Potential therapeutic effects of curcumin: Relationship to microtubule-associated proteins 2 in Aβ1–42 insult

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www.elsevier.com/locate/brainres

Research Report

Potential therapeutic effects of curcumin: Relationship to microtubule-associated proteins 2 in Aβ1–42 insult Zijian Xiaoa , Liming Lina , Zhonghua Liua , Fengtao Jia,c , Weiyan Shaob , Minjuan Wanga , Ling Liuc , Shengliang Lia , Feng Lia,⁎, Xianzhang Bub,⁎ a

Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, Guangdong, China c Department of Anesthesiology, The Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China b

A R T I C LE I N FO

AB S T R A C T

Article history:

Curcumin can bind senile plaques and promote disaggregation of existing amyloid deposits

Accepted 3 September 2010

and prevent aggregation of new amyloid deposits. Curcumin can also reverse distorted and

Available online 16 September 2010

curvy neurites around senile plaques and repair the neuritic abnormalities. We

Keywords:

anything to do with the changes of expression of microtubule-associated protein 2 (MAP2),

Curcumin

but curcumin could reverse damaged neurites by upregulation of MAP2 expression. In

β-amyloid protein

present study we designed and chemically synthesized curcumin and its six derivatives.

Microtubule-associated protein 2

After screening the protective effect of curcumin and derivatives, we found that the viability

SK-N-SH cell

of SK-N-SH cell model induced by Aβ1–42 was significantly increased by curcumin and Cur1,

Alzheimer's disease

and the expression of MAP-2 protein was obviously up-regulated in immunocytochemical

hypothesized whether altered neurite morphologies resulting from Aβ production had

staining and Western blot. The cell morphologies, including the number of neurites, neurite growth and neurite extension, were significantly improved. Cur1 showed more significant protective effect on SK-N-SH cells than curcumin. Our study revealed for the first time that the neuroprotective effect of curcumin and curcumin derivatives not only directly depends on their special chemical constitution, but they can resist to Aβ damage by up-regulation of MAP-2 expression. In view of the special advantages of curcumin and Cur1, we reasonably believe that curcumin and Cur1 may be considered as an ideal therapeutic agent for the treatment of AD. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

The accumulation of amyloid-β (Aβ) and senile plaques is widely believed to contribute to pathogenesis of Alzheimer's disease (AD) (Selkoe, 2001; Hardy and Selkoe, 2002; Walsh

et al., 2002). The recent studies found that curcumin could bind senile plaques in brain sections of patients with Alzheimer's disease or in a transgenic mouse model of AD. In vivo studies showed that curcumin could promote disaggregation of existing amyloid deposits and prevent aggrega-

⁎ Corresponding authors. E-mail addresses: [email protected] (F. Li), [email protected] (X. Bu). Abbreviations: Aβ, β-amyloid; AD, Alzheimer's disease; BSA, Bovine Serum Albumin; DMEM, Dulbcco's Modified Eagle Medium; DMSO, dimethyl sulphoxide; IOD, integrated optical density; MAP2, microtubule-associated protein 2; MS, mass spectrometry; MTT, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFTs, neurofibrillary tangles; NMR, nuclear magnetic resonance; OD, optical density; PBS, phosphate buffered saline; SPSS, Statistical Product and Service Solutions 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.09.019

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tion of new amyloid deposits, even reduce the size of remaining deposits (Garcia-Alloza et al., 2007), and further, Kim and Park (2002) demonstrated that curcumin and curcumin derivatives could inhibit the fAβ (fibrillar β-amyloid) formation from fresh Aβ and destabilize preformed fAβ in vitro, alternatively, not only curcumin and curcumin derivatives could decrease fAβ, but reduce the stability of fAβ which led to fAβ degradation, thus curcumin plays an important role in neuroprotective effect by resistance to neurotoxicity of Aβ. On the other hand, senile plaques are associated with synaptic loss and abnormal neurite morphology (Perl, 2010), which results in a disruption of synaptic integration in AD. Curcumin can reverse distorted and curvy neurites located in the surrounding of senile plaques, and repair the neuritic abnormalities induced by Aβ insult (Garcia-Alloza et al., 2007). Microtubule-associated protein 2(MAP2) is a neuronal cytoskeletal component, which takes part in maintaining cellular architecture and internal organization, with clear involvement of defining cell shape, in cell division and cellular processes, such as neurite extension (Yamaguchi et al., 2008). Alterations and deterioration of cytoskeleton is related to malfunction of nerve cells in brain tissue of AD (Johnson and Jope, 1992). Nukina and Ihara (1986) revealed that MAP2 monoclonal antibody could mark neurofibrillary tangles (NFTs), and MAP2 polyclonal antibody also labeled abnormal neurites around senile plaques in AD. We hypothesized whether altered neurite morphologies resulting from Aβ production had anything to do with the changes of expression of MAP2; whether curcumin could influence the expression of MAP2 lead to reversion of distorted and curvy neurites. We also wondered whether the chemical constitution of curcumin gives its certain advantages in the treatment of AD. Curcumin's compact and symmetrical phenol groups make it better brain-permeable and able to cross the blood brain barrier (Wang et al., 2010) and its compact and symmetric structure may also be suitable for specifically binding to free Aβ and subsequently inhibiting polymerization of Aβ into fAβ. The autofluorescence of curcumin may reliably label senile plaques and NFTs in AD, thus it facilitates to observe the changes of senile plaques and NFTs in brain tissue (GarciaAlloza et al., 2007). Although the recent studies supported the evidence that curcumin could reduce plaques, and partially restore the altered neurite structure, what special chemical constitution of curcumin is responsible for its neuroprotective properties might, however, be unknown, because most of curcumin used in the study, purchased from Chemical Corporation, had the identical chemical structure. In the work reported here, we designed and chemically synthesized curcumin and six kinds of curcumin derivatives for determining the relationship between the certain chemical constitution of curcumin and neuroprotective effect. We assessed the changes of expression of MAP2 in human neuroblastoma SKN-SH cells after treatment with curcumin and curcumin derivatives. Our study revealed for the first time that the neuroprotective effect of curcumin and curcumin derivatives not only directly depends on their special chemical constitution, but they can up-regulate the expression of MAP2 which led to resistance to Aβ damage. These results suggest that targeted modification of certain chemical constitution of curcumin would greatly improve its neuroprotective effect,

by which we might develop a class of curcumin drug as treatment for Alzheimer's disease.

2.

Results

2.1.

Synthesis of curcumin and curcumin derivatives

Curcumin and six curcumin derivates (Cur1–6) were designed in present study. For structure and activity relationship (SAR) analysis, the 3-methoxyl group of curcumin was removed to give Cur1; the methylation form of Cur1 and curcumin (Cur2 and Cur3 respectively) were designed to investigate the contributions of hydroxyl group; another methoxyl were added in the Cur3 giving Cur4 and meanwhile, all substituents were removed in Cur5 to further explore the functions of different substituents on the benzene rings; in Cur6, we replaced the benzene rings on both ends of curcumin with furan rings to investigate the effects of different aromatic group to the bioactivity. (Tab.1). All compounds were synthesized and characterized according to our previous study (Qiu et al., 2008).

2.2.

Neuronal damage induced by Aβ42

The cell viability was evaluated by MTT assay. Cell viability = (experimental group OD value – blank control group OD value)/ (control group OD value- blank control group OD value) × 100%. The cell viability of SK-N-SH cells was significantly decreased to 18.53%, 36.73%, 60.99%, 79.40% and 95.34% (Fig. 1) respectively after a 24-h exposure to Aβ1–42 of the different concentrations (40, 20, 10, 5, 2.5 μg/ml). The neuronal damage induced by Aβ1–42 was correlated with Aβ1–42 in a dose-dependent manner. In present study, Aβ1–42 group (10 μg/ml) served as neuronal damage model mentioned above for subsequent experiments.

2.3.

Protective effect of curcumin and curcumin derivatives

2.3.1.

Improving cell viability

The viability of SK-N-SH cells was reduced to approximately 60.99% of baseline after a 24-h exposure to Aβ1–42 at higher concentration (10 μg/ml). Curcumin and six derivates showed a different protective effect on SK-N-SH cells (Fig. 2). Curcumin and Cur1 among the derivates of curcumin could prevent SKN-SH cells from the cytotoxicity of Aβ1–42 and improve cell

Fig. 1 – MTT assay of cell viability after exposure to different concentrations of Aβ1–42.

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between curcumin group and Aβ1–42 group (P < 0.01) or between Cur1 group and Aβ1–42 group (P < 0.001). In addition, Cur1 exhibited better antagonistic effect on Aβ42-induced neurotoxicity than curcumin (P < 0.05) (Fig. 6). We proposed that Aβ1–42 inhibited significantly the expression of high molecular weight protein (MAP2), and curcumin and Cur1 could lead to the obvious upregulation of expression of MAP2, especially Cur1 showed more significant upregulation than curcumin, this result was consistent with those seen in the immunocytochemistry of MAP2 mentioned above.

Fig. 2 – MTT assay of cell viability after treatment with curcumin and its derivatives. *Curcumin group compared with the control group (P < 0.05). **Cur1 group compared with the control group (P < 0.05), also with Curcumin group (P < 0.001).

viability, especially Cur1 showed more significant protective effect (33.5 %) on SK-N-SH cells than curcumin (23.3%). There was statistical difference between curcumin group and Aβ1– 42 group (P < 0.05), and also significant difference between Cur1 group and Aβ1–42 group (P < 0.001). However, five derivates except Cur1 showed no obviously improving cell viability and protective effects on SK-N-SH cells.

2.3.2.

Protective effect on cytoskeleton

SK-N-SH cells were apoptotic/dying after exposure to Aβ1–42 for 24 h, and the morphologic alteration of MAP2-positive cells was found, such as a short neurites, decreased number of neuritis and MAP2 defect in the cellular processes. We also found some naked nucleus without the cytoplasm and some cells with a little cytoplasm. These results implied the loss of cytoskeletal components during the incubation of Aβ1–42. Following treatment with curcumin and Cur1, the number of neurites, neurite growth and neurite extension were observed and cellular morphologies were significantly improved (Fig. 3). Integrated optical density (IOD) showed that there is significant difference between curcumin group and Aβ1–42 group (P < 0.001) or between Cur1 group and Aβ1–42 group (P < 0.001). In addition, Cur1 exhibited better antagonistic effect on Aβ42induced neurotoxicity than curcumin (P < 0.001) (Fig. 4). These results suggested that curcumin and Cur1 were likely related to the upregulation of intracellular MAP2 expression, especially Cur1 showed more significant upregulation than curcumin.

2.3.3.

Western blot analysis of MAP2

Biochemical characterization was revealed by western blot that the mature MAP2a (300 kd) and 2b (280 kd), high molecular weight protein, were expressed in all groups including control, curcumin and Aβ1–42 groups, but the MAP2a and 2b protein levels were downregulated in Aβ1–42 group, while they were abundantly expressed in curcumin and Cur1 groups. This result indicated that the upregulation of MAP2a and 2b could be correlated with the effective antagonism of curcumin or Cur1 toward Aβ1–42 (Fig. 5). The relative band density showed that there is significant difference

3.

Discussion

Many central nervous system diseases are related to abnormal cytoskeleton components (Dustin and Brion, 1988), and microtubule-associated transport barriers may lead to aggregation of many proteins responsible for the cellular efflux and neurotransmitter transport, which results in the many dysfunctions in neurodegenerative diseases, such as Alzheimer disease, Parkinson's disease and Lewy body disease. Accumulation of Aβ in the brain causes changes in neuritic processes in individuals with these diseases (Petratos et al., 2008), so the synaptic and axono-dendritic degeneration begins at the distal neuritic ends. Earlier studies found that the mAb against MAP2, 5F9, might recognize a determinant present in NET, MAP2 monoclonal antibody could clearly mark NFTs in most infected region of brain tissue of patients with AD, and MAP2 polyclonal antibody might label abnormal neurites around senile plaques (Nukina and Ihara, 1986), suggesting that MAP2 may be involved in the occurrence of neurodegenerative diseases caused by neurotoxic effect of Aβ. In Alzheimer disease process, which there is a mechanism might interfere with the role of stable microtubules of MAP2, leading to the cytoskeletal changes and neuronal death, and the existence of certain factors on the role of MAP2 could affect the transport of organelles, resulting in microtubule-associated transport barriers. On the other hand, the signs of neurodegeneration are associated to a marked loss or reduction of immunoreactivity for MAP2. Several studies (Marcinkiewicz, 2002; Lain et al., 2005; Tohda et al., 2008; Shankar et al., 2009) including from our laboratory found that MAP2 protein was markedly reduced after Aβ insult. Western blot analysis in present study further revealed that the expression of MAP2a and MAP2b were decreased significantly in SK-N-SH cells upon exposure to Aβ1–42. MAP2 is a heat-stable phosphoprotein and belongs to structural microtubule-associated protein family (Morales et al., 2008; Sánchez et al., 2000; Cristofanilli et al., 2004; Buddle et al., 2003; Guo et al., 2001). MAP2 can be divided into high molecular weight (HMW) of MAP2a (280–300 KD) and MAP2b (270–280 KD), low molecular weight (LMV) of MAP2c (70 KD) and MAP2d (75 KD). In the brain of mammalian, MAP2a and MAP2b are neuron-specific protein, while MAP2c and MAP2d are primarily seen in glial cells (Vouyiouklis and Brophy, 1995; Richter-Landsberg and Gorath, 1999). In the present study, the reduction of MAP2a and MAP2b in Western blot, together with the decreased immunoreactivity for the dendritic marker MAP2 in immunocytochemical staining, indicate that the

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Fig. 3 – (A) Fluorescent immunocytochemistry of MAP2 and Hoechst 33258. a: Control group. SK-N-SH cells were cultured in normal medium. b: Aβ group. The damage of SK-N-SH cells was induced by Aβ1–42 for 24 h. “↑” stands for naked nucleus without the cytoplasm which contains MAP2 proteins (8.6%). “▲” stands for the cells with little cytoplasm (14.3%). c: Curcumin group. SK-N-SH cells were treated with curcumin for 24 h after induced by Aβ1–42. Curcumin significantly improved the cellular morphology. d: Cur1 group. SK-N-SH cells were treated with Cur1 after induced by Aβ1–42. The protective effect of Cur1 on cellular morphology was more obvious than curcumin. (B) Autofluorescence images of curcumin and Cur1. a: Control group. SK-N-SH cells were treated with curcumin, and without Aβ1–42. b: Aβ group. The damage of SK-N-SH cells was induced by Aβ1–42 for 24 h. Stop reaction after treatment with curcumin for 2 h. c: Curcumin group. SK-N-SH cells were treated with curcumin for 24 h after induced by Aβ1–42. Number of neurites, neurite growth, neurite extension were significantly increased. d: Cur1 group. SK-N-SH cells were treated with Cur1 for 24 h after induced by Aβ1–42. The cellular morphology was more obviously improved than those seen in curcumin group.

stability of microtubules, microtubule polymerization and cytoskeletal function have been affected. This mechanism was proven in our further experiments, for example, the cell viability assay revealed that SK-N-SH cells showed a significant impairment in dose-dependent and time-dependent

decrease in cell activity after Aβ insult, attentively, the reduction of cell viability was simultaneously seen associated to an important alteration in decreased MAP2 expression and the occurrence of abnormal neurite morphology. Therefore we reasonably thought that altered neurite morphologies

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Fig. 4 – Quantitative analysis of fluorescence intensity of MAP2. IOD was significantly declined in Aβ42 group, but elevated in curcumin and Cur1 groups. There is a significant difference between curcumin group and Aβ42 group (P < 0.001), and between Cur1 group and Aβ42 group (P < 0.001), and there is still a significant difference (P < 0.001), as compared curcumin group with Cur1 group.

Fig. 6 – Quantitative analysis of western blot for MAP2. The relative band density was significantly decreased in Aβ42 group, but increased in curcumin and Cur1 groups. There is a significant difference between curcumin group and Aβ42 group (P < 0.01), and between Cur1 group and Aβ42 group (P < 0.001), but there is a difference (P < 0.05), as compared curcumin group with Cur1 group.

resulting from Aβ production have anything to do with the reduction of expression of MAP2. Further experiments were necessary to clarify whether curcumin could revert to normal level of MAP2, and what chemical constitution of curcumin was responsible for improving abnormal neurite morphology, and what intracellular changes were, at least in part, attributable to the neuroprotective factor of curcumin against Aβ-dependent damage. Unlike cholinesterase inhibitors (i.e. donepezil hydrochloride), used clinically for the treatment of Alzheimer's disease, they do not prevent or reverse the underlying neurodegeneration in abnormal neurite morphology induced by amyloid β. The abundant reports supported that curcumin can promote disaggregation of existing amyloid deposits and prevent aggregation of new amyloid deposits (Yang et al., 2004; Lim et al., 2001; Zhang et al., 2006), thus it has a strong (role of specific) resistance to amyloid β, and also straighten the neurites of higher curvature near or even far from the senile plaque, and reverse neuritic abnormalities (Garcia-Alloza et al., 2007). Therefore, curcumin reported here could be considered as a neuroprotective agent for MAP2 against Aβdependent damage. Curcumin and curcumin derivatives are composed of conjugation of β-diketone chain and two benzene rings, and each of the benzene ring also connects to some methoxyls and hydroxyls. Through structure–activity

relationship analysis, our studies were consistent with the idea (Kim and Park, 2002) that neither the degree of conjugation of β-diketone chain nor the 3-methoxy group in the benzene ring plays an important role in the neuroprotective effect of curcumin. Instead, the β-diketone moiety appears to be important for protective mechanism of curcumin. In addition to β-diketone moiety, we also paid close attention to a different roles of 4-hydroxy or 3-methoxy (termed substituent groups) on the benzene ring of curcumin derivatives. According to structure–activity relationship theory, it is generally thought that hydroxyl groups may readily affect the biological activity of drug, which is partially attributable to hydroxyl groups etherificated or esterificated easily, led to the reduction of its activity. However, Ligeret et al. (2004) found that ortho-hydroxy-e-donor methoxy group may enhance the stability of phenolic hydroxyl group and its function of antioxidation. Considering that all of nine kinds of curcumin derivatives contain 4-hydroxy on the benzene ring in Kim's study, which are different from curcumin derivatives in our experiments, we analyzed that 4-hydroxy is the key group of resistance to Aβ toxicity, but 3-methoxy may greatly suppress 4-hydroxyl, thereby indirectly affect the effect of curcumin derivatives on anti-Aβ neurotoxicity. In the present study we designed and chemically synthesized curcumin and six kinds of curcumin derivatives. As compared with the curcumin, curcumin derivatives show some alterations in the chemical constitution. Their differences each other are mainly reflected in different substituent groups at both ends of benzene ring. The 3-methoxy of Cur1 was removed, but 4-hydroxyl group retained. The 4-hydroxyl group of Cur2 was etherified, simultaneously with 3-methoxy removed. The 4-hydroxyl group of Cur3 was etherified. The 4-hydroxyl group of Cur4 was etherified, also in the meanwhile, 5-methoxy increased. All substituents, linked on the benzene ring, of Cur5 were removed. The two benzene rings, on both the molecular sides of Cur6, were substituted by two furan rings. The quantitative immunocytochemical analysis showed that the changes of chemical constitution of curcumin and curcumin derivatives were followed by the morphological alterations of SK-N-SH cells and cytoplasmatic distribution of MAP2.

Fig. 5 – Western blot analysis of MAP2a and 2b isoforms. Lane 1: Control group. Lane 2: Curcumin group. Lane 3: Aβ group. Lane 4: Cur1 group. MAP2a (300 kd) and 2b (280 kd) isoforms were expressed in all groups, but MAP2a and 2b isoforms were downregulated in Aβ1–42 group, while they were abundantly expressed in curcumin and Cur1 groups.

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Among the curcumin and six kinds of curcumin derivatives, curcumin and Cur1 pretreated SK-N-SH cells, respectively, showed a significant increase in cell activity. The quantitative studies in MAP2-positive staining revealed a significant percent raise. Increased MAP2 immunoreactivity (IR) was seen in the dendritic and perikaryal portions of SK-NSH cells, and the cell bodies of well-spread shapes and profusion of long neurites were almost reverted to normal condition before treated with Aβ1–42. The results supported the evidence that curcumin and Cur1 not only may promote the expression of MAP2, particularly MAP2a isoforms, but prevent down-regulation of MAP2 protein induced by Aβ1–42. Increased MAP2 protein can accelerate the growth rate and repair of microtubules, thus help to restore normal physiological function of nerve cells. We further compared the differences in neuroprotection between curcumin and Cur1, finding that Cur1 had a stronger effect on resistance to Aβinduced damage than curcumin, which might be at least in part, attributable to the role of 4-hydroxyl group on the benzene ring, because 3-methoxy on the benzene ring of Cur1 had been removed. We also found a change in protective action of Cur1, less effective at resisting Aβ1–42 was observed if loss of 4-hydroxyl group on the benzene ring of Cur1 occurred. When two benzene rings, on both the molecular sides of Cur6, were substituted by two furan rings, neuroprotective factor against Aβ-dependent damage disappeared immediately, suggesting again that 4-hydroxyl group on the benzene ring might be a key group of resistance to Aβ neurotoxicity. Although both of Cur3 and Cur4 contained 3methoxy groups on the benzene ring respectively, the former had two 3-methoxy, the latter had three 3-methoxy, but both of them were not resistant to Aβ neurotoxicity. Curcumin, which contained one 3-methoxy, had a weaker resistance to Aβ than Cur1, 3-methoxy of which was removed, indicating that ortho-3-methoxy might greatly suppress 4-hydroxyl functions, led to the decrease in the role of 4-hydroxyl group. Therefore we speculated that 4-hydroxyl group might be primarily responsible for the neuroprotection of curcumin and curcumin derivatives in relation to anti-Aβ toxicity. This structure might be suitable for specific binding to fAβ and subsequent destabilization of the β-sheet-rich conformation of Aβ molecules in fAβ. Among the curcumin and six kinds of curcumin derivatives, curcumin and Cur1 can obviously promote the expression of MAP2. Increased MAP2 proteins may be implicated in the reorganization of the cytoskeleton in the neurodegeneration of AD, indicated that they have played a significant neuroprotective effect on improving abnormal neurite morphology and restoring normal physiological function of nerve cells. The chemical constitution of curcumin and curcumin derivatives may be responsible for their neuroprotective action, in addition to the important role of β-diketone moiety, 4-hydroxyl group on the benzene ring might be a key group of resistance to Aβ neurotoxicity, while 3-methoxy could indirectly affect the beneficial effect of curcumin derivatives in related to suppressing 4-hydroxyl. In view of the special advantages of curcumin and Cur1, such as the penetration of blood-brain barrier and disaggregation of amyloid β, etc., we reasonably believe that curcumin and Cur1 may be considered as an ideal therapeutic agent for the treatment of AD.

4.

Experimental procedures

4.1.

Chemicals

Human SK-N SH cells from Experimental Animal Center of Sun Yat-sen University were used in this study. Culture media and FBS were purchased from Gibco. Trypsin, MTT, DMSO and Aβ1–42 were purchased from Sigma-Aldrich. Antibody to marker proteins [anti-microtubule associated protein-2A&B (MAP2)], raised in rabbits, was purchased from Chemicon. Rhodamine goat anti- rabbit IgG were also purchased from Chemicon. HRP goat anti-rabbit IgG and anti-β-actin were purchased from Boshide. Bisbenzimide trihydrochloride Hoechst 33258 was from Alexis.

4.2.

Experiment groups

Aβ42 group: The use of Aβ1–42 peptides was based upon previous studies, because Aβ42 is very easier aggregation to form plaque deposition than Aβ40, and can induce the neuritic and synaptic toxicity. Added 40，20，10，5，2.5 μg/ml Aβ1–42 into SK-N-SH cells, respectively, and incubated them for 24 h after SK-N-SH cells were cultured for 3 days in normal medium. We chose Aβ1–42 (10 μg/ml) from the different concentrations of Aβ1–42 to establish damaged cell model of Aβ1–42 for subsequent experiments, because, in addition to 10 μg/ml Aβ1–42, other concentrations could cause serious injury to cells or too minor injury to be obviously seen. Curcumin and curcumin derivatives group: Dissolved curcumin and curcumin derivatives in DMSO respectively. Added 10 μg/ml curcumin and its six derivatives (Cur1, Cur2, Cur3, Cur4, Cur5, Cur6) respectively, meanwhile, Aβ1–42 (10 μg/ml) was yet added. Selected 10 μg/ml curcumin and Cur1 for the follow-up experiments, because, in addition to curcumin and Cur1, other curcumin derivatives were much less effective at protecting cells. Control group: SK-N-SH cells were cultured in normal medium (DMEM/F12 containing 10% FBS). Solvent control group: We added 4‰ DMSO of curcumin-free or cur1free into SK-N-SH cells to investigate whether DMSO solvent for curcumin and curcumin derivatives had toxic effect on cells.

4.3.

Synthesis of curcumin and curcumin derivatives

Curcumin and its six derivatives were designed and chemically synthesized in our lab. Curcumin was prepared according to Pedersen method (Pedersen et al., 1985) with slight modification. Boric anhydride (0.35 g, 5 mmol) was suspended in 10 mL EtOAc in the presence of acetylacetone (1.00 g, 10 mmol), and the mixture was stirred for 3 h at 70 °C. After removing the solvent, the resultant white solid was washed with hexane and then, 20 ml EtOAc, 4-hydroxy-3-methoxybenzaldehyde (3.04 g, 20 mmol), and tributyl borate (4.60 g, 20 mmol) were added, and the mixture was stirred for a further 30 min. Butylamine (73 mg, 1 mmol) dissolved in EtOAc was added dropwise for 15 min. The mixture reacted at 70 °C for 24 h. Then 1N HCl was added to adjust the pH to 5, and the solution was heated for 30 min at 60 °C. EtOAc was used to extract the product from the water layer. By using the similar procedure described above for

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Table 1 – MS and NMR analysis of curcumin and curcumin derivatives.

curcumin , Cur1, Cur2, Cur3, Cur4 and Cur 5 were prepared from (4-hydroxybenzaldehyde), (4-methoxybenzaldehyde), (3,4dimethoxybenzaldehyde), (3, 4, 5-trimethoxybenzaldehyde) and benzaldehyde respectively. Cur6 was identified as a new compound and prepared by using the similar procedure described above for curcumin, and the 4-hydroxy-3-methoxybenzaldehyde in curcumin was replaced by 5-methylfuran-2carbaldehyde (Qiu et al., 2008). A VarianUNITY INOVA 500 MHz spectrometer was used to record the 1H NMR spectra in which TMS was the internal standard. The MS analysis was performed on a Finnigan LCQ Deca XP ion trap mass spectrometer (Table 1).

4.4.

MTT assay of cell viability

MTT assay can test the cell survival and cell growth. In living cells, succinate dehydrogenase contained in mitochondria can reduce exogenous MTT to the water-insoluble MTT bluepurple crystalline formazan and deposits in the cells, dead cells have no such function. After formazan was dissolved by Dimethyl sulfoxide (DMSO), OD value can be measured by ELIASA at 490 nm wavelength, which can indirectly reflect the number of living cells. MTT assay worked as follows, added 20 μl MTT (5 mg/ml) and continued to incubate for 4 h in groups mentioned above, then added 150 μl DMSO into every

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well and measured with the experimental wavelength at 490 nm and the reference wavelength at 570 nm for detection of OD value in every well.

significant. All values were presented as means ± standard error (SE).

4.5.

Acknowledgments

Immunocytochemical staining

Cells were fixed with formaldehyde (4% paraformaldehyde, freshly depolymerized, in 0.1 M sodium phosphate buffer pH 7.4) for 15 min at room temperature and washed three times in phosphate buffered saline (10 mum phosphate buffer in 150 mM NaCl). Blocked with blocking buffer [5% (w/v) bovine serum albumin (BSA), 0.1% (w/v) Triton X-100 in Tris buffered saline (TBS) pH 7.4] for 1 h at room temperature. After blocking, cells were incubated with the primary antibody (anti-MAP2 1:500) in dilute solution [2% BSA, 0.3% Triton X-100 in TBS pH 7.4] at 4 °C overnight, and then the cells were washed and incubated with the secondary antibody conjugated to Rhodamine for 2 h at room temperature. The cells were washed three times in PBS, and then reacted with Hoechst 33258 colorant for 15 min at room temperature. After cells were sealed, the immunofluorescence staining of MAP2 was observed under Zeiss Axio Imager Z1 microscope (Carl Zeiss, Heidelberg, Germany). The cell body and the most proximal parts of the processes were outlined for digitalization of area and of fluorescence intensity of MAP2 immunoreactivity. The densitometric analysis was done by using Images Pro Plus 5.0.1. The integrated optical density (IOD) is the total of each light density in the measured region, which is related to the total content of MAP2-positive material.

4.6.

Western blot

The protein levels of MAP2, as well as MAP2a and 2b isoforms were evaluated by Western blot analysis. Samples were taken from whole cell culture in groups. The proteins of 5 μl sample were separated using gel electrophoresis. In order to make the proteins accessible to antibody detection, they were moved from within the gel onto a membrane made of nitrocellulose in 4 °C refrigerator at 500 mA for 5 h. Blocking of non-specific binding was achieved by placing the membrane in a dilute solution of protein-typically Bovine Serum Albumin (BSA). After blocking, the nitrocellulose membranes were incubated with MAP2 polyclonal antibody (1:2000) or β-actin monoclonal antibody (1:1000) at 4 °C overnight. After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to horseradish peroxidase-labelled fluorescence second antibody (1:1000) at room temperature for 1 h. The fluorescence labels allowed the placement of X-ray film against nitrocellulose membrane as it was exposed to the labels and created dark regions which correspond to the protein bands of MAP2 or β-actin. The densitometric analysis, relative bank density, was done by using Images Pro Plus 5.0.1.

4.7.

Statistical analysis

All data including MTT assays, immunofluorescence staining and Western blot was statistically analyzed by Statistical Product and Service Solutions (SPSS) 16.0. Differences among the groups were analyzed by one-way analysis of variance (ANOVA). t Test was used between each two groups. In all statistical analyses, P < 0.05 was regarded as statistically

The authors thank the School of Pharmaceutical Sciences, Sun Yat-sen University, for designing and chemically synthesizing curcumin and curcumin derivates, as well as mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis.

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