Neuroprotective effect of luteolin on amyloid β protein (25–35)-induced toxicity in cultured rat cortical neurons

Neuroprotective Effect of Luteolin on Amyloid b Protein (25–35)-Induced Toxicity in Cultured Rat Cortical Neurons

Hao-Yuan Cheng,1 Ming-Tsuen Hsieh,1 Fan-Shiu Tsai,1 Chi-Rei Wu,1 Chuan-Sung Chiu,1,2 Min-Min Lee,3 Hong-Xi Xu,4 Zhong-Zhen Zhao5 and Wen-Huang Peng1*1Graduate Institute of Chinese Pharmaceutical Sciences, College of Pharmacy, China Medical University, Taichung, Taiwan, R.O.C.2Hsin Sheng College of Medical Care and Management, Taoyuan, No.115, Fulin Li, Pingzhen City, Taoyuan County 324, Taiwan, R.O.C.3Department of Health and Nutrition Biotechnology, Asia University, No. 500, Lioufeng Road, Wufeng, Taichung County 41354, Taiwan, R.O.C.4Chinese Medicine Laboratory, Hong Kong Jockey Club Institute of Chinese Medicine, Hong Kong, China5School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region, P.R. China

The present study was carried out to investigate the neuroprotective effect of luteolin on amyloid b (Ab) (25–35)-induced neurotoxicity using cultured rat cortical neurons. After exposure of primary cultures of rat cortical cells to 10 mM Ab (25–35) for 48 h, cortical cell cultures exhibited marked apoptotic death. Pretreatment with luteolin (1, 10 mM) signifi cantly protected cortical cell cultures against Ab (25–35)-induced toxicity. Luteo-lin (1, 10 mM) showed a concentration-dependent inhibition on 10 mM Ab (25–35)-induced apoptotic neuronal death, as assessed by MTT assay. Furthermore, luteolin reduced apoptotic characteristics by DAPI staining. For Western blot analysis, the results showed that the protective effect of luteolin on Ab (25–35)-induced neurotoxicity was mediated by preventing of ERK-p, JNK, JNK-p, P38-p and caspase 3 activations in rat primary cortical cultures. Taken together, the results suggest that luteolin prevents Ab (25–35)-induced apop-totic neuronal death through inhibiting the protein level of JNK, ERK and p38 MAP kinases and caspase 3 activations. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: luteolin; Aβ; cortical neurons; MAP kinases; caspase 3.

INTRODUCTION

It is well known that neurodegeneration of the amyloid β peptide (Aβ) plays a major part in the memory dys-function observed in the early stages of Alzheimer’s disease (AD). AD shows a signifi cant extent of oxida-tive damage associated with a marked accumulation of amyloid β peptide (Aβ), the main constituent of senile plaques in brain, as well as deposition of neurofi brilary tangles and neurophil threads. In AD, neurodegenera-tion primarily affects certain types of neurons, particu-larly those in the cortex and hippocampus. The loss of neurons and synapses leads to cognitive impairment and the development of dementia (Coyle et al., 1983). It was shown that the addition of purifi ed Aβ peptide and its active fragment Aβ (25–35) can induce apoptosis in a variety of cell types in vitro (Eckert et al., 1998; Mattson et al., 1998). Direct injection of Aβ into specifi c brain areas induces neuronal cell death and has been a widely used animal model of AD (Cantarella et al., 2003). These fi ndings raise the possibility that Aβ may be responsible for apoptotic cell death observed in the brains of AD patients. Therefore, the search for

neuroprotective drugs is an important step toward the development of effective treatment strategies for neurodegenerative disorders.

The fl ower bud of Lonicera japonica Thunb. (Capri-foliaceae) has been shown to possess antibacterial, anti-pyretic and antiinfl ammatory effects and is used to cure infl uenza, upper respiratory infection and tonsillitis (Zhao, 2004). Luteolin, a 3′,4′,5,7-tetrahydroxyfl avone, belongs to fl avonoid compounds and is a main active constituent of Lonicerae Flos. In modern pharmaco-logical studies, luteolin possesses a high DNA protec-tive effect in the presence of H2O2 (Romanova et al., 2001), antiinfl ammatory, antioxidant, anti-AP1 activa-tion (Hirano et al., 2006) and phytoestrogen-like activi-ties (Dall’Acqua and Innocenti, 2004), and is a free radical scavenger (Brown and Rice-Evans, 1998; Sadzuka et al., 1997). A previous study found that luteo-lin attenuates SCOP-induced amnesia (Tsai et al., 2007).

On the other hand, mitogen-activated protein kinases (MAPKs) play an important role in the phosphoryla-tion of tau in AD brain and the subsequent appearance of pathology. MAPKs are important intermediates in signaling pathways that transduce extracellular stimula-tion into intracellular responses. There are three identi-fi ed subfamilies of MAPKs: (i) c-Jun N-terminal protein kinase (JNK), (ii) extracellular signal regulated protein kinase 1 and 2 (ERK1 and 2) and (iii) p38 MAPK. Recently, certain members of MAPK have been shown to play important roles in neuronal apoptosis in response to environmental stresses and apoptotic agents

* Correspondence to: W. H. Peng, Graduate Institute of Chinese Phar-maceutical Sciences, College of Pharmacy, China Medical University, 91 Hsueh Shih Road, Taichung, Taiwan, R.O.C.E-mail: [email protected]

Received 07 July 2008Revised 14 April 2009

Copyright © 2009 John Wiley & Sons, Ltd. Accepted 21 May 2009

PHYTOTHERAPY RESEARCHPhytother. Res. 24: S102–S108 (2010)Published online 16 July 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2940

NEUROPROTECTIVE EFFECT OF LUTEOLIN S103

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: S102–S108 (2010)DOI: 10.1002/ptr

(Hedergen et al., 1998). Caspases play a pivotal role in apoptosis (Nicholson and Thornberry, 1997). Among the members of caspases, caspase 3 has been suggested to play an important role in several models of apoptosis (Woo et al., 1998). However, the effect of luteolin on cultured rat cortical cell death by inhibition of the acti-vation of JNK and ERK/p38 MAP kinase pathways through antioxidant mechanisms has not been studied.

The aim of the present study was to investigate the neuroprotective action of luteolin against Aβ (25–35)-induced neuronal death, and the underlying mechanism in cultured rat cortical neurons. Furthermore, this study also evaluated Aβ (25–35)-mediated cytotoxicity and apoptosis was correlated closely with inducibility of all three different MAP kinases and the caspase 3 pathway.

MATERIALS AND METHODS

Chemicals. Aβ (25–35) was purchased from Sigma-Aldorich (Vienna, Austria). Luteolin (Fig. 1), poly-d-lysine, boric acid, cytosine arabinoside, 4′,6-diamidino-2- phenylindole (DAPI), 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), DNase I, papain, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO) and cytokine were pur-chased from Sigma-Aldrich (St Louis, MO, USA). Neurobasal medium, Dulbecco’s modifi ed Eagle’s medium (DMEM), penicillin streptomycin, B-27 supplement, trypan blue and horse serum were obtained from Gibco. Hank’s balanced salt solution (HBSS) was obtained from Hyclone. Anti-ERK1/2, anti- phosphor-ERK1/2, anti-p38, anti-phospho-p38, anti-JNK and anti-phospho-JNK were obtained from Calbiochem-Novabiochem Corporation (La Jolla, CA, USA). Anti-caspase 3 was obtained from PharMingen International (Tokyo, Japan).

Cell culture. Cultures of rat primary cortex neurons were prepared from embryonic day 18 Sprague-Dawley (BioLASCO Taiwan Co., Ltd) rat embryos as described (Nishikawa et al., 2000; Kume et al., 2002). The neocor-texes of rat embryos were dissected and placed in cold Hank’s balanced salt solution (HBSS). After removal of meninges, the tissues were minced and incubated at 37 ºC for 15 min in Ca2+/Mg2+-free HBSS containing 0.25% trypsin and 0.2 mg/mL DNase I, and the cell suspensions were centrifuged (300 × g for 10 min). The resulting pellets were resuspended in the 1 : 1 mixture of DMEM and F12 supplemented with 20% heat- inactivated FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were plated into 96-well poly-d-lysine coated dishes at a density of 1.5 × 105/mL.

Cytosine arabinoside (10 μm) was added to the culture 24 h after plating to prevent nonneuronal cell prolifera-tion. The cultures were incubated at 37 ºC in 5% CO2. After 48 h, the medium was replaced with neurobasal medium supplemented with B-27 and penicillin (without l-Glu/Gibco). Only mature cultures (10 days in vitro) were used for experiments.

Drug treatment and measurement of neurotoxicity. Aβ (25–35) was dissolved in sterile water (stock solution: 1 mg/mL) at a concentration of 100 μm (fi nal 10 μm). On the day of the experiment, luteolin was added to the culture medium for 2 h before Aβ (25–35) induction and during exposure for 48 h for MTT assay.

Cell viability was measured by MTT reduction essen-tially as described (Mandel et al., 2003). MTT was dis-solved at a concentration of 5 mg/mL in sterile PBS. Following a total of 24 h of treatment, 12 μL of the 5 mg/mL solution of MTT was added to each well, and the incubation continued for 2 h. Then 100 μL of the cell lysis buffer (20% SDS/50% N,N-dimethylfor-mamide, pH 4.7) was added. The absorbance was read at 570 nm on a microplate reader. To assess cell viabil-ity, cell counts using trypan blue exclusion were also performed.

DAPI staining of apoptotic cells. To detect morpho-logical evidence of apoptosis, cell nuclei were visualized following DNA staining with the fl uorescent dye DAPI (4′,6-diamidino-2-phenylindole). Cells were rinsed with phosphate-buffered saline (PBS), fi xed for 20 min in formalin, rinsed with PBS and then incubated for 10 min with DAPI (1 μg/mL) (Nardi et al., 1997). After washing with PBS, the cultures were examined using fl uorescent microscopy (Microphot FX, Nikon) and the percentage of apoptotic nuclei with condensed or fragmented chro-matin was evaluated.

Western blotting analysis. Activation of three major MAPKs was assayed by Western blotting with phosphorylated-ERK, phosphorylated-p38, phosphorylated-JNK-specifi c and anti-caspase 3 rabbit IgG antibody as described in the manufacturer’s proto-col (Lu et al., 2004). Cell extracts were prepared as described by Mesmer and Brune (1997). Briefl y, the cells were suspended in lysis buffer (50 mm Tris, 5 mm EDTA, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm PMSF, 10 mg/mL leupeptin, 10 μg/mL of aprotinin) on ice for 30 and then sonicated. Cell lysates were centrifuged at 4000 × g, 5 min at 4 ºC, and the protein concentration in the resultant supernatant was determined using a Bio-Rad Protein Assay kit according to the manufac-turer’s protocol. Total cellular proteins were resolved by SDS–PAGE (10% (w/v) gel) under reducing condi-tions. The gels were transferred electrophoretically to polyvinylidene difl uoride (PVDF) membrane and the immobilized proteins were then incubated with the anti-bodies as described above. After incubation with horse-radish peroxidase-conjugated anti-rabbit IgG antibodies, the membrane was developed with a chemilumines-cence detection kit. Duplicate fi lters were probed with ERK MAPK-specifi c antibody, p38 MAPK-specifi c antibody, JNK-specifi c antibody and caspase 3 anti-body, recognizing both phosphorylated and unphos-phorylated forms of each MAPK to verify total MAPK and caspase 3 protein levels.Figure 1. Chemical structure of luteolin.


S104 H.-Y. CHENG ET AL.

Protein assay. Protein content was measured by the method of Lowry et al. (1951) with bovine serum albumin as a standard.

Statistical analysis. All the data obtained were analysed using one-way analysis of variance (Alaei et al., 2006), followed by Scheffe’s multiple range test. Western blot-ting data were analysed using a unpaired Student’s t-test to assess differences between control and treated groups. The criterion for statistical signifi cance was p < 0.05 in all the evaluations.

RESULTS

Luteolin protects neurons against Ab (25–35)-induced cell death

The neuroprotective activity of luteolin was evaluated by assessing the viability of cultured cortical neuronal cells injured with Aβ (25–35); survival was assessed using the MTT assay. Exposure of the cortical cultures to 10 μm of Aβ (25–35) for 48 h markedly reduced the cell viability by about 55% compared with normal corti-cal cells. A dose of luteolin (1 and 10 μm) showed sig-nifi cant neuroprotective activities against Aβ (25–35) induced toxicity (Fig. 2). It was noteworthy that cellular viability was maintained at 80% and 85% in Aβ (25–35)-induced neurotoxicity by treatment with luteolin. These results indicated that luteolin prevented Aβ (25–35)-induced neurotoxicity in primary cultures of rat cortical cells.

DAPI staining of apoptotic cells

As shown in Fig. 3, DAPI nuclear staining was observed by fl uorescent microscopy. Aβ (25–35) treated neuronal

Cel

l via

bili

ty (

%)

Luteolin (μM)control 0 0.1 1 10

0

20

40

60

80

100

120

###

**

##

10 μM Aβ (25−35)

Figure 2. Protective effect of luteolin on Aβ (25–35)-induced toxicity in rat cortical cells. Cortical cells were pretreated with luteolin for 2 h and then were treated with 10 μM of Aβ (25–35) for 48 h. Cell viability was assessed by measuring the MTT reduction. Values are the mean ± SEM expressed as the percent-age of control value. ** p < 0.01 compared with control group. ## p < 0.01, ### p < 0.001 compared with Aβ (25–35) group (n = 3).

(B)

(A)

(C)

Figure 3. Cortical cells were pretreated with luteolin for 2 h and then were treated with 10 μM of Aβ (25–35) for 48 h. Fixed and stained with DAPI assay and investigated under a fl uorescence microscope (original magnifi cation ×200). (A) control group; (B) treated with 10 μM of Aβ (25–35) for 48 h; (C) pretreated with 10 μM of luteolin for 2 h and then treated with 10 μM of Aβ (25–35) for 48 h.



cultures demonstrated signifi cant nuclear rounding and shrinkage compared with the control cultures as indi-cated by DAPI staining. However, luteolin (10 μm) did not induce the appearance of rapid cell swelling, nuclear rounding and shrinkage. Typical photographs of DAPI staining showing the inhibitory effects of luteolin on Aβ (25–35) induced apoptotic-like cell death.

Luteolin inhibits Ab (25–35)-induced ERK, JNK and p38 phosphorylation

The study provided the fi rst evidence for the involve-ment of ERK, JNK and p38 activations in Aβ (25–35)-induced cortical neurotoxicity based on the following reasons. While the protein levels of ERK and p38 were unaffected, the phosphorylation levels of ERK and p38 (ERK-p and p38-p) were transiently and markedly increased (Figs 4, 5). The ERK and p38 phosphorylation levels were inhibited by luteolin. The JNK and JNK-p were signifi cantly activated after being treated with Aβ (25–35). The activations of JNK and JNK-p were also completely prevented by luteolin (10 μm) (Fig. 6). These results suggest that ERK-p, p38-p, JNK and JNK-p were transiently activated and were involved in the Aβ (25–35)-induced cell death in cultured rat cortical cells.

Luteolin inhibits Ab (25–35)-induced caspase 3 activation

Caspase 3, the 32 kDa protease constitutively expressed by many cell types and tissues, is implicated in apoptosis promoted by different death stimuli. To elucidate the apoptotic neuronal cell death, caspase-3 immunoreactiv-ity was measured after treatment with 10 μm Aβ (25–35). In 10 μm Aβ (25–35) treated cells, the caspase 3 activity markedly increased compared with control cultures.

Con Aβ Luteolin Con Aβ Luteolin

ERK ERK-p0

50

100

150

200

250

300

VEH Aβ (25-35) Aβ (25-35) + Luteolin

***

###

Per

cent

age

of V

EH

Figure 4. Immunoblot analysis of luteolin on Aβ (25–35)-induced protein levels change of ERK and ERK-P in rat cortical cells. Cortical cells were pretreated with 10 μM of luteolin for 2 h and then were treated with 10 μM of Aβ (25–35) for 48 h. *** p < 0.001 compared with control group. ### p < 0.001 compared with Aβ (25–35) group (n = 3).


P38 P38-p

Per

cent

age

of V

EH

0

500

1000

1500

2000

2500

3000

3500

VEH Aβ (25-35)

Aβ (25-35) + Luteolin

***

###

Figure 5. Immunoblot analysis of luteolin on Aβ (25–35)-induced protein levels change of p38 and p38-p in rat cortical cells. Corti-cal cells were pretreated with 10 μM of luteolin for 2 h and then were treated with 10 μM of Aβ (25–35) for 48 h. *** p < 0.001 compared with control group. ### p < 0.001 compared with Aβ (25–35) group (n = 3).


JNK JNK-p0

50

100

150

200

250

VEH Aβ (25-35)

Aβ (25-35) + Luteolin

***

###

***

Per

cent

age

of V

EH

###

Figure 6. Immunoblot analysis of luteolin on Aβ (25–35)-induced protein levels change of JNK and JNK-P in rat cortical cells. Cortical cells were pretreated with 10 μM of luteolin for 2 h and then were treated with 10 μM of Aβ (25–35) for 48 h. *** p < 0.001 compared with control group. ### p < 0.001 compared with Aβ (25–35) group (n = 3).

Luteolin (10 μm) signifi cantly blocked the Aβ (25–35)-induced increase of caspase 3 immunoreactivity (Fig. 7). The results suggest that luteolin reduced the processes of activation of caspase induced by Aβ (25–35).

DISCUSSION

The present study has been shown neuroprotection in cultured rat cortical neurons and the possibility of



preventing and/or treating memory defi cits such as those seen in AD. Oriental herbal medicines have been used for treating neurodegenerative disorders such as senile dementia in Asians. However, there is a little scientifi c evidence for their effectiveness. There is increasing evidence which suggests that intraneuronal Aβ accumulation may be an early event in AD (Takahashi et al., 2002).

In order to indicate a viable cell culture, immunocy-tochemistry with the neuronal marker, MAP-2, and the glial marker, GFAP, was used. This modifi ed culture method produces a viable and essentially pure neuronal population (Silva et al., 2006). It was found that the primary cultures of rat cortical cells were more than 90% neurons (data not shown). Therefore, based on these fi ndings, it is possible to dissect temporally differ-ent mechanisms of Aβ (25–35)-induced cell death.

For the MTT assay, luteolin signifi cantly protected primary cultures of rat cortical cells from Aβ (25–35) cell death (Fig. 2). In addition, DAPI is known to form fl uorescent complexes with natural double-stranded DNA. Cells containing densely stained and fragmented chromatin were identifi ed as apoptotic using a Nikon fl uorescence microscope with a DAPI fi lter. In the present study, exposure of the cortical cell cultures to 10 μm Aβ (25–35) induced cell apoptosis. Pretreatment with luteolin reduced apoptotic characteristics by DAPI staining (Fig. 2). Therefore, it was demonstrated that luteolin signifi cantly reduced Aβ (25–35)-induced apoptosis. Many neurodegenerative conditions, such as dementia, are closely related to free radical overloading and intracellular oxidative stress (Polidori et al., 2007).

Several lines of evidence support the use of the amyloid β-peptide associated oxidative stress model and cell death by free radical scavengers to study neurotoxicity in Alzheimer’s disease. In vivo and in vitro studies have concluded that the various benefi cial effects of luteolin and plants containing it were mainly due to its antioxi-dant effect (Peterson and Dwyer, 1998). On the other hand, polyphenolic antioxidants are scavengers of free radicals and modifi ers of various enzymatic functions. The structure of luteolin is fl avonoid and contains two phenolic structures. Therefore, we believe that the action of luteolin against Aβ (25–35)-induced cell apop-tosis is closely associated with the antioxidant proper-ties of luteolin.

MAPKs have been implicated in a wide array of physiological processes including cell growth, differen-tiation and apoptosis. Activation of each MAPK appears to regulate distinct cellular responses. For example, ERKs mediate cell proliferation and differentiation and protect the cells from apoptotic cell death (Jarpe et al., 1998; Wang et al., 1998), whereas JNK and p38 MAPK inhibit cell proliferation and may promote apoptotic cell death (Kyriakis and Avruch, 1996). Furthermore, another possible outcome of MAPK phosphorylation is the activation of several key transcription factors (Widmann et al., 1999). Accordingly, the balance between the ERK pathway and the stress-activated JNK and p38 MAPK pathways has been proposed to be a fundamental determinant of cell survival or apoptosis. In this report, while the protein levels of ERK and p38 were unaffected, phosphorylation levels of ERK and p38 (ERK-p and p38-p) were markedly increased before an obvious increase of apoptotic-like death induced by Aβ (25–35). Contrarily, ERK and p38 phosphorylation levels were inhibited by luteolin, both of which also prevented the apoptotic-like cell death. After Aβ (25–35) exposure of 48 h, JNK and JNK-p were mildly increased compared with the control group. Meanwhile, it was found that the variations of subcellular distribu-tion of JNK and JNK-p were also prevented by luteolin. The Aβ (25–35)-induced cortical neurotoxicity might be mediated partially by the activation of ERK, JNK-p and p38. From the results, luteolin prevented Aβ (25–35)-induced apoptotic neuronal death by inhibiting the protein level of JNK, ERK and p38 MAP kinase activa-tion. Xagorari et al. pointed out that luteolin inhibits LPS-induced TNF-α release by inhibiting the extracel-lular-regulated kinases (ERK) and p38 MAPK path-ways in macrophages, and blocks LPS-induced activation of NF-κβ, which is critically required for infl ammatory gene expression (Xagorari et al., 2002). On the basis of review, we suggested that luteolin which can inhibit the infl ammatory effect may attenuate the cell death by Aβ (25–35).

In the caspase family, which consists of more than 10 homologues, caspase 3 has been suggested to play an important role in Aβ (25–35)-induced apoptosis (Cardoso et al., 2002). In particular, analysis of caspase-3-defi cient mice revealed a decrease in apoptosis in the developing brain, suggesting that caspase-3 is necessary for apoptosis of developing neurons (Kuida et al., 1996). In AD brains, the protein level of caspase-3 was also increased (Shimohama et al., 1999). Caspase-3 is a key protease which is activated during the early stages of apoptosis and, like other members of the caspase family, is synthesized as an inactive proenzyme which is pro-

Luteolin Con Aβ

Caspase 3

β-actin

caspase 30

50

100

150

200

250

300

350 ControlAβ (25-35) Aβ (25-35) + Luteolin

**

###Perc

enta

ge o

f Con

trol

Figure 7. Immunoblot analysis of luteolin on Aβ (25–35)-induced protein levels change of caspase 3 in rat cortical cells. Cortical cells were pretreated for 2 h with 10 μM luteolin and then were further treated with 10 μM of Aβ (25–35) for 48 h. ** p < 0.01 compared with control group. ### p < 0.001 compared with Aβ (25–35) group (n = 3).



cessed in cells undergoing apoptosis by self-proteolysis and/or cleavage by other proteases. The study examined whether the Aβ (25–35) induced the activation of caspase 3 in this culture system. In the present study, it was found that Aβ (25–35) induced activation of caspase 3. When compared with Aβ (25–35) treatment alone, pretreatment of luteolin attenuated the Aβ (25–35)-induced caspase 3 activity. From these results, it is sug-gested that luteolin exhibited a neuroprotective effect by preventing activation of caspase 3.

Estrogenic compounds have been shown to protect neurons from a variety of toxic stimuli in vitro and in vivo and the depletion of estrogen at menopause has been associated with increased risk of neurodegenera-tive diseases. Luteolin, belongs to the fl avonoids, pos-sesses phytoestrogen mimicking actions and has been shown to have a positive effect on the cognitive function in females (Lee et al., 2005). Our previous study found that luteolin attenuated the defi cits of passive avoidance performance induced by SCOP (Tsai et al., 2007). Simi-larly, the gonadal hormone 17-estradiol is known to affect the organization of the developing brain (Arai et al., 1996) and also to improve memory and to offer protection against Alzheimer’s disease (van Duijn, 1999) as well as other forms of neuronal degeneration (Linford and Dorsa, 2002). 17-Estradiol reduced the number of apoptotic neurons and reduced the number of neurons containing active caspase-3. It is well established that specifi c receptors for estrogen are found in the hippocampus, a brain structure important for memory. Analyses of the activity–structure rela-tionships of luteolin and related compounds indicate that the presence of the C2-C3 double bond on the C ring (Huang et al., 1999), conjugated with the 4-oxo functional group (Fotsis et al., 1997), are critical for this biological activity. Luteolin (3′ 4′,5,7- tetrahydroxyfl avone) contains catechol hydroxyl groups on the A and B rings that may form quinones capable of binding covalently to proteins. The non-exchangeable occupancy of type II estrogen sites by luteolin is likely to involve covalent attachment (Marka-verich and Gregory, 1993). Early reports also indicate that estradiol has comparable antiapoptotic properties

in primary cortical neurons and that these properties are mediated through estrogen receptors. From these reviews, it was deduced that luteolin may be able to bind covalently to estrogen receptors. On the other hand, antiestrogens act by inhibiting estrogen receptor (ER) function. Unlike raloxifene and tamoxifen which exhibit both antagonist an agonist properties, ICI 182,780 (ICI) is considered a ‘pure’ anti-estrogen devoid of any ago-nistic activities. Diwakar et al. pointed out that when male mouse brain was preincubated with ICI 182,780 (1 nm) for 0.5 h followed by incubation with L-BOAA (β-N-oxalyl-amino-l-alanine, an excitatory amino acid which acts as an agonist of the AMPA subtype of gluta-mate receptors), (1 nm) for 1 h, the neuroprotection was abolished (Diwakar et al., 2006). Furthermore, in animal experiments, ICI 182,780 did not have any effect, indi-cating the inherent differences in response to estrogen receptor antagonists between male and female animals (Shiau et al., 1998). From these reviews, we believe that the estrogen receptor antagonist in the neural protec-tion systems plays a negative role. In this study, there-fore, we have not explored this issue. As for the role of estrogen, estrogen antagonist and other receptors, such as neurotransmitters, on the reversals of impairment caused by luteolin, further investigations are warranted. Therefore, we suggest that the effect of luteolin against Aβ (25–35)-induced cell apoptosis is closely associated with estrogen receptor properties.

In conclusion, luteolin may protect against Aβ (25–35) induced apoptosis by the prevention of ERK-P, JNK, JNK-P, p38-P and caspase 3 activation in rat primary cortical neuron. The neurprotective effects of luteolin may provide a potential therapeutic approach for pre-venting and/or treating the neurodegenerative diseases of AD.

Acknowledgements

This study was supported by grants from the National Sciences Council for the fi nancial support of this manuscript under contract no. NSC96-2320-B-039-016 and the China Medical University for the fi nancial support of this manuscript under contract no. CMU96-079 and CMU96-085.

REFERENCES

Alaei H, Borjeian L, Azizi M, Orian S, Pourshanazari A, Hanninen O. 2006. Treadmill running reverses retention defi cit induced by morphine. Eur J Pharmacol 536: 138–141.

Arai Y, Sekine Y, Murakami S. 1996. Estrogen and apoptosis in the developing sexually dimorphic preoptic area in female rats. Neurosci Res 25: 403–407.

Brown JE, Rice-Evans CA. 1998. Luteolin-rich artichoke extract protects low density lipoprotein from oxidation in vitro. Free Radic Res 29: 247–255.

Cantarella G, Uberti D, Carsana T, Lombardo G, Bernardini R, Memo M. 2003. Neutralization of TRAIL death pathway pro-tects human neuronal cell line from beta-amyloid toxicity. Cell Death Differ 10: 134–141.

Cardoso SM, Swerdlow RH, Oliveira CR. 2002. Induction of cyto-chrome c-mediated apoptosis by amyloid β 25–35 requires functional mitochondria. Brain Res 931: 117–125.

Coyle JT, Price DL, Delong MR. 1983. Alzheimer’s disease: a disorder of central cholinergic innervation. Science 219: 1184–1190.

Dall’Acqua S, Innocenti G. 2004. Antioxidant compounds from Chaerophyllum hirsutum extracts. Fitoterapia 75: 592–595.

Diwakar L, Kenchappa RS, Annepu J, Saeed U, Sujanitha R, Ravindranath V. 2006. Down-regulation of glutaredoxin by estrogen receptor antagonist renders female mice suscep-tible to excitatory amino acid mediated complex I inhibition in CNS. Brain Res 1125: 176–184.

Eckert A, Cotman CW, Zerfass R, Hennerici M, Muller WE. 1998. Lymphocytes as cell model to study apoptosis in Alzheimer’s disease: vulnerability to programmed cell death appears to be altered. J Neural Transm 54 (Suppl.): 259–267.

Fotsis T, Pepper MS, Aktas E et al. 1997. Flavonoids, dietary-derived inhibitors of cell proliferation and in vitro angiogen-esis. Cancer Res 57: 2916–2921.

Hedergen T, Claret FX, Kallunki T et al. 1998. Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury. J Neurosci 18: 5124–5135.

Hirano T, Higa S, Arimitsu J et al. 2006. Luteolin, a fl avonoid, inhibits AP-1 activation by basophils. Biochem Biophys Res Commun 340: 1–7.

Huang YT, Hwang JJ, Lee PP et al. 1999. Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis-associated properties in A431 cells



overexpressing epidermal growth factor receptor. Br J Pharmacol 128: 999–1010.

Jarpe MB, Widmann C, Knall C et al. 1998. Anti-apoptotic versus pro-apoptotic signal transduction: Checkpoints and stop signs along the road to death. Oncogene 17: 1475–1482.

Kuida K, Zheng TS, Na S et al. 1996. Decreased apoptosis in the brain and premature lethality in CPP32-defi cient mice. Nature 384: 368–372.

Kume T, Nishikawa H, Taguchi R et al. 2002. Antagonism of NMDA receptors by sigma receptor ligands attenuates chemical ischemia-induced neuronal death in vitro. Eur J Pharmacol 455: 91–100.

Kyriakis JM, Avruch J. 1996. Sounding the alarm: protein kinase cascades activated by stress and infl ammation. J Biol Chem 271: 24313–24316.

Lee YB, Lee HJ, Sohn HS. 2005. Soy isofl avones and cognitive function. J Nutr Biochem 16: 641–649.

Linford NJ, Dorsa DM. 2002. 17β-Estradiol and the phytoestro-gen genistein attenuate neuronal apoptosis induced by the endoplasmic reticulum calcium-ATPase inhibitor thapsigar-gin. Steroids 67: 1029–1040.

Lowry OH, Rosebrough NJ, Farr AL et al. 1951. Protein measure-ment with the Folin phenol reagent. J Bio chem 193: 265–275.

Lu XH, Bradley RJ, Dwyer DS. 2004. Olanzapine produces trophic effects in vitro and stimulates phosphorylation of Akt/PKB, ERK1/2, and the mitogen-activated protein kinase p38. Brain Res 1011: 58–68.

Mandel S, Reznichenko L, Amit T, Youdim MB. 2003. Green tea polyphenol (-)-epigallocatechin-3-gallate protects rat PC12 cells from apoptosis induced by serum withdrawal indepen-dent of PI3-Akt pathway. Neurotox Res 5: 419–424.

Markaverich BM, Gregory RR. 1993. Preliminary assessment of luteolin as an affi nity ligand for type II estrogen- binding sites in rat uterine nuclear extracts. Steroids 58: 268–274.

Mattson MP, Partin J, Begley JG. 1998. Amyloid beta-peptide induces apoptosis-related events in synapses and den-drites. Brain Res 807: 167–176.

Messmer UK, Brune B. 1997. Attenuation of p53 expression and Bax down-regulation during phorbol ester mediated inhibi-tion of apoptosis. Br J Pharmacol 121: 625–634.

Nardi N, Avidan G, Daily D, Zilkha-Falb R, Barzilai A. 1997. Bio-chemical and temporal analysis of events associated with apoptosis induced by lowering the extracellular potassium concentration in mouse cerebellar granule neurons. J Neurochem 68: 750–759.

Nicholson DW, Thornberry NA. 1997. Caspases: killer proteases. Trends Biochem Sci 22: 299–306.

Nishikawa H, Hashino A, Kume T, Katsuki H, Kaneko S, Akaike A. 2000. Involvement of direct inhibition of NMDA receptors in the effects of j-receptor ligands on glutamate neurotoxic-ity in vitro. Eur J Pharmacol 404: 41–48.

Peterson J, Dwyer J. 1998. Flavonoids: dietary occurrence and biochemical activity. Nutr Res 18: 1995−2018.

Polidori MC, Griffi ths HR, Mariani E, Mecocci P. 2007. Hallmarks of protein oxidative damage in neurodegenerative diseases: focus on Alzheimer’s disease. Amino Acids 32: 553–559.

Romanova D, Vachalkova A, Cipak L, Ovesna Z, Rauko P. 2001. Study of antioxidant effect of apigenin, luteolin and quer-cetin by DNA protective method. Neoplasma 48: 104–107.

Sadzuka Y, Sugiyama T, Shimoi K, Kinae N, Hirota S. 1997. Protective effect of fl avonoids on doxorubicin-induced car-diotoxicity. Toxicol Lett 92: 1–7.

Shiau AK, Barstad D, Loria PM et al. 1998. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95: 927–937.

Shimohama S, Tanino H, Fujimoto S. 1999. Changes in caspase expression in Alzheimer’s disease: comparison with development and aging. Biophys Res Commun 256: 381–384.

Silva RF, Falcao AS, Fernandes A, Gordo AC, Brito MA, Brites D. 2006. Dissociated primary nerve cell cultures as models for assessment of neurotoxicity. Toxicol Lett 163: 1–9.

Takahashi RH, Nam EE, Edgar M, Gouras GK. 2002. Alzheimer beta-amyloid peptides: normal and abnormal localization. Histol Histopathol 17: 239–246.

Tsai FS, Peng WH, Wang WH et al. 2007. Effects of luteolin on learning acquisition in rats: Involvement of the central cho-linergic system. Life Sci 80: 1692–1698.

van Duijn CM. 1999. Hormone replacement therapy and Alzheimer’s disease. Maturitas 31: 201–205.

Wang X, Martindale JL, Liu Y, Holbrook NJ. 1998. The cellular response to oxidative stress: Infl uences of mitogen- activated protein kinase signalling pathways on cell survival. Biochem J 333: 291–300.

Widmann C, Gibson S, Jarpe MB, Johnson GL. 1999. Mitogen activated protein kinase: Conservation of a three-kinase module from yeast to human. Physiol Rev 79: 143–180.

Woo M, Hakem R, Soengas MS et al. 1998. Essential contribu-tion of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev 12: 806–819.

Xagorari A, Roussos C, Papapetropoulos A. 2002. Inhibition of LPS-stimulated pathways in macrophages by the fl avonoid luteolin. Br J Pharmacol 136: 1058–1064.

Zhao ZZ. 2004. An Illustrated Chinese Material Medica in Hong Kong. Chung Hwa Book Co., Ltd: Hong Kong, 66.

Documents

Neuroprotective effect of luteolin on amyloid β protein (25–35)-induced toxicity in cultured rat cortical neurons