lowast

Dagger Dagger

BSO

ACEQCT

SBU0126

0

10

20

30

40

TUN

EL-p

ositi

ve ce

lls (

of t

otal

cells

)

(b)

P-ERK

ERK

P-p38

p38

BSO (2h)

CTL

VEH

ACE

QCT

ACE+

U0126

QCT

+U0126

(c)

Daggerlowast

Dagger

0

50

100

150

200

250

D

CF-D

A in

tens

ity

BSO (4h)

CTL

VEH AC

E

QCT

ACE+

U0126

QCT

+U0126(d)





0 15 30 60 240BSO (min)

(C)

(M)

120

PKC-

120576

(a)

(C)

(M)

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

PKC-

120576

120576V1

-2

(b)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

120576V1

-2

BSO (2h)

(c)


BSO (24h)

lowast

0

20

40

60

80

LDH

rele

ase (

m

ax)

120576V1-2

(d)

TUN

EL-p

ositi

ve ce

lls


BSO (24h)

lowast

0

10

20

30

40

( o

f tot

al ce

lls)

120576V1-2

(e)


BSO (4h)

lowast

0

100

150

200

250

D

CF-D

A in

tens

ity

120576V1-2

(f)









Competing Interests


Acknowledgments


References







































3 Results









lowast

VEH 10

10

20

30

40

50

60

LDH

rele

ase (

m

ax)

QCT (120583M)BSO (24 h)

(a)

CTL

lowast



VEH 1QCT (120583M)

0

5

10

15

20

25

30

TUN

EL-p

ositi

ve ce

lls (

of t

otal

cells

)

BSO (24h)

(b)


4h

0h

(c)

0 2 41 8Time (h)


lowast

lowast

lowast lowast

80

100

120

140

160

180

DCF

-DA

inte

nsity

(d)





P-ERK

ERK

P-JNK

JNK

P-p38

p38

0 15 30 60 120 240BSO (min)

(a)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

(b)

lowast

lowast

CTL

VEH AC

E

QCT SB

U0126

BSO (4h)

0

50

100

150

200

250

D

CF-D

A in

tens

ity

(c)






DaggerDagger

lowast

0

10

20

30

40

50

60

70LD

H re

leas

e (

of m

ax)





minus + + + + + + + + +BSO

ACEQCT

SBU0126

(a)





minus + + + + + + + + +

lowast

Dagger Dagger

BSO

ACEQCT

SBU0126

0

10

20

30

40

TUN

EL-p

ositi

ve ce

lls (

of t

otal

cells

)

(b)

P-ERK

ERK

P-p38

p38

BSO (2h)

CTL

VEH

ACE

QCT

ACE+

U0126

QCT

+U0126

(c)

Daggerlowast

Dagger

0

50

100

150

200

250

D

CF-D

A in

tens

ity

BSO (4h)

CTL

VEH AC

E

QCT

ACE+

U0126

QCT

+U0126(d)





0 15 30 60 240BSO (min)

(C)

(M)

120

PKC-

120576

(a)

(C)

(M)

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

PKC-

120576

120576V1

-2

(b)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

120576V1

-2

BSO (2h)

(c)


BSO (24h)

lowast

0

20

40

60

80

LDH

rele

ase (

m

ax)

120576V1-2

(d)

TUN

EL-p

ositi

ve ce

lls


BSO (24h)

lowast

0

10

20

30

40

( o

f tot

al ce

lls)

120576V1-2

(e)


BSO (4h)

lowast

0

100

150

200

250

D

CF-D

A in

tens

ity

120576V1-2

(f)









Competing Interests


Acknowledgments


References








































lowast

VEH 10

10

20

30

40

50

60

LDH

rele

ase (

m

ax)

QCT (120583M)BSO (24 h)

(a)

CTL

lowast



VEH 1QCT (120583M)

0

5

10

15

20

25

30

TUN

EL-p

ositi

ve ce

lls (

of t

otal

cells

)

BSO (24h)

(b)


4h

0h

(c)

0 2 41 8Time (h)


lowast

lowast

lowast lowast

80

100

120

140

160

180

DCF

-DA

inte

nsity

(d)





P-ERK

ERK

P-JNK

JNK

P-p38

p38

0 15 30 60 120 240BSO (min)

(a)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

(b)

lowast

lowast

CTL

VEH AC

E

QCT SB

U0126

BSO (4h)

0

50

100

150

200

250

D

CF-D

A in

tens

ity

(c)






DaggerDagger

lowast

0

10

20

30

40

50

60

70LD

H re

leas

e (

of m

ax)





minus + + + + + + + + +BSO

ACEQCT

SBU0126

(a)





minus + + + + + + + + +

lowast

Dagger Dagger

BSO

ACEQCT

SBU0126

0

10

20

30

40

TUN

EL-p

ositi

ve ce

lls (

of t

otal

cells

)

(b)

P-ERK

ERK

P-p38

p38

BSO (2h)

CTL

VEH

ACE

QCT

ACE+

U0126

QCT

+U0126

(c)

Daggerlowast

Dagger

0

50

100

150

200

250

D

CF-D

A in

tens

ity

BSO (4h)

CTL

VEH AC

E

QCT

ACE+

U0126

QCT

+U0126(d)





0 15 30 60 240BSO (min)

(C)

(M)

120

PKC-

120576

(a)

(C)

(M)

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

PKC-

120576

120576V1

-2

(b)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

120576V1

-2

BSO (2h)

(c)


BSO (24h)

lowast

0

20

40

60

80

LDH

rele

ase (

m

ax)

120576V1-2

(d)

TUN

EL-p

ositi

ve ce

lls


BSO (24h)

lowast

0

10

20

30

40

( o

f tot

al ce

lls)

120576V1-2

(e)


BSO (4h)

lowast

0

100

150

200

250

D

CF-D

A in

tens

ity

120576V1-2

(f)









Competing Interests


Acknowledgments


References







































P-ERK

ERK

P-JNK

JNK

P-p38

p38

0 15 30 60 120 240BSO (min)

(a)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

(b)

lowast

lowast

CTL

VEH AC

E

QCT SB

U0126

BSO (4h)

0

50

100

150

200

250

D

CF-D

A in

tens

ity

(c)






DaggerDagger

lowast

0

10

20

30

40

50

60

70LD

H re

leas

e (

of m

ax)





minus + + + + + + + + +BSO

ACEQCT

SBU0126

(a)





minus + + + + + + + + +

lowast

Dagger Dagger

BSO

ACEQCT

SBU0126

0

10

20

30

40

TUN

EL-p

ositi

ve ce

lls (

of t

otal

cells

)

(b)

P-ERK

ERK

P-p38

p38

BSO (2h)

CTL

VEH

ACE

QCT

ACE+

U0126

QCT

+U0126

(c)

Daggerlowast

Dagger

0

50

100

150

200

250

D

CF-D

A in

tens

ity

BSO (4h)

CTL

VEH AC

E

QCT

ACE+

U0126

QCT

+U0126(d)





0 15 30 60 240BSO (min)

(C)

(M)

120

PKC-

120576

(a)

(C)

(M)

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

PKC-

120576

120576V1

-2

(b)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

120576V1

-2

BSO (2h)

(c)


BSO (24h)

lowast

0

20

40

60

80

LDH

rele

ase (

m

ax)

120576V1-2

(d)

TUN

EL-p

ositi

ve ce

lls


BSO (24h)

lowast

0

10

20

30

40

( o

f tot

al ce

lls)

120576V1-2

(e)


BSO (4h)

lowast

0

100

150

200

250

D

CF-D

A in

tens

ity

120576V1-2

(f)









Competing Interests


Acknowledgments


References







































DaggerDagger

lowast

0

10

20

30

40

50

60

70LD

H re

leas

e (

of m

ax)





minus + + + + + + + + +BSO

ACEQCT

SBU0126

(a)





minus + + + + + + + + +

lowast

Dagger Dagger

BSO

ACEQCT

SBU0126

0

10

20

30

40

TUN

EL-p

ositi

ve ce

lls (

of t

otal

cells

)

(b)

P-ERK

ERK

P-p38

p38

BSO (2h)

CTL

VEH

ACE

QCT

ACE+

U0126

QCT

+U0126

(c)

Daggerlowast

Dagger

0

50

100

150

200

250

D

CF-D

A in

tens

ity

BSO (4h)

CTL

VEH AC

E

QCT

ACE+

U0126

QCT

+U0126(d)





0 15 30 60 240BSO (min)

(C)

(M)

120

PKC-

120576

(a)

(C)

(M)

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

PKC-

120576

120576V1

-2

(b)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

120576V1

-2

BSO (2h)

(c)


BSO (24h)

lowast

0

20

40

60

80

LDH

rele

ase (

m

ax)

120576V1-2

(d)

TUN

EL-p

ositi

ve ce

lls


BSO (24h)

lowast

0

10

20

30

40

( o

f tot

al ce

lls)

120576V1-2

(e)


BSO (4h)

lowast

0

100

150

200

250

D

CF-D

A in

tens

ity

120576V1-2

(f)









Competing Interests


Acknowledgments


References







































0 15 30 60 240BSO (min)

(C)

(M)

120

PKC-

120576

(a)

(C)

(M)

CTL

VEH

ACE

QCT

SB U0126

BSO (2h)

PKC-

120576

120576V1

-2

(b)

P-ERK

ERK

P-p38

p38

CTL

VEH

ACE

QCT

120576V1

-2

BSO (2h)

(c)


BSO (24h)

lowast

0

20

40

60

80

LDH

rele

ase (

m

ax)

120576V1-2

(d)

TUN

EL-p

ositi

ve ce

lls


BSO (24h)

lowast

0

10

20

30

40

( o

f tot

al ce

lls)

120576V1-2

(e)


BSO (4h)

lowast

0

100

150

200

250

D

CF-D

A in

tens

ity

120576V1-2

(f)









Competing Interests


Acknowledgments


References











































Competing Interests


Acknowledgments


References


























































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