European Journal of Pharmacology 670 (2011) 392–398

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European Journal of Pharmacology

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Molecular and Cellular Pharmacology

The neuroprotective effects of phytoestrogen α-zearalanol on β-amyloid-inducedtoxicity in differentiated PC-12 cells

Yilong Dong a,b, Nan Yang a, Yanyong Liu a, Qing Li a, Pingping Zuo a,⁎a Institute of Basic Science, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, Chinab School of Life Science, Yunnan University, 2 Cuihu Bei Road, Kunming, Yunnan 650091, China

⁎ Corresponding author at: Department of PharmacoChinese Academy of Medical Sciences, 5 Dong Dan SaTel.: +86 10 6529 6404; fax: +86 10 6529 6404.

E-mail address: zuopp_pumc@126.com (P. Zuo).

0014-2999/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.ejphar.2011.09.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 May 2011Received in revised form 29 August 2011Accepted 7 September 2011Available online 21 September 2011

Keywords:α-ZearalanolOxidative stressApoptosis

Although favorable effects of estrogen replacement therapy on Alzheimer's disease on postmenopausalwomen have been recognized, an associated increased incidence of uterine and breast tumors has jeopar-dized the clinical use of estrogen. Phytoestrogen α-zearalanol (α-ZAL) is a reductive product of the Gibber-ella zeae metabolite and abundant in plants and vegetables, which has been shown to protect cell injury withlow side-effects on uterine and breast. This study was designed to evaluate the neuroprotective effects ofα-ZAL, on the cultured differentiated PC-12 cells, while 17β-estradiol (17β-E2) has been used as an estrogenpositive control. Following a 24 h exposure of the cells to amyloid β-peptide fragment 25–35 (Aβ25–35), a sig-nificant reduction in cell survival and activities of total superoxide dismutase (SOD) and glutathione perox-idase (GSH-Px), as well as increased of malondialdehyde (MDA) were observed. However, preincubation ofthe cells with α-ZAL or 17β-E2 prior to Aβ25–35 exposure elevated the cell survival and SOD and GSH-Px ac-tivities, and decreased the level of MDA. In addition, Aβ25–35 caused a significant cell apoptosis and increasedapoptotic rate, accompanied by decreasing of bcl-2 expression and increasing bax, caspase-3 expression, pre-treatment of the cells with α-ZAL or 17β-E2 ameliorated these changes induced by Aβ25–35. Taken together,these data indicated that the phytoestrogen α-ZAL may effectively antagonize Aβ25–35-induced cell toxicityby attenuating oxidative stress and apoptotic cell death, in a manner similar to 17β-E2. Our results suggestedthat α-ZAL can be used as a potential substitute of 17β-E2 in postmenopausal women for Alzheimer's diseaseprevention.

logy, Institute of Basic Science,n Tiao, Beijing 100005, China.

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Alzheimer's disease is a neurodegenerative disorder characterizedclinically by progressive dementia and pathologically by intraneuronalneurofibrillary tangles, extracellular deposition of amyloid β peptides(Aβ) and phosphorylation of tau protein (Forman et al., 2004; Mattson,2004). Although the cause of Alzheimer's disease is still unknown, re-searches have indicated that postmenopausal women appear to be atan increased risk for Alzheimer's disease compared with the same agemen or premenopausal women (Miech et al., 2002; Zandi et al., 2002).Postmenopausal depletion of endogenous estrogens may contribute tothis risk. Several studies have reported that estrogen replacement ther-apy may reduce the risk of developing Alzheimer's disease in postmen-opausal women (Asthana et al., 1999; Canderelli et al., 2007; Ohkuraet al., 1994; Shaywitz et al., 2003; Valen-Sendstad et al., 2010). Estro-gens may exert several neuroprotective effects on the brain, includinginhibition of Aβ formation, diminishing hyperphosphorylation of tau

protein, reduction of oxidative stress-related cell damage, and protec-tion of neurons against the noxious consequences of chronic inflamma-tory reaction (Simpkins et al., 2009). However, the fact that estrogenmay predisposewomen to amuch higher incidence of breast and endo-metrial cancers has undoubtedly compromised or jeopardized the clin-ical application of estrogen (Chlebowski et al., 2010). Thus, the searchfor safe and effective estrogen substitutes becomes a practical issue.

Recently, a plant-derived phytoestrogen, α-zearalanol (α-ZAL) hasbeen proposed as a potential replacement for estrogen. α-ZAL is a re-ductive product of the Gibberella zeae metabolite zearalenone (ZEN)(Fig. 1), which is abundant in plants and vegetables including soybean,wheat, grape, radish, celery, spinach and apple (Zöllner et al., 2000).α-ZAL is rapidly metabolized in the body with few residues left in organssuch asmuscle, heart, liver, pancreas, kidney, and blood (Kleinova et al.,2002). It has been observed to protect humanumbilical vein endothelialcell against damage induced by homocysteine or oxidized low densitylipoprotein (oxLDL) via its antioxidant property (Duan et al., 2008; Xuet al., 2008). In addition, Zheng and colleagues have reported that α-ZALmay inhibit proton F0F1-ATPase activity of rat brain mitochondrial,which leads to protective actions and antitumor property. (Zheng andRamirez, 1999) Also, there were evidences shown that the effect of α-ZAL on breast oncogene expression (Deng et al., 2010) and uterine

Fig. 1. Chemical structure of zearalenone (ZEN, left panel) and α-zearalanol (α-ZAL,right panel).

393Y. Dong et al. / European Journal of Pharmacology 670 (2011) 392–398

overgrowth is less than estrogen (Dai et al., 2004a); furthermore,α-ZALmay increase the gene expression of anti-tumor BIN1 in breast tissue(Deng et al., 2004), meaning that α-ZAL was able to alleviate the inci-dence of mammary gland tumor. Previously, our published resultshave shown that α-ZAL may effectively alleviate synapse loss and neu-ronal damage induced by Aβ just like estrogen (Dong et al., 2006, 2007),indicating that α-ZAL can be used as a “safe-estrogen” in postmeno-pausal women for Alzheimer's disease prevention dependent on itslow side-effect compared with estrogen. However, the mechanism ofα-ZAL against Aβ-induced cytoxicity is still unclear. Therefore, in thepresent study, we investigated the effect of α-ZAL on Aβ25–35-inducedneurotoxicity in differentiated PC-12 cells in vitro and underlying mo-lecular mechanisms when 17β-estradiol (17β-E2) has been used as anestrogen positive control.

2. Materials and methods

2.1. Materials

Aβ25–35, 17β-E2, nerve growth factor (NGF), MTT, RNase A and propi-dium iodide (PI) were purchased from Sigma-Aldrich (Saint Louis, MO,USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum(FBS) and horse serum (HS)were obtained fromGibco Invitrogen Corpo-ration (Carlsbad, CA, USA). The protease inhibitor mixture and BCA Pro-tein Assay Kit was purchased from Pierce Biotechnology (Rockford, IL,USA). The reagent kits for the measurement of MDA, SOD, and GSH-Pxwere purchased from Nanjing Jiancheng Bioengineering Institute (Nan-jing, China). Bcl-2, bax, caspase-3 and GAPDH antibodies were pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). α-ZALwas obtained as a gift from Prof. Shunling Dai at Perking UnionMedicalCollege. All other chemicals usedwere of the highest grade commercial-ly available.

2.2. Cell culture and treatment

Rat pheochromocytoma cells (PC12 cells, obtained from Cell Cen-ter, Institute of Basic Science, Chinese Academy of Medical Sciences)were cultured in DMEM supplemented with 5% FBS, 10% HS, 2 mMglutamine, 100 U/ml penicillin and 100 μg/ml streptomycin in a 5%CO2 humidified atmosphere at 37 °C and grown until 80% confluence.Cells were differentiated with 50 ng/ml NGF for 5 days and used for amaximum of 25 passages. α-ZAL or 17β-E2 was dissolved in ethanoland added to cell culture media at designated concentrations, thefinal concentration of ethanol≤0.1%. 12 h later, cells were exposedto Aβ25–35 for 24 h. Then, the cells will be collected for further re-search. Prior to α-ZAL or 17β-E2 treatment, the media were replacedwith fresh DMEM without FBS in order to remove endogenous estro-gen. Aβ25–35 peptide stock solution of 1 mM was prepared in steriledistilled water, stored at −20 °C, and incubated for 72 h at 37 °C toaggregate before use.

2.3. Cell viability assay

Cells were plated in 96-well plates, after various treatments, cellviability was determined by MTT assay. MTT was applied to the cul-tures at a final concentration of 0.5 mg/ml for 4 h at 37 °C. The medi-um was then aspirated, and 100 μl dimethyl sulfoxide (DMSO) wasadded to solubilize the colored formazan product. Absorbance was

determined at 578 nm on a scanning multiwell plate reader (Dyna-tech Lab, USA) after agitating the plates.

2.4. Measurement of MDA content and antioxidant enzyme activities

For assay of lipid peroxide and antioxidants, the cultures werewashed with ice-cold PBS and then pooled in 0.1 M PBS–0.05 mMEDTA-buffered solution and homogenized. The homogenate was cen-trifuged at 11,000×g for 20 min at 4 °C, after which the protein con-centration was determined by the BCA Protein Assay Kit, using bovineserum albumin (BSA) as a reference standard. The supernates werecollected and stored at −80 °C until use. The levels of MDA and anti-oxidant enzyme activities include total SOD and GSH-Px was deter-mined according to the instructions for the reagent kits.

2.5. Transmission electron microscopy

For electron microscopic analysis of apoptosis, the cells were har-vested by trypsinization and washed twice with cold PBS, then fixedin 2.5% glutaraldehyde for 2 h at room temperature. After beingwashedwith PBS, the cells were post-fixed in 1% osmium tetroxide containing0.1% potassium ferricyanide for 1 h at 4 °C, dehydrated through gradedalcohol, and embedded in Epon 812 for subsequent sectioning. Theultra-thin sections (65 nm) were stained with uranyl acetate and leadcitrate, and examined under transmission electron microscope.

2.6. Flow cytometry assay

For flow cytometry quantitative analysis of apoptosis, the cells wereharvested by trypsinization andwashed twicewith cold PBS, then fixedin 70% ethanol for 30 min at 4 °C. After beingwashedwith PBS, the cellswere collected by centrifugation at 800×g for 5 min at 4 °C, then resus-pended in a solution containing 10 g/l RNase A and 20 mg/l PI at roomtemperature for 30 min. Specimens were analyzed using flow cytome-try. The cells in the subdiploid peak were considered apoptotic.

2.7. Western blotting

Cells after treatment were washed three times with cold PBS andlysed using cold RIPA buffer (150 mMNaCl, 1%NP-40, 0.5% sodiumdeox-ycholate, 0.1% SDS, 5 mM ethylenediamine tetraacetic acid, 50 mM Tris–HCl, pH 8.0) supplemented with protease inhibitor mixture, then centri-fuged at 11,000×g for 10 min at 4 °C. The supernatant will be collectedand protein concentration was estimated by BCA Protein Assay Kit,using BSA as the standard. Equal amounts of protein were subjected toSDS-PAGE. The proteins were then transferred to nitrocellulose mem-branes. After transfer, the membranes were blocked with Tris-bufferedsaline (TBS) containing 0.1% Tween-20 (TBST) and 5% non-fat-milk for2 h at room temperature and then overnight at 4 °C with primary anti-bodies: anti-bcl-2 (1:1000), anti-bax (1:500), anti-caspase-3 (1:500)and anti-GAPDH (1:2000). After washing, themembranes were incubat-ed with goatradish peroxidase-conjugated secondary antibody (1:5000),followed by detection using enhanced chemiluminescence. Scanned im-ages of the developedblotswere quantifiedusing densitometry functionsin Image-Pro Express 4.0.

2.8. Statistical analysis

Data were expressed as mean±standard deviation (S.D.). Statisti-cal analyses were performed with one-way ANOVA followed by SPSSsoftware. A criterion for statistical confidence of P≤0.05 (two tailed)was adopted. Data from triplicate performances and all experimentswere performed using 3 separate cultures to confirm reproducibility.

394 Y. Dong et al. / European Journal of Pharmacology 670 (2011) 392–398

3. Results

3.1. α-ZAL protected PC-12 cells against Aβ25–35-induced cytotoxicity

To determine the toxicity of Aβ25–35-induced, cultured PC-12 cellswere exposed to Aβ25–35 (1–20 μM) for 24 h and the cell viability wasassessed by the MTT reduction assay. Aβ25–35 induced cell death in adose-dependent manner and the 50% inhibiting concentration (IC50)was 12.7 μM(Fig. 2A). Based on the result, 10 μMwas selected as the op-timal Aβ25–35 concentration for subsequent experiments. Cells were thentreated with various concentrations (10−6, 10−7 and 10−8 M) of α-ZALor 17β-E2 for 12 h, after which the cells were incubated with 10 μMAβ25–35 for 24 h. The cell viability was measured using an MTT assay.As shown in Fig. 2B, pretreatment withα-ZAL or 17β-E2 significantly in-creased the viability of cells against Aβ25–35-induced cytotoxicity. Al-though the protective effect of α-ZAL is less than 17β-E2, there is nostatistical difference in cell viability when cells are treated with α-ZALor 17β-E2 at same concentration. Since 10−7 M α-ZAL and 17β-E2show suitable effects (the cell viability was 72–75% of control), 10−7 Mwas chosen for the further experiments.

3.2. α-ZAL suppressed PC-12 cells apoptosis induced by Aβ25–35

We observed the nuclear morphologic changes associated withapoptosis by transmission electron microscopy. As shown in Fig. 3A,the cells untreated with Aβ25–35 exhibited the normal ultrastructuralmorphology of cytoplasm, nuclei and completely nuclear membrane.24 h after treatment of Aβ25–35, cells showed typical apoptosis character-ized by nuclear chromatin condensation, nuclear membrane splitting,fragmentation of nuclei, and formation of apoptotic bodies (Fig. 3A). Toinvestigate the anti-apoptotic effect of α-ZAL, the apoptotic rate wasdetected by flow cytometry. When the cells were cultured with 10 μM

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Fig. 2. Protective effect of α-ZAL on Aβ25–35-induced cytotoxicity in cultured differentiatedPC12 cells. A: PC-12 cells were treated with the indicated concentrations (1–20 μM) ofAβ25–35 for 24 h. B: PC-12 cells were pretreated with different concentrations of 17β-E2 orα-ZAL (10−6–10−8 M) for 12 h and then incubated with Aβ25–35 (10 μM) for an additional24 h. Viability of cells was assessed by MTT assay. Percentage of cell viability was relativeto the untreated control cells. Values represent asmean±S.D. of 3 independent experiments.*Pb0.05, **Pb0.01 versus control; #Pb0.05, ##Pb0.01 versus Aβ25–35-treated cells.

Aβ25–35, a visible subdiploid peak (apoptosis peak) was observed andthe apoptotic rate increased sharply from 2.08% to 25.94% comparedwith control. At the same time, cells were pre-treated with 10−7 M17β-E2 or α-ZAL for 12 h before being exposed to Aβ25–35, the percent-age of apoptotic cells was 10.22%, 11.95%, respectively (Fig. 3B and C).The results indicated the anti-apoptotic effect of α-ZAL against theAβ25–35-induced toxicity of cultured PC-12 cells.

3.3. α-ZAL attenuated lipid peroxidation and rescued loss of antioxidantenzyme activities in Aβ25–35-treated PC-12 cells

Enzymes important in regulating oxidative stress are altered in Alz-heimer's disease. To investigate themechanisms involved in the protec-tive effects of α-ZAL, the level of total SOD, GSH-Px and MDA weremeasured by using a commercial assay kit. As shown in Table 1, incuba-tion with 10 μM Aβ25–35 for 24 h significantly increased the contents ofMDA (114.2%) in cells as compared to control. Moreover, preincubationwith 17β-E2 or α-ZAL attenuated the contents of MDA by 34.9%, 32.8%respectively when compared to cells treated with Aβ25–35. In addition,exposure to 10 μM Aβ25–35 significantly decreased the activities ofSOD (60.2%) and GSH-Px (58.7%) in cells as compared to control. How-ever, pretreatment with 17β-E2 orα-ZAL showed a significant increasein the activities of SOD (53.7%, 48.8%) and GSH-Px (55.4%, 46.7%) whencompared to cells treatedwith Aβ25–35. Taken together, these results in-dicated that pretreatmentwithα-ZAL prevented lipid peroxidation andattenuated the changes in SOD and GSH-Px activities induced by thetreatment of Aβ25–35.

3.4. α-ZAL prevented apoptosis-related protein changes in Aβ25–35 treatedPC-12 cells

Bcl-2, bax and caspase-3 levels were assessed by Western blottingto determine whether these key regulators of apoptosis were in-volved in the neuroprotective effect of α-ZAL. The results showedthat the decreased bcl-2 and increased bax protein levels in Aβ25–35

treated cells compared with untreated cells (Fig. 4). The pretreatmentof cells with 17β-E2 or α-ZAL reversed the alternations of bcl-2 andbax expressions induced by Aβ25–35. With Aβ25–35, we also observedthat the increased of cleaved caspase-3 protein level, 17β-E2 orα-ZAL effectively blocked the activated caspase-3 expression inducedby Aβ25–35 (Fig. 4).

4. Discussion

Clinical and experimental evidences have demonstrated that estro-gen replacement therapy may be beneficial to Alzheimer's disease pre-vention on postmenopausal women (Asthana et al., 1999; Canderelliet al., 2007; Ohkura et al., 1994; Shaywitz et al., 2003; Valen-Sendstadet al., 2010), however, the use of estrogen has been imperiled due tothe uncertain increased risk of developing breast aswell as endometrialcancers in women taking estrogen replacement therapy. Our publisheddata have shown that treatmentwith phytoestrogenα-ZAL can provideprotection against neuronal injury in vivo and in vitro, but the side ef-fect on breast and uterus is less than estrogen (Dong et al., 2006,2007). The present study was carried out to investigate the mechanismof neuroprotective effects of phytoestrogen α-ZAL, while 17β-E2 hasbeen used as an estrogen positive control. In this study, we provide evi-dence that exposure of cultured differentiated PC-12 cells to Aβ25–35

(10 μM) for 24 h caused apoptotic cell death, as determined by MTTassay, morphological examination and flow cytometry analysis. Whenthe cells were preincubated with 17β-E2 or α-ZAL, the cell viability,and apoptotic ratio were all improved. Furthermore, cells treated withAβ25–35 showed that decreased levels of the antioxidant enzymes ofSOD and GSH-Px, but increased lipid peroxidation as shown by MDA ac-cumulation. Aβ25–35 also induced down-regulation anti-apoptoticbcl-2, up-regulation pro-apoptotic bax and caspase-3; all of the

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Fig. 3. Protective effects of α-ZAL on Aβ25–35-induced apoptosis in cultured PC-12. A. Morphology change of apoptosis in PC-12 cells induced by Aβ25–35 under transmission electronmicroscope. i: normal nuclei of control cells, ×8000; ii: condensation of nuclear chromatin and iii: wrinkling of nuclear membrane, fragmentation of nuclei, and formation of ap-optotic bodies when cells treated with Aβ25–35 for 24 h (arrow in Figure), ×4000. B: apoptotic rate were assessed by flow cytometry. Apoptotic peak was indicated by arrow. C: thepercentage of apoptotic cells in each group. *Pb0.05, **Pb0.01 versus control; #Pb0.05, ##Pb0.01 versus Aβ25–35-treated cells.

395Y. Dong et al. / European Journal of Pharmacology 670 (2011) 392–398

Table 1Effects of α-ZAL on lipid peroxidation and antioxidant enzyme activities in Aβ25–35-treated cultured PC-12 cells.

Treatment MDA SOD GSH-Px

(nM/mg protein) (U/mg protein) (U/mg protein)

Control 0.91±0.12 85.16±13.07 57.52±8.95Aβ25–35 1.95±0.09b 33.88±9.57b 23.78±6.92b

17β-E2+Aβ25–35 1.27±0.13c 52.06±13.73d 36.96±6.50d

α-ZAL+Aβ25–35 1.31±0.13c 50.41±12.53d 34.89±8.02d

Cells were pretreated with 10−7 M 17β-E2 or α-ZAL for 12 h and then incubated withAβ25–35 (10 μM) for an additional 24 h. Data were presented as mean±S.D.; aPb0.05,bPb0.01 versus control; cPb0.05, dPb0.01 versus Aβ25–35-treated cells.

396 Y. Dong et al. / European Journal of Pharmacology 670 (2011) 392–398

above Aβ25–35-induced changes were significantly reduced whencells preincubated with 17β-E2 or α-ZAL. The results from the pre-sent study showed that the effect involved inα-ZALmediated neuro-protection in a manner similar to that of 17β-E2.

Aβ is a soluble protein 40–42 amino acid residues in length that sig-nificantly contributes to the initiation and/or progression of neurodegen-erative changes in Alzheimer's disease (Yankner, 1996) The predominantforms of Aβ in the human brain are Aβ (1–40) and Aβ (1–42), which iscausal in activating a biochemical cascade that leads to cell death(Hölscher, 2005; Korczyn, 2008). However, Aβ (25–35) fragment, physi-ologically present in elderly people, is themore toxic region and has beenfound to play a relevant role in Alzheimer's disease and is universallyused in research (Millucci et al., 2010). Although the mechanism bywhich Aβ induces neurodegeneration remains controversial, there is aconfluence of opinion suggesting that oxidative stress is mediated by re-active oxygen species (ROS)which are deeply involved in the neurotoxiceffects of Aβ (Canevari et al., 2004). Furthermore, alongwith the accumu-lation of excessive ROS, protein oxidation (Castegna et al., 2002; Sultanaet al., 2006), lipid peroxidation (Butterfield and Lauderback, 2002), DNAand RNA oxidation (Lovell et al., 1999; Nunomura et al., 1999) will be in-duced, which are associated with apoptotic cell death and result in

Control A 25-35 17 -E2+ A -ZAL +A

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Fig. 4. Effects of α-ZAL on the expression of bcl-2, bax, and caspase-3 in cultured PC-12cells. Cells were pretreated with 10−7 M 17β-E2 or α-ZAL for 12 h and then incubatedwith Aβ25–35 (10 μM) for an additional 24 h. A: assessment of bcl-2, bax and caspase-3 pro-tein levels by western blotting. B: quantitative analysis of protein levels by densitometry.Densitometric analysis is mean±S.D. of 3 independent experiments. *Pb0.05, **Pb0.01versus control; #Pb0.05, ##Pb0.01 versus Aβ25–35-treated cells.

cognitive dysfunctions (Zhu et al., 2006). Thus, modulation of oxidativestress induced by Aβ has been speculated to be a potential therapy forAlzheimer's disease. It has been reported that 17β-E2 may alleviate oxi-dative stress by facilitating the expression of antioxidant enzyme, includeSOD andGSH-Px (Borrás et al., 2005). To assess the effect ofα-ZAL on ox-idative stress, we choose to monitor the change of MDA, SOD and GSH-Px.MDA is an end product of lipid peroxidation, and has been consideredas a late biomarker of oxidative stress and cellular damage (Vaca et al.,1988). Our present study showed that Aβ25–35 caused amarked decreasein cell survival which is accompanied by increased oxidative stress asshown by accumulation of MDA. However, increase in cell death andMDA level induced by Aβ25–35 was significantly attenuated when thecells were pretreated with 17β-E2 or α-ZAL. SOD and GSH-Px are well-known parameters of the antioxidative enzymes, which can react withROS and neutralize them to alleviate oxidative stress. The decrease ofSOD and GSH-Px has been shown in Alzheimer's disease patients(Padurariu et al., 2010) and relates to accelerate oxidative stress-in-duced neuronal death (Higgins et al., 2010). Our findings showed thatSOD and GSH-Px activity by Aβ25–35 treatment was significantly de-creasedwhen comparedwith the untreated cells, and the decreased ac-tivity was significantly increased by 17β-E2 or α-ZAL pretreatment.These results suggest that α-ZAL protects cells against oxidative stressinduced toxicity; the effect was similar to that of 17β-E2.

Studies on postmortem tissues provide direct morphological andbiochemical evidence including the presence of DNA damage, nuclearapoptotic bodies, and other markers of apoptosis (Lassmann et al.,1995; Smale et al., 1995) implicating that neurons in the brain of Alz-heimer's disease patients degenerate via an apoptotic mechanism(Culmsee and Landshamer, 2006) On the other hand, several investi-gators have demonstrated that oxidative stress is involved in the ap-optotic mechanism of Aβ-mediated neurotoxicity (Behl et al., 1994;Miranda et al., 2000), and that oxidative stress has been consideredas a pivotal trigger for the development of Aβ-induced apoptoticcell death (Datta et al., 2002; Fukui et al., 2005). In this report, wehave shown that differentiated PC-12 cells exposed to 10 μM Aβ25–35

demonstrated a typical apoptosis, including nuclear chromatin conden-sation, nuclearmembrane splitting, fragmentation of nuclei, and forma-tion of apoptotic bodies bymorphological observation. Flow cytometricanalysis of apoptotic cells demonstrated a significant increase of apo-ptotic rate from 2.08% in untreated cells to 25.94% in cells treated withAβ25–35 while excessive oxidative stress has happened as stated before.However, preincubation with 17β-E2 or α-ZAL, apoptotic rate was sig-nificantly decreased. The result indicates thatα-ZAL possesses a similaranti-apoptotic effect as 17β-E2.

One putative mechanism of estrogen anti-apoptosis is regulation ofthe Bcl-2 family, pivotal regulators of apoptosis that include both proteinsthat promote cell survival (e.g., bcl-2, bcl-xL, and bcl-w) and others thatantagonize it (e.g., bax, bad andbim). Up-regulation of anti-apoptotic pro-teins, such as bcl-2 (Nilsen andDiaz Brinton, 2003) or down-regulation ofproapoptotic bax (Sharma and Mehra, 2008) may contribute to the pro-tective effects of estrogen. Bcl-2 is predominantly localized on the outermitochondrialmembrane, andmediates an anti-apoptotic effect by stabi-lizing the mitochondrial membrane, inhibiting mitochondrial permeabil-ity transition pore ability and preventing mitochondrial cytochrome crelease during oxidative stress (Kowaltowski et al., 2000; Shimura et al.,2000). In contrast, bax is predominantly localized in the cytosol, andupon activation, translocates to the mitochondria and triggers the lossof mitochondrial membrane potential, which leads to the release of cyto-chrome c (Priault et al., 1999). The release of cytochrome c contributes tothe activation of caspase-9, which in turn causes activation of caspase-3and subsequent apoptosis (Cardone et al., 1998; Zornig et al., 2001). Con-sistent with the emerging literature, we found that, there was down-regulation of bcl-2 and up-regulation of bax expression in PC-12 cellsafter treatment with Aβ25–35 for 24 h, but pretreatment of cells with17β-E2 reversed these changes, the same antagonism of apoptosis ef-fects also has been observed when cells were pretreated with α-ZAL

397Y. Dong et al. / European Journal of Pharmacology 670 (2011) 392–398

before Aβ25–35 insult. We further determined the expression of activat-ed caspase-3 and show that Aβ25–35 increased the level of caspase-3,while 17β-E2 and α-ZAL treatment attenuated the expression of acti-vated caspas-3. Our findings from the present study strongly suggestthatα-ZALmay be against Aβ25–35-induced apoptosis, and that the reg-ulation of bcl-2, bax and caspase-3 protein expression may play an im-portant role in the suppressive effect of α-ZAL on cell death just like17β-E2. Although the cellular mechanisms ofα-ZAL remain to be eluci-dated, there is evidence to support thatα-ZAL owns the ability to inter-act with estrogen receptors sinceα-ZAL has the benzene ring structureresembling 17β-E2, while its the affinity of binding to the estrogen re-ceptor is estimated to be only one-tenth of that for 17β-E2 (Dai et al.,2004b), maybe this is one of the reasons why α-ZAL regulates the ex-pression of gene and/or protein. Thus, the cross-action between α-ZALand estrogen receptor is worth more research.

In summary, our results demonstrate thatα-ZAL protects differen-tiated PC-12 cells against Aβ25–35-induced cytotoxicity and that itsprotective effect at least in part, is attributable to its antioxidant prop-erty, as demonstrated by prevention of lipid peroxidation, rescue lossof antioxidant enzyme activities, and modulation of the expression ofapoptotic protein. The findings indicated that protective effects of α-ZAL is similar to 17β-E2, and it seems that α-ZAL can be used as a po-tential alternative for estrogen in Alzheimer's disease prevention inpostmenopausal women. However, clinical efficacy and potential tox-icity of α-ZAL in larger trials require further assessment before rec-ommendations regarding their use can be established.

Disclosure

The authors declared no conflict of interest.

Acknowledgment

Grant: this work was supported by the grant from the Nation BasicResearch Program of China (Project no. 2007CB507404).

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