research article alpha-lipoic acid attenuates cerebral...

Research ArticleAlpha-Lipoic Acid Attenuates Cerebral Ischemia andReperfusion Injury via Insulin Receptor andPI3KAkt-Dependent Inhibition of NADPH Oxidase

Yinhua Dong Hongxin Wang and Zefeng Chen

Department of Neurology The Affiliated Fourth Centre Hospital of Tianjin Medical University Tianjin 300140 China

Correspondence should be addressed to Yinhua Dong dongyinhua2012163com

Received 27 September 2014 Revised 14 February 2015 Accepted 17 February 2015

Academic Editor Małgorzata Kotula-Balak

Copyright copy 2015 Yinhua Dong 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

Alpha-lipoic acid (ALA) has various pharmacological effects such as antioxidative anti-inflammatory and antiapoptotic propertiesIn the present study administration of ALA (40mgkg ip) for 3 days resulted in a significant decrease in neuronal deficit scoreand infarct volume and a significant increase in grip time and latency time in Morris water maze at 48 h after middle cerebralartery occlusion and reperfusion (MCAOR) in rats ALA also reduced the increased TUNEL-positive cells rate and the enhancedcaspase-3 activity induced by MCAOR However the underlying mechanisms remain poorly understood In this study we foundthat ALA could activate insulin receptor and PI3KAkt signaling pathways inhibit the expression and activity of NADPH oxidaseand subsequently suppress the generation of superoxide and the augment of oxidative stress indicators including MDA proteincarbonylation and 8-OHdG In conclusion ALA attenuates cerebral ischemia and reperfusion injury via insulin receptor andPI3KAkt-dependent inhibition of NADPH oxidase

1 Introduction

Ischemic stroke is a major cause of disability and the secondcause of death worldwide [1] Despite considerable progressin the understanding of the pathophysiology of ischemicstroke in recent years therapeutic options have until nowbeen limited The only approved drug for ischemic stroke isrecombinant tissue plasminogen activator [2] Neverthelesseven though blood flow is restored timely reperfusion canparadoxically exacerbate brain injury because of neuronaloxidative stress

Mechanistically oxidative stress resulting from the over-production of reactive oxygen species (ROS) is implicatedin the pathophysiology of cerebral ischemia and reperfusion(CIR) injury Based on this hypothesis ROS-scavengingantioxidants have been speculated to be neuroprotectiveagainst ischemic stroke However numerous ROS-scaveng-ing antioxidants have shown disappointing results in clinicaltrials Inhibiting ROS generation is a novel therapeuticapproach to suppress oxidative stress at its root [3] Howeverthe sources of ROS in CIR injury are largely unknown

Among the sources of ROS only NADPH oxidase canprimarily produce ROS as the primary production in CIRinjury [4] Previous study has demonstrated that NOX KOmice showed less brain infarction compared with wild-type(WT) mice after MCAOR [5]Therefore NADPH oxidase isa promising therapeutic target for ischemic stroke Moreoverthe activity of NADPHoxidase is reportedly regulated by sev-eral signaling pathways such as insulin receptor PI3KAktand MAPKs pathways [6ndash8]

Recent studies have demonstrated that endogenous anti-oxidants such as superoxide dismutase glutathione andalpha-lipoic acid (ALA) have neuroprotective effects [9ndash11]Several studies have indicated that ALA possesses numerousbiological effects including antioxidative anti-inflammatoryand antiapoptotic properties [12 13] ALA is reported toprovide neuroprotection against CIR injury via inhibitingoxidative stress [14 15] However whether the neuropro-tective effects of ALA against oxidative stress are due toinhibiting NADPH oxidase remains to be investigated ALAis reported to be a directly binding activator of the insulinreceptor [16] whether activation of insulin receptor induced

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2015 Article ID 903186 9 pageshttpdxdoiorg1011552015903186

2 International Journal of Endocrinology

by ALA is responsible for its neuroprotection against CIRinjury remains to be clarified

In the present study a rat model of middle cerebral arteryocclusionreperfusion (MCAOR) was used to investigate theneuroprotective effects of ALA We demonstrated that ALAattenuated CIR injury via insulin receptorAkt-dependentinhibition of NADPH oxidase

2 Methods

21 Materials ALA paraformaldehyde and 235-triphen-yltetrazolium chloride (TTC) were purchased from Sigma-Aldrich (MO USA) Tissue protein extraction kits the bicin-choninic acid assay (BCA) kits the primary antibodies andthe respective secondary antibodies were purchased fromSanta Cruz Biotechnology (CA USA) The malondialdehyde(MDA) detection kits and caspase-3 activity assay kits wereobtained from Nanjing Jiancheng Bioengineering Institute(Nanjing China) The ELISA kits for protein carbonyl and8-hydroxydeoxyguanosine (8-OHdG) were purchased fromCell Biolabs (CA USA)

22 ALA Solution Preparation ALA (80mgmL) was dis-solved in 10 of ethanol and sterilely filtered ALA solutionswere prepared immediately before use

23 Animals All animal protocols were performed in com-pliance with the National Institute of Health Guide for theCare and Use of Laboratory Animals and approved by theAnimal Ethics Committee of Tianjin Medical University Allefforts weremade tominimize the injuries experienced by theanimals as well as the number of animals used In this studyadult male Sprague-Dawley rats (Vital River LaboratoriesBeijing China) weighing 280ndash300 g were usedThe rats werehoused in a temperature (22∘C plusmn 1∘C) and humidity (60 plusmn10) controlled environment with a 12 12 h light-dark cycleand given free access to food andwaterThe rats (119899 = 60) wererandomly assigned to 3 groups that is the sham group (119899 =20) the MCAOR group (119899 = 20) and the ALA + MCAORgroup (119899 = 20) For theALA+MCAOR group ALA (40mgkg) was daily administered intraperitoneally (ip) to rats for 3days For the sham and MCAOR groups the rats were giventhe same amount of ethanol

24 Cerebral MCAOR Surgery Cerebral MCAOR was per-formed in rats following a previously described method [17]with slight modification by the same skilled investigatorBriefly the surgical procedure was performed under sterileconditions with autoclaved surgical instruments and mate-rials The rats were anesthetized using a face mask with35 halothane and maintained with 10ndash20 halothane in70 N

2O and 30 O

2 The common carotid artery internal

carotid artery (ICA) and external carotid artery (ECA) werecarefully exposed A 4-0 suture (Ethicon Inc NJ USA)was inserted into ICA through ECA and was gently pushedfurther to occlude the middle cerebral artery (MCA) Two hafter MCA occlusion reperfusion was achieved by carefulsuture removalThe incision was sewed after reperfusion was

confirmed using a laser Doppler flowmeter The rats in thesham group underwent the same surgical procedure withoutocclusion To relieve pain and discomfort in the postoperativeperiod topical lidocaine gel was applied on the wound ofrats The cranial temperature and rectal temperature weremaintained at 37 plusmn 05∘C using a feedback-regulated water-heating system Regional cerebral blood flow (rCBF) meanarterial blood pressure (MABP) pH arterial blood gases andblood glucose levels were monitored throughout surgery

25 Neurological Deficit Score At 48 h after reperfusion theneurological deficit (119899 = 20 for each group) was scored ina blinded fashion with regard to the experimental groupsaccording to a previously described method [18] with slightmodification 0 no deficit 1 mild forelimb weakness 2severe forelimb weakness 3 compulsory circling 4 nospontaneouswalkingwith depressed consciousness level and5 death

26 Grip Test At 48 h after reperfusion forepaw grip test(119899 = 20 for each group) was performed to evaluate forceand fatigability of rats by two investigators who are blinded tothe experimental groups A metallic rope was stretched hori-zontally 50 cm over a cotton padThe rats were suspended onthe rope by forepaws and the grip time was recorded

27 Rota-Rod The motor and coordination of rats wasdetected using Rota-rod Before surgery the rats (119899 = 20for each group) were familiarized with the apparatus At 48 hafter reperfusion the rats were placed on the rotating shaftand the rotate speed was increased from 4 to 40 rmin Thelatency time was recorded automatically by the apparatuswhen the rat fell off the rotating shaft

28 Cerebral Infarct Volume Cerebral infarct volume wasdetermined by TTC staining At 48 h after reperfusion therats (119899 = 10 for each group) were decapitated The brainswere removed quickly and then frozen at minus80∘C for 15minand then sliced into consecutive 2mm coronal sections Thebrain slices were incubated with 2 TTC at 37∘C for 30minand then incubated with 10 paraformaldehyde overnightThe images were captured and the corrected infarct volumewas calculated according to a previously described method[19]

29 Terminal Deoxynucleotidyl Transferase dUTP Nick EndLabeling (TUNEL) Apoptotic neurons were detected byTUNEL using ApopTag Fluorescein In Situ Apoptosis Detec-tion Kit (Millipore MA USA) in compliance with the man-ufacturerrsquos protocol At 48 h after reperfusion the rats (119899 = 5for each group) were decapitated The brains were removedrapidly embedded and sliced in a cryostat (20120583m thickness100 120583m intervals) After deparaffinization and rehydrationthe brain slices were incubated with proteinase K (20120583gmL)for 15min and incubated in 3 H

2O2for 5min The

brain slices were then incubated with working-strength TdTenzyme at 37∘C for 1 h in a humidified chamber After rinsingin the stopwash buffer the brain slices were incubated

International Journal of Endocrinology 3

with the working-strength antidigoxigenin conjugate for30min The nucleus was counterstained by 410158406-diamidino-2-phenylindole (DAPI 01 120583gmL)The images were capturedusing a fluorescence microscope (Leica Germany)

210 Western Blot The whole cell lysates of cortical samples(119899 = 5 for each group) were obtained using tissue pro-tein extraction kits supplemented with protease inhibitors(Roche IN USA) The total protein concentration wasdetermined using BCA kits An equal amount of protein wasseparated by electrophoresis in 10 sodium dodecyl sulfatepolyacrylamide gels and then transferred to nitrocellulosemembranes After blocking with 5 nonfat milk the blotswere incubated with the primary antibodies overnight at 4∘CAfter washing with TBST the blots were then incubated withthe respective secondary antibodies for 2 h and developedby chemiluminescence Relative optical densities of the blotswere quantified by densitometry

211 NADPH Oxidase Activity Assay NADPH oxidase activ-ity was detected by superoxide dismutase-inhibitable cyto-chrome c reduction assay [20] Briefly tissue homogenate(119899 = 5 per group) was distributed in 96-well plates (200 120583gwell) NADPH (100 120583molL) and cytochrome c (500 120583molL)were added in the presence or absence of SOD (200UmL)and then incubated for 30minThe absorbancewasmeasuredat 550 nm using amicroplate reader NADPH oxidase activitywas expressed as the reduced cytochrome c per mg proteinper min as relative absorbance units (RAU)

212 Detection of Superoxide Production Lucigenin-en-hanced chemiluminescence was employed to measure theproduction of superoxide [21] Briefly cortical homogenate(119899 = 5 per group) was distributed in white 96-well plates(100 120583gwell) Lucigenin (5 120583molL) and NADPH (100120583molL) were separately added immediately before reading using amicroplate luminometer The level of superoxide productionwas expressed as the percentage of the sham group

213 Detection of Oxidative Stress and Apoptosis RelatedParameters The total protein concentration of the corticalhomogenate (119899 = 5 for each group) was determined usingBCA kits The levels of MDA and caspase-3 activity weredetected using respective assay kits The levels of proteincarbonyl and 8-OHdG were determined by respective ELISAkits The levels of caspase-3 activity protein carbonyl and 8-OHdG were expressed as the percentage of the sham group

214 Statistical Analysis Data were expressed as means plusmnstandard error of the mean (SEM) The results were analyzedby one-way ANOVA followed by Tukeyrsquos test or two-wayANOVA followed by Bonferronirsquos multiple comparison test119875 lt 005 was considered statistically significant

3 Results

31 Physiologic Parameters As shown in supplementaryTable 1 in Supplementary Material available online at

httpdxdoiorg1011552015903186 before occlusion dur-ing occlusion and after reperfusion no significant intergroupdifference was noted in the physiologic parameters includingcranial temperature rectal temperature MABP glucose pHPO2 and PCO

2(119875 gt 005) and no significant difference was

observed in rCBF between MCAOR and ALA + MCAORgroups

32 The Cytotoxicity and Dose Selection of ALA To explorethe cytotoxicity of ALA ALA (20 40 and 80mgkg ip)was administered to rats for 3 days As seen in supplemen-tary Figure 1 ALA (20 40 and 80mgkg) did not affectthe caspase-3 activity NADPH oxidase activity NADPHoxidase-derived superoxide production and the levels ofMDA protein carbonyl and 8-OHdG in cortex of rats Toinvestigate the protective effects of ALA against CIR injuryALA (20 40 and 80mgkg ip) was administered to ratsfor 3 days before surgery As seen in supplementary Figure 2at the dose of 40mgkg ALA provided the maximum neu-roprotection against MCAOR-induced neurologic deficitsand cerebral infarction Moreover neurologic deficits andcerebral infarction induced by MCAOR were attenuated bypretreatment but not posttreatment with ALA

33 ALAAmelioratedMCAOR-InducedNeurological DeficitsNeurological function was evaluated by neurological deficitscore grip time test and Rota-rod test at 48 h after reper-fusion Compared to the sham group MCAOR-treated ratsdisplayed a significant increase in neurological deficit score(Figure 1(a) 119875 lt 001) a remarkable decrease in grip time(Figure 1(b) 119875 lt 001) and a striking reduction in Rota-rodlatency time (Figure 1(c) 119875 lt 001) These results indicatedthat MCAOR induced a significant neurological deficit inrats However ALA significantly reversed these MCAOR-induced changes in neurological function (Figures 1(a) 1(b)and 1(c) 119875 lt 001)

34 ALA Attenuated MCAOR-Induced Cerebral InfarctionCerebral infarctionwas determined by TTC stainingThe ratsin the sham group displayed no cerebral infarction Howevera significant increase in cerebral infarct volume was observedin MCAOR-treated rats (Figures 1(d) and 1(e) 119875 lt 001)Conversely ALA remarkably reduced the augmented cerebralinfarct volume induced by MCAOR (Figures 1(d) and 1(e)119875 lt 001)

35 ALA Reduced MCAOR-Induced Neuronal ApoptosisDNA fragmentation and caspase-3 activity were detectedto investigate whether ALA could affect MCAOR-inducedneuronal apoptosisThe number of TUNEL-positive cells wassignificantly higher in the cortex of MCAOR-treated rats at48 h after reperfusion compared with the sham group (Fig-ures 2(a) and 2(b) 119875 lt 001) Consistent with these obser-vations caspase-3 activity in the cortex of MCAOR-treatedrats was also significantly enhanced compared to the shamgroup (Figure 2(c) 119875 lt 001) In contrast ALA considerablyinhibited MCAOR-induced neuronal apoptosis (Figure 2119875 lt 001)


Neu

rolo

gica

l defi

cit s

core

4

3

2

1

0

Sham MCAOR

lowastlowast

ALA + MCAOR

(a)

Grip

tim

e (s)

400

300

200

100

0

lowastlowast

Sham MCAOR ALA + MCAOR

(b)

Late

ncy

time (

s)

400

300

200

100

0

lowastlowast


(c)

Cor

rect

ed in

frac

t vol

ume (

mm3)

300

250

200

150

100

50

0

lowastlowast


(d)


(e)

Figure 1 ALA improved neurological functions and attenuated MCAOR-induced infarct volume (a) ALA attenuated MCAOR-inducedneurological deficit (b)ALAamelioratedMCAOR-induced reduction of grip time (c)ALAattenuatedMCAOR-induced decrease in latencytime in Morris water maze (d) Quantitative analysis of cerebral infarct volume (e) ALA inhibited MCAOR-induced cerebral infarct Forthe ALA +MCAOR group ALA (40mgkg ip) was daily administered to rats for 3 days For the sham andMCAOR groups the rats weregiven the same amount of ethanol Data were expressed as means plusmn SEM 119875 lt 001 compared with the sham group lowastlowast119875 lt 001 comparedwith the MCAOR group


Sham MCAOR ALA + MCAORD

API

TUN

ELM

erge

(a)

lowastlowast


TUN

EL-p

ositi

ve ce

lls ra

te (

)

80

60

40

20

0

(b)

lowastlowast


Casp

ase-3

activ

ity (

of c

ontro

l)

500

400

300

200

100

0

(c)

Figure 2ALAattenuatedMCAOR-inducedneuronal apoptosis (a)DNA fragmentation in apoptotic neuronal cells was detected byTUNELarrows represent TUNEL-positive cells bar = 50120583m (c) Quantitative analysis of TUNEL-positive cells rate For the ALA +MCAOR groupALA (40mgkg ip) was daily administered to rats for 3 days For the sham and MCAOR groups the rats were given the same amount ofethanol Data were expressed as means plusmn SEM 119875 lt 001 compared with the sham group lowastlowast119875 lt 001 compared with the MCAOR group

36 ALA Inhibited NADPH Oxidase through Activation ofInsulin Receptor and PI3KAkt Signaling Pathway p47-phoxand gp91-phox two subunits of NADPH oxidase weresignificantly higher in the cortex of MCAOR-treated ratsthan that of the sham group However the increased expres-sion of p47-phox and gp91-phox induced by MCAOR was

effectively inhibited by ALA Interestingly ALA could inducethe phosphorylation of Akt Importantly all these effectsmediated by ALA could be abolished by coadministrationwith PI3K specific inhibitor wortmannin (WM 03mgkgip) or insulin receptor inhibitor HNMPA-(AM)

3(5mgkg

ip) for 3 days (Figures 3(a) and 3(b) 119875 lt 001) Moreover


p-insulin receptor 120573

Total insulin receptor 120573

p-Akt

Total Akt

P47-phox

gp91-phox

120573-actin

MCAOR

+

MCAOR

ALA+

MCAORSham MCAOR

ALA ALA+

+

+

WM HNMPA

(a)

p-IR120573

IR120573

ratio

( o

f sha

m)

400

300

200

100

0

p-A

ktA

kt ra

tio (

of s

ham

)250

200

150

100

50

0

400

300

200

100

0

gp91

-pho

x ex

pres

sion

( o

f sha

m)

400

300

200

100

0

ShamMCAORALA + MCAOR

WM + ALA + MCAORHNMPA + ALA + MCAOR







$$$$$$$$

lowastlowast lowastlowast

lowastlowastlowastlowast


p47-

phox

expr

essio

n (

of s

ham

)

(b)

Figure 3 ALA inhibited NADPH oxidase through activation of insulin receptor and PI3KAkt signaling (a) The expression of p-insulinreceptor total insulin receptor p-Akt total Akt p47-phox and gp91-phox (b) Quantitative analysis of the expression of proteins For theALA + MCAOR group ALA (40mgkg ip) was daily administered to rats for 3 days For the sham and MCAOR groups the rats weregiven the same amount of ethanol Data were expressed as means plusmn SEM 119875 lt 001 compared with the sham group lowastlowast119875 lt 001 comparedwith the MCAOR group $$119875 lt 001 compared with the ALA + MCAOR group




$$$$


NA

DPH

oxi

dase

activ

ity (R

AU)

80

60

40

20

0

VAS2870 + MCAOR

(a)

$$$$


Supe

roxi

de (

of s

harm

)

600

400

200

0



VAS2870 + MCAOR

(b)

$$$$


MD

A (n

mol

mg

prot

ein)

40

30

20

10

0



VAS2870 + MCAOR

(c)

$$ $$


Prot

ein

carb

onyl

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(d)

$$$$


8-O

HdG

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(e)

Figure 4 ALA suppressedMCAOR-induced oxidative stress (a) ALA inhibitedMCAOR-induced upregulation ofNADPHoxidase activity(b) ALA suppressed MCAOR-induced superoxide generation and the increment of oxidative stress indicators including MDA (c) proteincarbonyl (d) and 8-OHdG (e) For the ALA + MCAOR group ALA (40mgkg ip) was daily administered to rats for 3 days For the shamand MCAOR groups the rats were given the same amount of ethanol Data were expressed as means plusmn SEM 119875 lt 001 compared with thesham group lowastlowast119875 lt 001 compared with the MCAOR group $$119875 lt 001 compared with the ALA + MCAOR group


ALA could induce the phosphorylation of insulin receptorwhich was abrogated by HNMPA-(AM)

3but not WM

These results suggested that ALA inhibited NADPH oxidasethrough activation of insulin receptor andPI3KAkt signalingpathway

37 ALA Suppressed MCAOR-Induced Oxidative Stress Anoticeable increment of NADPH oxidase activity and aremarkable augment of superoxide productionwere observedin the cortex of MCAOR-treated rats (Figures 4(a) and 4(b)119875 lt 001) In accordancewith the increasedNADPHoxidase-derived superoxide production MCAOR resulted in a sig-nificant increase in the levels of MDA protein carbonyl and8-OHdG (Figures 4(c) 4(d) and 4(e) 119875 lt 001) In contrastALA could effectively suppress MCAOR-induced oxidativestress in the cortex of rats (Figure 4 119875 lt 001) Howeverall these effects mediated by ALA could be abrogated bycoadministration with NADPH oxidase inhibitor VAS2870(2mgkg ip) or PI3K specific inhibitor wortmannin (WM03mgkg ip) or insulin receptor inhibitor HNMPA-(AM)

3

(5mgkg ip) for 3 days (Figure 4 119875 lt 001)

4 Discussion

In the present study we reported the neuroprotective effectsof ALA against CIR injury We found that administrationof ALA (40mgkg ip) for 3 days significantly amelioratedbrain injury induced byMCAOR in rats In accordance withprevious studies [14 15] MCAOR-induced neurologicaldeficits were effectively attenuated by ALA as determinedby grip test and Rota-rod test Furthermore ALA couldmarkedly reduce MCAOR-induced infarct volume and neu-ronal apoptosis However themolecularmechanismwherebyALA exerts its neuroprotection is largely unknown

Oxidative stress induced by excessive production of ROSplays a crucial role in the pathogenesis of CIR injury [22]Excessive ROS directly oxidize several biomacromoleculessuch as lipids proteins and DNA and indirectly activatesignaling pathways resulting in cellular dysfunction and cellapoptosis [23] Thus suppressing ROS production may bea novel strategy for neuroprotection against CIR injury Inthis study ALA could inhibit MCAOR-induced superoxidegeneration Subsequently the elevated levels of oxidativestress indicators including MDA protein carbonyl and 8-OHdG induced by MCAOR were suppressed by ALANevertheless the underlying mechanism by which ALAsuppresses MCAOR-induced oxidative stress merits deeperinvestigation

Multiple studies have proved that NADPH oxidase isthe major source of ROS during ischemic stroke [24 25]To implore the mechanism underlying the neuroprotec-tive effects of ALA we focused on NADPH oxidase Inthis study ALA significantly inhibited MCAOR-inducedupregulation of p47-phox and gp91-phox At the same timeMCAOR-induced increased NADPH oxidase activity wasalso abolished by ALA Importantly ALA-mediated neu-roprotection against oxidative stress was completely abol-ished by VAS2870 These results suggested that inhibiting

NADPH oxidase might play a pivotal role in ALA-mediatedneuroprotection against CIR injury However the molecularmechanism bywhich ALA inhibits NADPHoxidase is largelyunknown

Since ALA has been reported to directly modify insulinreceptor [26] the possibility that ALA activates the insulinreceptor in CIR injury was evaluated As expected ALAelicited a pronounced phosphorylation of insulin receptorsuggesting that ALA could activate insulin receptor Con-sidering that PI3KAkt signaling pathway is a prototypicalevent downstream of insulin receptor whether ALA couldactivate PI3KAkt pathway was further evaluated The phos-phorylated Akt was significantly increased in ALA-treatedmice which indicated that ALA could activate PI3KAktpathway Importantly ALA-mediated PI3KAkt activationwas abrogated by an insulin receptor specific inhibitorHNMPA-(AM)

3or a PI3KAkt specific inhibitor wortman-

nin These results suggested that ALA induced insulinreceptor-dependent activation of PI3KAkt signaling path-ways Interestingly the ALA-mediated inhibition of NADPHoxidase and suppression of oxidative stress were also abol-ished by HNMPA-(AM)

3or wortmannin suggesting that

activating insulin receptor and PI3KAkt signaling pathwaysby ALA results in NADPH oxidase inhibition Neverthelessthe critical components downstream of PI3KAkt throughwhich ALA inhibits NADPH oxidase remain unclear

In conclusion ALA provides neuroprotective effectsagainst MCAOR-induced brain injury The mechanismunderlying this neuroprotection involves insulin receptorand PI3KAkt-dependent inhibition of NADPH oxidase

Conflict of Interests

The authors declare that they have no competing interests

References

[1] G ADonnanM FisherMMacleod and SMDavis ldquoStrokerdquoThe Lancet vol 371 no 9624 pp 1612ndash1623 2008

[2] J E Cohen and R R Leker ldquoIntravenous thrombolytic therapyfor acute ischemic strokerdquoTheNewEngland Journal ofMedicinevol 365 no 10 pp 964ndash965 2011

[3] K A Radermacher K Wingler P Kleikers et al ldquoThe 1027thtarget candidate in stroke will NADPH oxidase hold uprdquoExperimental amp Translational Stroke Medicine vol 4 no 1article 11 2012

[4] J-M Li N P Gall D J Grieve M Chen and A M ShahldquoActivation of NADPH oxidase during progression of cardiachypertrophy to failurerdquo Hypertension vol 40 no 4 pp 477ndash484 2002

[5] H Chen G S Kim N Okami P Narasimhan and P HChan ldquoNADPH oxidase is involved in post-ischemic braininflammationrdquo Neurobiology of Disease vol 42 no 3 pp 341ndash348 2011

[6] J Du L M Fan A Mai and J-M Li ldquoCrucial roles of Nox2-derived oxidative stress in deteriorating the function of insulinreceptors and endothelium in dietary obesity of middle-agedmicerdquo British Journal of Pharmacology vol 170 no 5 pp 1064ndash1077 2013


[7] C-Y Lu Y-C Yang C-C Li K-L Liu C-K Lii and H-W Chen ldquoAndrographolide inhibits TNF120572-induced ICAM-1 expression via suppression of NADPH oxidase activa-tion and induction of HO-1 and GCLM expression throughthe PI3KAktNrf2 and PI3KAktAP-1 pathways in humanendothelial cellsrdquo Biochemical Pharmacology vol 91 no 1 pp40ndash50 2014

[8] D Pandey and D J R Fulton ldquoMolecular regulation ofNADPH oxidase 5 via the MAPK pathwayrdquo American Journalof PhysiologymdashHeart and Circulatory Physiology vol 300 no 4pp H1336ndashH1344 2011

[9] W Kim D W Kim D Y Yoo et al ldquoNeuroprotective effectsof PEP-1-CuZn-SOD against ischemic neuronal damage in therabbit spinal cordrdquo Neurochemical Research vol 37 no 2 pp307ndash313 2012

[10] K Mizuno T Kume C Muto et al ldquoGlutathione biosynthesisvia activation of the nuclear factor E2-related factor 2 (Nrf2)mdashantioxidant-response element (ARE) pathway is essential forneuroprotective effects of sulforaphane and 6-(methylsulfinyl)hexyl isothiocyanaterdquo Journal of Pharmacological Sciences vol115 no 3 pp 320ndash328 2011

[11] M Sayin P Temiz A Var and C Temiz ldquoThe dose-dependentneuroprotective effect of alpha-lipoic acid in experimentalspinal cord injuryrdquo Neurologia i Neurochirurgia Polska vol 47no 4 pp 345ndash351 2013

[12] A Ahmadi N Mazooji J Roozbeh Z Mazloom and JHasanzade ldquoEffect of alpha-lipoic acid and vitamin E supple-mentation on oxidative stress inflammation and malnutritionin hemodialysis patientsrdquo Iranian Journal of Kidney Diseasesvol 7 no 6 pp 461ndash467 2013

[13] Y Yang W Wang Y Liu et al ldquo120572-lipoic acid inhibits highglucose-induced apoptosis in HIT-T15 cellsrdquo DevelopmentGrowth amp Differentiation vol 54 no 5 pp 557ndash565 2012

[14] B J Connell M Saleh B V Khan and TM Saleh ldquoLipoic acidprotects against reperfusion injury in the early stages of cerebralischemiardquo Brain Research vol 1375 pp 128ndash136 2011

[15] W M Clark L G Rinker N S Lessov S L Lowery and MJ Cipolla ldquoEfficacy of antioxidant therapies in transient focalischemia in micerdquo Stroke vol 32 no 4 pp 1000ndash1004 2001

[16] B Diesel S Kulhanek-HeinzeMHoltje et al ldquo120572-Lipoic acid asa directly binding activator of the insulin receptor protectionfrom hepatocyte apoptosisrdquo Biochemistry vol 46 no 8 pp2146ndash2155 2007

[17] S S Raza M M Khan A Ahmad et al ldquoNeuroprotectiveeffect of naringenin is mediated through suppression of NF-120581B signaling pathway in experimental strokerdquoNeuroscience vol230 pp 157ndash171 2013

[18] E Z Longa P R Weinstein S Carlson and R CumminsldquoReversible middle cerebral artery occlusion without craniec-tomy in ratsrdquo Stroke vol 20 no 1 pp 84ndash91 1989

[19] L Belayev O F Alonso R BustoW Zhao andM D GinsbergldquoMiddle cerebral artery occlusion in the rat by intraluminalsuture neurological and pathological evaluation of an improvedmodelrdquo Stroke vol 27 no 9 pp 1616ndash1623 1996

[20] J-M Li A M Mullen S Yun et al ldquoEssential role of theNADPH oxidase subunit p47119901ℎ119900119909 in endothelial cell superoxideproduction in response to phorbol ester and tumor necrosisfactor-120572rdquo Circulation Research vol 90 no 2 pp 143ndash150 2002

[21] M-A Barbacanne J-P Souchard B Darblade et al ldquoDetectionof superoxide anion released extracellularly by endothelial cells

using cytochrome c reduction ESR fluorescence and lucigenin-enhanced chemiluminescence techniquesrdquo Free Radical Biologyamp Medicine vol 29 no 5 pp 388ndash396 2000

[22] R Rodrigo R Fernandez-Gajardo R Gutierrez et al ldquoOxida-tive stress and pathophysiology of ischemic stroke novel ther-apeutic opportunitiesrdquo CNS amp Neurological DisordersmdashDrugTargets vol 12 no 5 pp 698ndash714 2013

[23] K Sinha J Das P B Pal and P C Sil ldquoOxidative stressthe mitochondria-dependent and mitochondria-independentpathways of apoptosisrdquo Archives of Toxicology vol 87 no 7 pp1157ndash1180 2013

[24] C EWalder S P GreenWCDarbonne et al ldquoIschemic strokeinjury is reduced inmice lacking a functional NADPHoxidaserdquoStroke vol 28 no 11 pp 2252ndash2258 1997

[25] H Chen Y S Song and P H Chan ldquoInhibition of NADPHoxidase is neuroprotective after ischemia-reperfusionrdquo Journalof Cerebral Blood Flow amp Metabolism vol 29 no 7 pp 1262ndash1272 2009

[26] D Konrad ldquoUtilization of the insulin-signaling network in themetabolic actions of alpha-lipoic acidmdashreduction or oxida-tionrdquo Antioxidants amp Redox Signaling vol 7 no 7-8 pp 1032ndash1039 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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


by ALA is responsible for its neuroprotection against CIRinjury remains to be clarified

In the present study a rat model of middle cerebral arteryocclusionreperfusion (MCAOR) was used to investigate theneuroprotective effects of ALA We demonstrated that ALAattenuated CIR injury via insulin receptorAkt-dependentinhibition of NADPH oxidase

2 Methods

21 Materials ALA paraformaldehyde and 235-triphen-yltetrazolium chloride (TTC) were purchased from Sigma-Aldrich (MO USA) Tissue protein extraction kits the bicin-choninic acid assay (BCA) kits the primary antibodies andthe respective secondary antibodies were purchased fromSanta Cruz Biotechnology (CA USA) The malondialdehyde(MDA) detection kits and caspase-3 activity assay kits wereobtained from Nanjing Jiancheng Bioengineering Institute(Nanjing China) The ELISA kits for protein carbonyl and8-hydroxydeoxyguanosine (8-OHdG) were purchased fromCell Biolabs (CA USA)

22 ALA Solution Preparation ALA (80mgmL) was dis-solved in 10 of ethanol and sterilely filtered ALA solutionswere prepared immediately before use

23 Animals All animal protocols were performed in com-pliance with the National Institute of Health Guide for theCare and Use of Laboratory Animals and approved by theAnimal Ethics Committee of Tianjin Medical University Allefforts weremade tominimize the injuries experienced by theanimals as well as the number of animals used In this studyadult male Sprague-Dawley rats (Vital River LaboratoriesBeijing China) weighing 280ndash300 g were usedThe rats werehoused in a temperature (22∘C plusmn 1∘C) and humidity (60 plusmn10) controlled environment with a 12 12 h light-dark cycleand given free access to food andwaterThe rats (119899 = 60) wererandomly assigned to 3 groups that is the sham group (119899 =20) the MCAOR group (119899 = 20) and the ALA + MCAORgroup (119899 = 20) For theALA+MCAOR group ALA (40mgkg) was daily administered intraperitoneally (ip) to rats for 3days For the sham and MCAOR groups the rats were giventhe same amount of ethanol

24 Cerebral MCAOR Surgery Cerebral MCAOR was per-formed in rats following a previously described method [17]with slight modification by the same skilled investigatorBriefly the surgical procedure was performed under sterileconditions with autoclaved surgical instruments and mate-rials The rats were anesthetized using a face mask with35 halothane and maintained with 10ndash20 halothane in70 N

2O and 30 O

2 The common carotid artery internal

carotid artery (ICA) and external carotid artery (ECA) werecarefully exposed A 4-0 suture (Ethicon Inc NJ USA)was inserted into ICA through ECA and was gently pushedfurther to occlude the middle cerebral artery (MCA) Two hafter MCA occlusion reperfusion was achieved by carefulsuture removalThe incision was sewed after reperfusion was

confirmed using a laser Doppler flowmeter The rats in thesham group underwent the same surgical procedure withoutocclusion To relieve pain and discomfort in the postoperativeperiod topical lidocaine gel was applied on the wound ofrats The cranial temperature and rectal temperature weremaintained at 37 plusmn 05∘C using a feedback-regulated water-heating system Regional cerebral blood flow (rCBF) meanarterial blood pressure (MABP) pH arterial blood gases andblood glucose levels were monitored throughout surgery

25 Neurological Deficit Score At 48 h after reperfusion theneurological deficit (119899 = 20 for each group) was scored ina blinded fashion with regard to the experimental groupsaccording to a previously described method [18] with slightmodification 0 no deficit 1 mild forelimb weakness 2severe forelimb weakness 3 compulsory circling 4 nospontaneouswalkingwith depressed consciousness level and5 death

26 Grip Test At 48 h after reperfusion forepaw grip test(119899 = 20 for each group) was performed to evaluate forceand fatigability of rats by two investigators who are blinded tothe experimental groups A metallic rope was stretched hori-zontally 50 cm over a cotton padThe rats were suspended onthe rope by forepaws and the grip time was recorded

27 Rota-Rod The motor and coordination of rats wasdetected using Rota-rod Before surgery the rats (119899 = 20for each group) were familiarized with the apparatus At 48 hafter reperfusion the rats were placed on the rotating shaftand the rotate speed was increased from 4 to 40 rmin Thelatency time was recorded automatically by the apparatuswhen the rat fell off the rotating shaft

28 Cerebral Infarct Volume Cerebral infarct volume wasdetermined by TTC staining At 48 h after reperfusion therats (119899 = 10 for each group) were decapitated The brainswere removed quickly and then frozen at minus80∘C for 15minand then sliced into consecutive 2mm coronal sections Thebrain slices were incubated with 2 TTC at 37∘C for 30minand then incubated with 10 paraformaldehyde overnightThe images were captured and the corrected infarct volumewas calculated according to a previously described method[19]

29 Terminal Deoxynucleotidyl Transferase dUTP Nick EndLabeling (TUNEL) Apoptotic neurons were detected byTUNEL using ApopTag Fluorescein In Situ Apoptosis Detec-tion Kit (Millipore MA USA) in compliance with the man-ufacturerrsquos protocol At 48 h after reperfusion the rats (119899 = 5for each group) were decapitated The brains were removedrapidly embedded and sliced in a cryostat (20120583m thickness100 120583m intervals) After deparaffinization and rehydrationthe brain slices were incubated with proteinase K (20120583gmL)for 15min and incubated in 3 H

2O2for 5min The

brain slices were then incubated with working-strength TdTenzyme at 37∘C for 1 h in a humidified chamber After rinsingin the stopwash buffer the brain slices were incubated








3 Results










Neu

rolo

gica

l defi

cit s

core

4

3

2

1

0

Sham MCAOR

lowastlowast

ALA + MCAOR

(a)

Grip

tim

e (s)

400

300

200

100

0

lowastlowast


(b)

Late

ncy

time (

s)

400

300

200

100

0

lowastlowast


(c)

Cor

rect

ed in

frac

t vol

ume (

mm3)

300

250

200

150

100

50

0

lowastlowast


(d)


(e)




API

TUN

ELM

erge

(a)

lowastlowast


TUN

EL-p

ositi

ve ce

lls ra

te (

)

80

60

40

20

0

(b)

lowastlowast


Casp

ase-3

activ

ity (

of c

ontro

l)

500

400

300

200

100

0

(c)




3(5mgkg





p-Akt

Total Akt

P47-phox

gp91-phox

120573-actin

MCAOR

+

MCAOR

ALA+

MCAORSham MCAOR

ALA ALA+

+

+

WM HNMPA

(a)

p-IR120573

IR120573

ratio

( o

f sha

m)

400

300

200

100

0

p-A

ktA

kt ra

tio (

of s

ham

)250

200

150

100

50

0

400

300

200

100

0

gp91

-pho

x ex

pres

sion

( o

f sha

m)

400

300

200

100

0









$$$$$$$$




p47-

phox

expr

essio

n (

of s

ham

)

(b)





$$$$


NA

DPH

oxi

dase

activ

ity (R

AU)

80

60

40

20

0

VAS2870 + MCAOR

(a)

$$$$


Supe

roxi

de (

of s

harm

)

600

400

200

0



VAS2870 + MCAOR

(b)

$$$$


MD

A (n

mol

mg

prot

ein)

40

30

20

10

0



VAS2870 + MCAOR

(c)

$$ $$


Prot

ein

carb

onyl

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(d)

$$$$


8-O

HdG

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(e)




3but not WM



3


4 Discussion













References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Results










Neu

rolo

gica

l defi

cit s

core

4

3

2

1

0

Sham MCAOR

lowastlowast

ALA + MCAOR

(a)

Grip

tim

e (s)

400

300

200

100

0

lowastlowast


(b)

Late

ncy

time (

s)

400

300

200

100

0

lowastlowast


(c)

Cor

rect

ed in

frac

t vol

ume (

mm3)

300

250

200

150

100

50

0

lowastlowast


(d)


(e)




API

TUN

ELM

erge

(a)

lowastlowast


TUN

EL-p

ositi

ve ce

lls ra

te (

)

80

60

40

20

0

(b)

lowastlowast


Casp

ase-3

activ

ity (

of c

ontro

l)

500

400

300

200

100

0

(c)




3(5mgkg





p-Akt

Total Akt

P47-phox

gp91-phox

120573-actin

MCAOR

+

MCAOR

ALA+

MCAORSham MCAOR

ALA ALA+

+

+

WM HNMPA

(a)

p-IR120573

IR120573

ratio

( o

f sha

m)

400

300

200

100

0

p-A

ktA

kt ra

tio (

of s

ham

)250

200

150

100

50

0

400

300

200

100

0

gp91

-pho

x ex

pres

sion

( o

f sha

m)

400

300

200

100

0









$$$$$$$$




p47-

phox

expr

essio

n (

of s

ham

)

(b)





$$$$


NA

DPH

oxi

dase

activ

ity (R

AU)

80

60

40

20

0

VAS2870 + MCAOR

(a)

$$$$


Supe

roxi

de (

of s

harm

)

600

400

200

0



VAS2870 + MCAOR

(b)

$$$$


MD

A (n

mol

mg

prot

ein)

40

30

20

10

0



VAS2870 + MCAOR

(c)

$$ $$


Prot

ein

carb

onyl

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(d)

$$$$


8-O

HdG

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(e)




3but not WM



3


4 Discussion













References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Neu

rolo

gica

l defi

cit s

core

4

3

2

1

0

Sham MCAOR

lowastlowast

ALA + MCAOR

(a)

Grip

tim

e (s)

400

300

200

100

0

lowastlowast


(b)

Late

ncy

time (

s)

400

300

200

100

0

lowastlowast


(c)

Cor

rect

ed in

frac

t vol

ume (

mm3)

300

250

200

150

100

50

0

lowastlowast


(d)


(e)




API

TUN

ELM

erge

(a)

lowastlowast


TUN

EL-p

ositi

ve ce

lls ra

te (

)

80

60

40

20

0

(b)

lowastlowast


Casp

ase-3

activ

ity (

of c

ontro

l)

500

400

300

200

100

0

(c)




3(5mgkg





p-Akt

Total Akt

P47-phox

gp91-phox

120573-actin

MCAOR

+

MCAOR

ALA+

MCAORSham MCAOR

ALA ALA+

+

+

WM HNMPA

(a)

p-IR120573

IR120573

ratio

( o

f sha

m)

400

300

200

100

0

p-A

ktA

kt ra

tio (

of s

ham

)250

200

150

100

50

0

400

300

200

100

0

gp91

-pho

x ex

pres

sion

( o

f sha

m)

400

300

200

100

0









$$$$$$$$




p47-

phox

expr

essio

n (

of s

ham

)

(b)





$$$$


NA

DPH

oxi

dase

activ

ity (R

AU)

80

60

40

20

0

VAS2870 + MCAOR

(a)

$$$$


Supe

roxi

de (

of s

harm

)

600

400

200

0



VAS2870 + MCAOR

(b)

$$$$


MD

A (n

mol

mg

prot

ein)

40

30

20

10

0



VAS2870 + MCAOR

(c)

$$ $$


Prot

ein

carb

onyl

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(d)

$$$$


8-O

HdG

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(e)




3but not WM



3


4 Discussion













References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















API

TUN

ELM

erge

(a)

lowastlowast


TUN

EL-p

ositi

ve ce

lls ra

te (

)

80

60

40

20

0

(b)

lowastlowast


Casp

ase-3

activ

ity (

of c

ontro

l)

500

400

300

200

100

0

(c)




3(5mgkg





p-Akt

Total Akt

P47-phox

gp91-phox

120573-actin

MCAOR

+

MCAOR

ALA+

MCAORSham MCAOR

ALA ALA+

+

+

WM HNMPA

(a)

p-IR120573

IR120573

ratio

( o

f sha

m)

400

300

200

100

0

p-A

ktA

kt ra

tio (

of s

ham

)250

200

150

100

50

0

400

300

200

100

0

gp91

-pho

x ex

pres

sion

( o

f sha

m)

400

300

200

100

0









$$$$$$$$




p47-

phox

expr

essio

n (

of s

ham

)

(b)





$$$$


NA

DPH

oxi

dase

activ

ity (R

AU)

80

60

40

20

0

VAS2870 + MCAOR

(a)

$$$$


Supe

roxi

de (

of s

harm

)

600

400

200

0



VAS2870 + MCAOR

(b)

$$$$


MD

A (n

mol

mg

prot

ein)

40

30

20

10

0



VAS2870 + MCAOR

(c)

$$ $$


Prot

ein

carb

onyl

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(d)

$$$$


8-O

HdG

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(e)




3but not WM



3


4 Discussion













References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















p-Akt

Total Akt

P47-phox

gp91-phox

120573-actin

MCAOR

+

MCAOR

ALA+

MCAORSham MCAOR

ALA ALA+

+

+

WM HNMPA

(a)

p-IR120573

IR120573

ratio

( o

f sha

m)

400

300

200

100

0

p-A

ktA

kt ra

tio (

of s

ham

)250

200

150

100

50

0

400

300

200

100

0

gp91

-pho

x ex

pres

sion

( o

f sha

m)

400

300

200

100

0









$$$$$$$$




p47-

phox

expr

essio

n (

of s

ham

)

(b)





$$$$


NA

DPH

oxi

dase

activ

ity (R

AU)

80

60

40

20

0

VAS2870 + MCAOR

(a)

$$$$


Supe

roxi

de (

of s

harm

)

600

400

200

0



VAS2870 + MCAOR

(b)

$$$$


MD

A (n

mol

mg

prot

ein)

40

30

20

10

0



VAS2870 + MCAOR

(c)

$$ $$


Prot

ein

carb

onyl

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(d)

$$$$


8-O

HdG

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(e)




3but not WM



3


4 Discussion













References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















$$$$


NA

DPH

oxi

dase

activ

ity (R

AU)

80

60

40

20

0

VAS2870 + MCAOR

(a)

$$$$


Supe

roxi

de (

of s

harm

)

600

400

200

0



VAS2870 + MCAOR

(b)

$$$$


MD

A (n

mol

mg

prot

ein)

40

30

20

10

0



VAS2870 + MCAOR

(c)

$$ $$


Prot

ein

carb

onyl

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(d)

$$$$


8-O

HdG

( o

f sha

m)

400

300

200

100

0



VAS2870 + MCAOR

(e)




3but not WM



3


4 Discussion













References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3but not WM



3


4 Discussion













References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article alpha-lipoic acid attenuates cerebral...

Documents