Neuroprotective effects of puerarin against beta-amyloid-induced neurotoxicity

Brain Research Bulletin 85 (2011) 212–218

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Neuroprotective effects of puerarin against beta-amyloid-induced neurotoxicity in PC12cells via a PI3K-dependent signaling pathway

Guihua Xing 1,Miaoxian Dong 1,Xiaoming Li,Yu Zou,Li Fan,Xiaoli Wang,Defu Cai,Chengchong Li,Li Zhou,Jicheng Liu,Yingcai Niu ?

The Institute of Medicine,Qiqihar Medical University,333BuKui Street,JianHua District,Qiqihar 161042,China

a r t i c l e i n f o Article history:

Received 21March 2011

Received in revised form 23March 2011Accepted 28March 2011Available online 5 April 2011Keywords:Apoptosis PI3K Puerarin

Beta-amyloid Bad

a b s t r a c t

Epidemiological data have indicated that estrogen replacement therapy (ERT)can decrease the risk of developing Alzheimer’s disease (AD).Phytoestrogens have been proposed as potential alternatives to ERT.The aim of the present study was to assess the neuroprotective effects of puerarin,a phytoestrogen isolated from Pueraria lobata,against the toxicity of beta-amyloid (A ?)in relation to the mitochondria-mediated cell death process,and to elucidate the role the activation of Akt and modulation of the pro-and antiapoptotic proteins in puerarin-induced neuroprotection.The present study shows that puerarin afforded protection against A ?-induced toxicity through inhibiting apoptosis in PC12cells.This result was also con?rmed by the activated caspase-3assay.P-Akt,Bcl-2and p-Bad expression increased after pretreatment with puerarin in PC12cells exposed to A ?25–35,whereas Bax expression and cytochrome c release decreased.Interestingly,these effects of puerarin against A ?25–35insult were abolished by wortmannin,an inhibitor of PI3K phosphorylation.These ?ndings suggest that puerarin prevent A ?-induced neurotoxicity through inhibiting neuronal apoptosis,and might be a potential preventive or therapeutic agent for AD.

? 2011 Elsevier Inc. All rights reserved.

1.Introduction

Alzheimer’s disease (AD)is an age-related neurodegenerative disorder af?icting an estimated 30million people worldwide [2].However,the cause of AD is still unknown and the treatment is therefore only palliative [27,38].Postmenopausal women may have a greater risk of developing AD than men,perhaps due to lower endogenous estrogen levels following menopause [15].AD is more prevalent in women than in men [25].Loss of ovarian steroids,particularly estrogens,at the menopause may increase the susceptibility of the aging brain to neurodegenerative disor-ders and be a risk factor for development of AD [43].There is also good clinical evidence that surgical removal of the ovaries before menopause increases the risk of dementia and cognitive impair-

Abbreviations:AD,Alzheimer’s disease;ERT,estrogen replacement therapy;SERMs,selective estrogen receptor modulators;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetraz-olium bromide;LDH,Lactate dehydrogenase;FACS,Fluorescence activated cell sorting;PBS,phosphate-buffered saline;PI,propidium iodide;Ct,threshold cycle;ELISA,Enzyme-linked immunosorbent assay;S.D.,stan-dard deviation;ANOVA,one way analysis of variance;p-Bad,Bcl-2associated death agonist;PTP,permeability transition pore;JNK,c-jun N-terminal kinase.?Corresponding author.Tel.:+864526720289;fax:+864526720289.E-mail address:nyc1968@https://www.360docs.net/doc/6d16944692.html, (Y.Niu).1

Contributed equally.ment in women [40].Epidemiological studies indicate that estrogen replacement therapy (ERT)lowers the risk of developing AD [13].Estrogen therapy is one of the most compelling potential strate-gies for the prevention of AD.Strong biologic evidence supports the bene?cial of estrogen on the brain,including neurotrophic effects,reducing beta-amyloid (A ?)accumulation,enhancing neurotrans-mitter release and action,and protecting against oxidative damage [4,39,48].In addition,both prospective and case-control studies found that women who took estrogen had up to a 50%lower risk of developing AD [18].Furthermore,in vitro studies have demon-strated neuroprotective effects of estrogens against glutamate-and ?-amyloid-induced neurotoxicity [31].ERT in post-menopausal women has been linked to a higher incidence of uterine and breast cancer,especially after long-term use [30].Consequently,the selec-tive estrogen receptor modulators (SERMs)that exert tissue speci?c estrogenic effects may provide the bene?ts of ERT without the risks [3].A group of natural SERMs are phytoestrogens,which are struc-turally similar to estrogen,and may serve as an alternative to ERT [32].

Although there are a few papers about the bene?cial effects of phytoestrogens on memory or the central nervous system [20],the effects of phytoestrogens on the central nervous system in humans are poorly understood.Puerarin (for its structure,see Fig.1),a naturally occurring iso?avone C-glycoside,was isolated from Puer-aria lobota.Puerarin which has been classi?ed as a phytoestrogen

0361-9230/$–see front matter ? 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.brainresbull.2011.03.024

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Fig.1.Chemical structure of puerarin.

can be highly effective against angiocardiopathy and cerebrovascu-lar diseases with properties of holding pharmacokinetics of rapid absorption from the intestine and presenting in brain organ tis-sue by speci?c transport pathways and low toxicity[34].Zhang and co-workers have shown that puerarin could attenuate A?25–35-induced PC12cell injure and apoptosis and could also promote the survival of PC12cells.Puerarin was also found to increase the Bcl-2/Bax ratio[47].However,the upstream and downstream signaling components of regulation by puerarin of Bcl-2family expression have also not been clearly resolved.It was recently reported that estrogen protects against A?-induced neurotoxicity via the activa-tion of Akt[9].Therefore,these?ndings led us to examine whether puerarin has a neuroprotective function,as estrogen does,and whether the effects are induced by suppressing neuronal apoptosis via the activation of PI3K/Akt signaling pathway.

2.Materials and methods

2.1.Cell culture

PC12cells,a rat pheochromocytoma,obtained from the cell bank of Institute of Biochemistry and Cell Biology,SIBS,CAS(Shanghai,China)were maintained in Dulbecco’s modi?ed Eagle medium(Hyclone,Logan,Utah,USA)supplemented with10%fetal bovine serum,50units/ml penicillin(Invitrogen,Carlsbad,CA,USA) and100mg/ml streptomycin(Invitrogen).The cells were seeded in?60mm dishes (Nalge Nunc Int.,Rochester,NY,USA)at1×104cells/cm2and maintained at37?C in a humidi?ed atmosphere of5%CO2.For neuronal differentiation,the cells were cultured with appropriate concentration of50ng/ml rat NGF for48h[19].There-after,the cells were washed repeatedly to remove NGF,and fresh medium replaced before all the experiments performed in the present study.

2.2.Determination of cell viability

Cell viability was measured by quantitative colorimetric assay with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)method as described previously[14].Brie?y,the cells were cultured at a density5×104cells per well in growth medium for24h in96-well plates,and then preincubated with or without0.01–1000?M or100?M puerarin(solubilized in1,2-propanediol)(98% purity by HPLC;Sino-Herb Company,Xian,China),which was followed24h later by exposure to20?M aggregated A?25–35(Sigma,St.Louis,MO,USA)prepared as described previously[33]for another24h or0–48h.Twenty?ve microliter/well of MTT solution(?nal concentration,500?g/ml)was added and cells were incubated at37?C for4h.Supernatants were then aspirated off and formazan crystals were dissolved with DMSO.The optical density of each well was determined at570nm using a microplate reader(Sa?re2,Tecan Group Ltd.,Maennedorf,Switzerland).

2.3.Determination of cytotoxicity

PC12cells were cultured as described above and then preincubated with or with-out puerarin at concentrations of50?M,100?M,and200?M,which was followed 1h later by exposure to20?M aggregated A?25–https://www.360docs.net/doc/6d16944692.html,ctate dehydrogenase (LDH)release was measured by cytotoxicity detection kit(Genmed,Westbury,NY) following the instructions provided by the manufacturer,and OD values were mea-sured at490nm with a microplate reader(Sa?re2,Tecan Group Ltd.,Maennedorf, Switzerland).Results were expressed as percentage of Triton X-100-induced LDH release.

2.4.Fluorescence activated cell sorting(FACS)analysis

The cells were cultured at a density1.5×105cells per well in growth medium for 24h in96-well plates,and then preincubated with100?M puerarin,which was fol-lowed1h later by exposure to20?M aggregated A?25–35.Wortmannin(Calbiochem, San Diego,CA,USA),an inhibitor of PI3K phosphorylation,was added to cells1h prior to puerarin at a?nal concentration of200nM.Annexin V assays were done using the Annexin V-FITC Apoptosis Detection Kit I(Becton Dickinson,San Jose,CA).Cells were washed twice with cold phosphate-buffered saline(PBS)and resuspended in binding buffer before addition of Annexin V-FITC and propidium iodide(PI).Cells were vortexed and incubated for15min in the dark at room temperature before analysis using a FACS Calibur?ow cytometer(BD Biosciences,San Jose,CA)and FlowJo software(Tree Star,San Carlos,CA).

2.5.RNA isolation and real-time PCR

The cells were treated as described above for the FACS analysis,and harvested by scraping into ice cold PBS24h later.Total RNA was isolated from cells using RNAiso Reagent kit(Takara Biotechnology,Dalian,China),and cDNA was synthe-sized with ExScript TM RT kit(Takara Biotechnology,Dalian,China)according to the manufacturer’protocol.PCR were performed by using SYBR?Premix Ex Taq TM in an ABI7300real-time PCR system(Applied Biosystems,CA).The following sequences were used as primers for real-time PCR ampli?cation:Bax sense primer,5 -AGA CAC CTG AGC TGA CCT TGG AG-3 ,and Bax antisense primer,5 -GTT GAA GTT GCC ATC AGC AAA CA;Bcl-2sense primer,5 -TGA ACC GGC ATC TGC ACA C-3 ,and Bcl-2antisense primer,5 -CGT CTT CAG AGA CAG CCA GGA G-3 ;and GAPDH sense primer,5 -GAC AAC TTT GGC ATC GTG GA-3 and GAPDH antisense primer,5 -ATG CAG GGA TGA TGT TCT GG-3 .The thermal pro?le was as follows:1cycle of95?C for10s;40cycles of5s at95?C and31s at60?C.Threshold cycle(Ct)data were collected using the Sequence Detection Software version1.2.3(Applied Biosystems, CA).The Ct represents the cycle number at which a?uorescent signal rises statis-tically above background.Real-time PCR assay was performed in triplicate for each sample to ensure reproducibility.The relative quanti?cation of gene expression was analyzed by the2– Ct method[23].The fold change in target gene cDNA relative to the GAPDH internal control was determined by:

Fold change=2? Ct,where Ct=(Ct target gene?Ct GAPDH)?(Ct control?Ct GAPDH) 2.6.Western blot

The cells were treated as described above for the FACS analysis.Cytoplasm proteins were isolated from PC12cells using Cytoplasmic Protein Extraction kit (Beyotime Biotechnology,Haimen,China),and protein concentrations were deter-mined using the BCA Protein Assay kit according to the protocol provided by the manufacturer(Beyotime Biotechnology,Haimen,China),then they were aliquoted and stored.One hundred microliter of supernatant was added to an equal volume of2×SDS sample buffer and boiled for5min at100?C.The samples were then stored at?80?C until analyzed.Equal amounts of protein(100?g/lane)were sepa-rated by15%SDS-polyacrylamide gel electrophoresis and then electrotransferred onto a nitrocellulose?lter membrane.After blocking for4h in a solution of8% nonfat dry milk in Tris-buffered saline containing0.1%Tween(pH7.6)at room tem-perature,membrane was then incubated overnight at4?C with primary antibody (caspase-3,phospho-Akt,and Akt antibody,Cell Signaling Technology Inc.,Beverly, MA;other antibody,Santa Cruz Biotechnology,Santa Cruz,CA)in concentrations of 1:1000(p-Akt),1:1000(Akt),1:1500(Bcl-2),1:1500(Bax),1:1000(p-Bad),1:1000 (Bad),and1:2500(GAPDH)in Tris-buffered saline with0.1%Tween20contain-ing8%nonfat dry milk.After washing four times,the membrane were incubated with Horseradish Peroxidase Labeled Anti-Mouse IgG(10000:1;Medical Biological Laboratory Co.,Nagoya,Japan)at room temperature for2h and again washed four times.The blots were developed using an ECL western blotting kit(Amersham Bio-sciences,Piscataway,NJ,USA)as recommended by the manufacturer.GAPDH was probed as an internal control to con?rm that an equal amount of protein was loaded in each lane.Band intensities were quanti?ed by an AlphaImager TM2200using the SpotDenso function of AlphaEaseFC TM Software version3.1.2(Witec,Littau, Switzerland).

2.7.Enzyme-linked immunosorbent assay(ELISA)

The cells were treated as described above for the FACS analysis.Cells were col-lected and fractionated.Cytosolic cytochrome c was determined by the Quantikine?rat/mouse cytochrome c assay kit(R&D systems,Minneapolis,MN,USA)within 96-well plates according to procedure given by the manufacturer.Cytosolic frac-tions were pipetted in triplicate onto a microplate precoated with rat/mouse cytochrome c.After a2h incubation and washing,substrate solution was added to each well.The reaction was stopped after30min,and the optical density was measured at450nm,corrected at540nm,using a microplate reader(Sa?re2,Tecan Group Ltd.,Maennedorf,Switzerland).Cytochrome c concentrations expressed as nanograns/milliliter were extrapolated from the standard curves generated using reconstitute the r/m cytochrome c standard.

2.8.Statistical analysis

All values in the?gures of present study indicate means±standard deviation (S.D.),and all determinations were repeated three times.The one way analysis

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Fig.2.Prevention of A?25–35-induced cell death by puerarin.(A)Puerarin reduces A?-induced PC12cells death in dose-dependent manner.PC12cells were pretreated with increasing concentrations(0.01–1000?M)of puerarin for1h,followed by exposure to aggregated A?25–35for24h,and cell viability was determined by MTT.

(B)Puerarin reduces A?-induced PC-12cells death in time-dependent manner.PC12 cells were pretreated with(black bars)or without(white bars)puerarin(100?M) for1h and exposed to aggregated A?25–35(20?M)for the indicated times,and cell viability was determined by MTT.(C)Effects of preconditioning with puerarin on LDH release in PC12cells subjected A?25–35insult.LDH release has been normalized to the maximal releasable amount obtained by incubating cells with1%Triton X-100for30min.Data obtained from three separate experiments and are expressed as mean±S.D.;*,P<0.05compared to A?25–35alone or that at the matched time point.

of variance(ANOVA)was used to evaluate the difference among multiple groups followed by a post hoc test(Student–Newman–Keuls)when variable distributions were normal.Otherwise,the nonparametric Mann–Whitney U test was used.The data were analyzed using SPSS13.0software(SPSS Inc.,Chicago,IL),and P<0.05 was considered statistically

signi?cant.Fig.3.Puerarin prevents A?25–35-induced apoptosis through a PI3K-dependent pathway.PC12cells were pretreated with100?M puerarin for1h and exposed to aggregated A?25–35(20?M)for24h.Two hundred nM wortmannin was added to cells1h prior to puerarin.Cells apoptosis was measured by labeling cells with annexin-V-FITC and counterstaining with PI.(A)Annexin-V-FITC/PI double stain-ing of PC12cells.The numbers indicate the percentage of cells in each quadrant (lower left:FITC?/PI?,intact cells;lower right:FITC+/PI?,apoptotic cells;upper left: FITC?/PI+,necrotic cells;upper right:FITC+/PI+,late apoptotic cells).(B)The bar chart describes the percentual distribution of apoptotic cells.Percentage of annexin V-positive cells analysis of FACS obtained from three separate experiments and are expressed as mean±S.D.;*,P<0.05compared to A?25–35alone.

3.Results

3.1.Neuroprotective ef?cacy of puerarin against Aˇ25–35

Cell viability detected by MTT revealed that puerarin signif-icantly prevented the PC12cells death induced by A?25–35in dose-dependent manner up to100?M and in time-dependent manner up to24h(Fig.2A and B).Thus,we selected24h time and20?M concentration of A?25–35and100?M concentration of puerarin for subsequent experiments.

We also determined LDH release as an indicator of cytotoxi-city.A colorimetric assay revealed that exposure to A?25–35alone induced a signi?cant increase in LDH release compared with control by124%.Relative to A?25–35alone,puerarin(50,100,or200?M) signi?cantly decreased A?25–35-induced LDH release by19–31%. Puerarin alone had no effect on LDH release from the PC12cells (Fig.2C).

3.2.Preventive effect of puerarin on Aˇ25–35-induced cell

apoptosis

Cell apoptosis was quanti?ed by staining cell with annexin-V-FITC/PI(Fig.3A).Quantitative analysis of Annexin V-positive cells

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Fig.4.Puerarin inhibiting the cytosolic cytochrome c levels in PC12cells.PC12cells were pretreated with100?M puerarin for1h,and exposed to aggregated A?25–35 (20?M)for24h.Two hundred nM wortmannin was added to cells1h prior to puer-arin.The level of cytosolic cytochrome c was measured by ELISA.Values obtained from three separate experiments and expressed as mean±S.D.;*,P<0.05compared to A?25–35alone.

revealed that treatment of PC12cells with20?M A?25–35for24h evoked marked cell apoptosis as indicated by the percentage of Annexin V-positive cells.Pretreatment of PC12cells with100?M puerarin,prior to A?25–35exposure,signi?cantly decreased cell apoptosis.Pretreatment with the wortmannin slightly increased A?25–35-induced cell apoptosis compared with A?25–35alone.More importantly,wortmannin abolished the protective effect of puer-arin against A?25–35-induced apoptosis(Fig.3B).

3.3.Preventive effect of puerarin on Aˇ25–35-induced cytochrome

c release

Cytochrome c concentration in cytosolic fractions was measured using a commercial ELISA kits.PC12cells treated with A?25–35for 24h showed an increase in the cytosolic cytochrome c levels.Pre-treatment with puerarin attenuated the A?25–35-induced increase in cytochrome c levels.Wortmannin inhibited the protective effect of puerarin against A?25–35-induced cytochrome c release(Fig.4).

3.4.Effects of puerarin on expression of Bax,phospho-Bcl-2 associated death agonist(p-Bad)and Bcl-2

Given that Bcl-2family proteins are important modulators of PC12cells apoptosis induced by A?25–35,we determined the effect of puerarin on Bax,p-Bad and Bcl-2protein levels,and the role of PI3K signal pathway.Western blot results(Fig.5A)shows that puerarin pretreatment(100?M for24h)increases Bcl-2and p-Bad protein levels in PC12cells which decreased signi?cantly with A?25–35treatment,whereas Bax were signi?cantly decreased.The wortmannin abolished the alteration of Bcl-2,p-Bad,and Bax levels exerted by puerarin(Fig.5B).Consistent with the results of protein levels,real-time PCR revealed that puerarin pretreatment signi?-cantly increase Bcl-2and decrease the Bax mRNA levels(Fig.5C). We further determined the activation of caspase-3by western blot analysis.Cells treated with A?25–35exhibited an increase in cleaved caspase-3,whose response was signi?cantly depressed by the puer-arin pretreatment(Fig.5D).

3.5.Effect of puerarin on Aˇ25–35-induced phosphorylation of Akt

We examined the effect of puerarin regulating A?25–35-induced Akt activation.Western blot results show that no appreciable Akt activation(phosphorylation)was induced by treatment with 20?M A?25–35.Puerarin pretreatment increased Akt phosphoryla-tion.Activation of Akt by the puerarin was blocked when the PC12 cells were preinculbated with wortmannin(Fig.5E).

4.Discussion

A?accumulation has been causally implicated in the neuronal dysfunction and neuronal loss that underlies the clinical manifes-tations of AD[10,37].Recently,the involvement of apoptosis has been corroborated by studies showing that A?alters expression of the Bcl-2family of apoptosis-related genes[46]and survival signal-ing pathways are required for protection of A?-mediated neuronal apoptosis[7].Phytoestrogens are candidate novel drugs for AD [16].This study shows that puerarin prevents A?25–35-induced PC12cells neurotoxicity and apoptosis.Inhibition of apoptotic by puerarin may involve modulation of the expression of pro-and antiapoptotic proteins,speci?cally Bax,p-Bad and Bcl-2.The neu-roprotective effects of puerarin are dependent upon activation of the PI3K survival pathway and phosphorylation of Akt.

Phytoestrogens are plant-derived molecules that structurally resemble endogenous estrogens containing a diphenolic chemical structure that can directly bind to estrogen receptors(ER)to regu-late gene expression mediated by estrogen response element[42]. Phytoestrogens have received considerable attention as potential alternatives to estrogen[8].Puerarin is one of the major phytoe-strogens isolated from Pueraria lobata,a chinese medicine known as Gegen.It was used to reduce febrile symptoms,dilat aeteria coro-naria and cerebral vessels,and decrease myocardial consumption of oxygen[12].We have previously demonstrated that puerarin possesses protective effects against PC12cells damage induced by MPP+in vitro[29].Other studies have also shown that puerarin possesses neuroprotection of puerarin in vivo[44].Previous work by Zhang showed puerarin signi?cantly inhibited A?25–35-induced apoptosis of PC12cells[47].These?ndings are consistent with the data of the present study showing that pretreatment of PC12 cells with100?M puerarin,prior to A?25–35peptide exposure, signi?cantly decreased cell apoptosis and LDH release.Actually, our results show that puerarin has not been able to inhibit A?-induced cell injury completely.It bears emphasis that our results and the proposed model do not exclude the involvement of any other mechanisms,such as JNK signaling pathways,in protection from A?-induced cell death by puerarin.Again,100?M puerarin may not be suf?cient to provide maximum levels pf protection against the A?-induced cell injury.

LDH is a stable cytoplasmic enzyme present in all cells,and is rapidly released into the cell culture supernatant when the plasma membrane is damaged.Thus,it can be used as a reliable biochem-ical index for neuronal plasma membrane damage[49].MTT is an index of mitochondrial viability because it is reduced by metaboli-cally active mitochondria[11].Our results indicated that puerarin exerted a signi?cant induction in PC12cells mitochondrial viabil-ity and were effective in preventing a decline in LDH release from PC12cells.We also found that the number of apoptotic cells in the PC12cells signi?cantly increases after A?25–35treatment,which suggests the role of apoptosis in the development of A?-induced neurodegeneration.Puerarin preconditioning signi?cantly reduced apoptosis in PC12cells.This result con?rmed by levels of activated caspase-3which are known to measure the later stages of cell death.

A?is thought to directly induce neuronal apoptosis.The Bcl-2 family of proteins regulates apoptosis by modulating mitochondrial permeability and the release of cytochrome c[28].The antiapop-totic protein Bcl-2resides in the outer mitochondrial wall and inhibits cytochrome c release.The proapoptotic proteins,such as Bad and Bax,are located in the cytosol but translocate to the mitochondria and form a proapoptotic complex with Bcl-2.This translocation is inhibited by phosphorylation of Bad,leading to its cytosolic sequestration[36].Thus,phosphorylation of Bad may pro-

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Fig.5.Puerarin modulation A ?25–35-induced alteration of Bcl-2family via PI3K-dependent pathway.PC12cells were pretreated with 100?M puerarin.Following 1h incubation,aggregated A ?25–35(20?M)was added.Wortmannin (200nM)was added to cultures 1h prior to A ?25–35.(A)Bax,Bcl-2,p-Bad,Bad,p-Akt,Akt,and caspase-3levels were determined by immunoblot analysis with antibody to Bax,Bcl-2,p-Bad,Bad,p-Akt,Akt,and caspase-3.The loading of the lanes was normalized to levels of GAPDH.(B)Quantitated results of Bax,p-Bad,and Bcl-2are presented relative to control.(C)Total RNA was isolated from PC12cells using RNAiso reagent and used for cDNA synthesis.The mRNA levels of Bax and Bcl-2were detected by real-time PCR.2? Ct analysis of PCR obtained from three separate experiments.(D)Quantitated results of cleaved caspase-3were presented relative to control.(E)Quantitated results of p-Akt were presented relative to control.All densitometric analysis of western blot obtained from three separate experiments,and data are expressed as mean ±S.D.,*,P <0.05compared to A ?25–35alone.#,P <0.05compared to A ?+puerarin treatment cells.

mote cell survival.The ratio of Bcl-2/Bax protein,being a critical factor of the apoptotic state of cells,has also been suggested to determine survival or death following A ?insult [21].Our results showed a pronounced increase in both mRNA and protein lev-els of Bax after 24h of incubation with A ?25–35.In contrast to the results obtained form Bax,mRNA and protein levels of Bcl-2were decreased.Thus,the Bcl-2/Bax ratio was lower in cells treated with A ?25–35.Puerarin preconditioning prevented these changes,thus inhibiting increase of Bax expression and decrease of Bcl-2,leading to an increased Bcl-2/Bax ratio.Pretreatment with wort-mannin inhibited the protective effects of puerarin on Bcl-2and Bax expression,suggesting that the effect of puerarin is mediated via the activation of PI3K/Akt signaling.There was no signi?cant difference in expression of total Bad among the various treatment groups.However,Bad phosphorylation was reduced in the A ?25–35treatment group.Puerarin preconditioning prevented the decrease in Bad phosphorylation,thus maintaining it in its nonactivated form.Again,pretreatment with wortmannin abolished the effect of puerarin,leading to a reduction in Bad phosphorylation.

Numerous components of the PI3K/Akt pathway play an impor-tant role in cell survival pathway [45].Studies have also reported that PI3K/Akt signal has an antiapoptotic activity through different

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mechanisms,e.g.,by activated Akt.It induces Bad phosphorylation which prevents its binding with Bcl-2and translocation into the mitochondria[26].The mitochondrial permeability transition pore (PTP)is a latent nonspeci?c pore in the inner membrane of the mitochondria that has been widely recognized to play an impor-tant role in apoptosis.PTP opening is important in mitochondrial events leading to programmed cell death,whereas inhibition of PTP prevents this pathologic insult thereby supporting the central role for PTP in cell survival[5].Since free Bcl-2is essential for main-taining the closed state of PTP,it is conceivable to consider that Akt phosphorylation mediates inhibition of mitochondrial PTP open-ing[35].If PTP remains closed,cytochrome c is con?ned to the mitochondrial intermembrane space and hence unable to trigger proapoptotic caspase activation,including caspase-3.These facts suggest that Akt activation regulates cell survival and apoptosis.Akt appears to be an important factor in the PI3K survival signal.The lit-erature relating role of PI3K/Akt pathway in A?toxicity,however, presents an inconsistent picture.Chiang and colleagues showed PI3K signaling is involved in A?-induced memory loss,and the inhibition of PI3K signaling rescued A?1–42-induced memory loss in vivo[6].Data from other investigators show that application of A?1–42reduced Akt activity,and consequently,elevated Akt activity is capable of reducing A?1–42-induced PC12cells death[24].Appar-ent discrepancies between their results may be due to differences in study design,such as in vivo or in vitro.The phosphatidyli-nositol3-kinase(PI3-K)/Akt pathway and the c-jun N-terminal kinase(JNK)pathway are thought to be involved in regulation of Bad[17].Soláet al.reported that A?induced a weak,rapid,and transient activation of Akt.Exposure to A?for24h resulted in sig-ni?cant levels of apoptosis,but no detectable Akt phosporylation [41].Previous work by Li et al.show A?1–40-induced decreased level of BAD phosphorylation[22,29].These?ndings are consis-tent with the results of present study.Our data demonstrate that PC12cells exposure to A?25–35for24h resulted in signi?cant levels of apoptosis,but A?did not increased the Akt phosphoryla-tion from basal levels.Bad phosphorylation,possible downstream effectors of Akt,was increased in PC12cells.The results suggest that Akt phosphorylation was not required for A?25–35-induced cell death.Other factors like c-jun N-terminal kinase(JNK)were found to be involved in targeting the apoptotic component of A?-induced cell death through modulation of pro-and antiapop-totic protein levels[1,46].This is an interesting question,which warrants future investigation.In addition,puerarin alone treated PC12cells improved phosphorylation of Akt.This effect was not accompanied by changes in Akt content(Data not shown).More importantly,wortmannin suppressed the protective effect of puer-arin in modulating A?25–35-induced apoptosis and blocked the Akt phosphorylation associated with puerarin.Further,A?25–35-mediated decrease of p-Bad,normally increased by puerarin,was no longer responsive after inhibition of PI3K signaling.Similar results were obtained in cytochrome c release studies.These data strongly suggest that puerarin-induced PI3K activation is necessary for protection from A?-induced cell death.However,our results do not exclude the possibility that other mechanisms may also mediate puerarin protection from A?-induced apoptosis,includ-ing the activation of other signaling pathways such as the c-JNK pathway.

In conclusion,the current investigation suggests that puerarin modulates A?-induced apoptosis by activating a PI3K-dependent pathway in association with Akt phosphorylation.Speci?c inhibitors of PI3K blocked Akt activation and almost completely abolished the protective effect of puerarin against A?-induced cell death.Akt phosphorylation in turn suppresses translocation of Bad, thus inhibiting cytochrome c release and caspase activation.Our ?ndings suggest that puerarin might be a potential drug for the AD to suppress neuronal cells apoptosis.Acknowledgment

This research was supported by the National Natural Science Foundation of China(No.81073080).

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