Osthole Reverses Beta-Amyloid Peptide Cytotoxicity on Neural Cells by Enhancing Cyclic AMP Response Element-Binding Protein Phosphorylation

Abstract

Accumulation of β-amyloid peptide (Aβ) in the brain plays an important role in the pathogenesis of Alzheimer’s disease (AD). Previous studies have demonstrated the neuroprotective role of osthole against oxygen and glucose deprivation in cortical neurons. However, the effects of osthole on Aβ-induced neurotoxicity in neural cells have rarely been reported. The current study was designed to investigate the protective effects of osthole on a cell model of AD insulted by exogenous Aβ_25-35 and the potential mechanism(s). In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, immunofluorescence analysis, apoptosis assay, reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques were used in primary cortical neurons and SH-SY5Y cells. Our data showed that osthole reduced intracellular Aβ levels in neural cells, which was associated with decreased BACE1 protein; osthole reversed exogenous Aβ_25-35-induced cell viability loss, apoptosis, and synapsin-1 reduction, which was related to the reestablishment of phosphorylation of cyclic AMP response element-binding protein (CREB). The collective evidence indicates that osthole possesses the ability to protect cortical neurons and SH-SY5Y cells against Aβ injury, and the underlying mechanism may be attributed to the enhancement of CREB phosphorylation.

Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder of the brain characterized by progressive cognitive loss and memory decline. The abnormal deposition of β-amyloid peptide (Aβ) is one of the most notable neuropathological hallmarks of AD. Aβ-Induced synaptic loss and subsequent neuronal death have been implicated as a major cause of the cognitive decline associated with AD.^1–4) Accumulating evidence suggests that multiple cellular targets and signaling pathways mediate Aβ-induced neurotoxicity, including N-methyl-D-aspartate (NMDA) receptors and cyclic AMP response element-binding protein (CREB).^5,6) CREB is a pro-survival transcription factor responsible for the expression of a large number of downstream genes,⁷⁾ such as brain-derived neurotrophic factor (BDNF), an important neurotrophic factor that possessed ability to promote neural protection, plasticity, and regeneration in a variety of conditions, such as central nervous system (CNS) injury, cerebral ischemia, and electrical stimulation.^8–10) It is well established that several protein kinase pathways converge to co-regulate CREB activity. Aβ has been shown to interfere with events downstream of activated CREB, especially gene expression of BDNF.¹¹⁾ Moreover, reduced phosphorylation of CREB (p-CREB) has been observed in postmortem brains of AD patients and in transgenic (Tg)-AD mice overexpressing Aβ.^12,13) It has been reported that enhanced CREB function in the CA1 region of the dorsal hippocampus rescues the spatial memory deficits in Tg mice.¹⁴⁾ Therefore, identification of CREB phosphorylation modulators may provide therapeutics that limit the toxic effects of Aβ.¹⁰⁾

Osthole, an active constituent of Angelica Pubescentis Radix (R. angelicae pubescentis) and Cnidium monnieri (L.) Cusson, has been reported to have diverse pharmacological activities,¹⁵⁾ such as anti-hepatitis, anti-inflammatory, and anti-allergic effects.^16–18) Previous studies showed that osthole had an effect on cognitive impairment and neural degeneration in the hippocampus, induced by chronic cerebral hypoperfusion in rats, and the potential mechanism may be related to anti-oxidation and anti-apoptotic actions.¹⁹⁾ A recent study also showed that osthole played a role in protecting neural cells against oxygen and glucose deprivation in rat cortical neurons, through regulating the expression of mitogen-activated protein kinase (MAPK) proteins.²⁰⁾ However, the effect of osthole on Aβ-induced neurotoxicity in neural cells has been rarely reported so far. To gain further insight into the biological role (s) of osthole, in this study we attempted to assess the neuroprotective effects of osthole on a cell model of AD induced by exogenous Aβ and elucidate the correlationship between neuroprotection and CREB phosphorylation.

MATERIALS AND METHODS

Primary Cortical Neurons and SH-SY5Y Cell Culture

Neurons were cultured from the cortices of newborn (days 0–3) mice (C57BL/6). Briefly, meninges-free cortices were isolated and digested with 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) at 37°C for 15 min. The cells (6×10⁵/mL) were plated on 96-well plates or 10-mm dishes with poly-L-lysine-coated tissue culture wells and maintained at 37°C in a humidified atmosphere (5% CO₂–95% air). Neurons were grown in Dulbecco’s modified Eagle’s medium (DMEM) high-glucose media with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 µg/mL streptomycin (1% P/S; all from Gibco Invitrogen Corporation, New York, NY, U.S.A.). The medium were replaced with DMEM containing 2.5 µM cytarabine in 3 d after seeding, which suppressed the growth of gliocytes and consistently provided neuronal cultures with 95% purity. After 14-d culture, neurons were assessed by immunostaining with antibody directed against the neuronal marker NF-M. We extended our observations by measuring p-CREB levels in response to a 15-min glutamate stimulus (100 µM).²¹⁾

The human neuroblastoma cell line, SH-SY5Y, was obtained from Capital Medical University (Beijing, China) and maintained in DMEM/F12 supplemented with 10% FBS and 1% P/S at 37°C in humidified 5% CO₂–95% air. The cells were passaged every 3 d until 75% confluence was achieved.

Preparation of Osthole

The osthole (7-methoxy-8-isopentenoxycoumarin, C₁₅H₁₆O₃, 244.39 Da, structure shown in Fig. 1, >98% purity) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (110822-200407; Beijing, China) and dissolved in dimethyl sulfoxide (DMSO, less than 0.1%, which had no effect on neuronal viability).

Fig. 1. The Chemical Structure of Osthole

Preparation of Aβ_25-35 Stock Solution

The Aβ_25-35 (Sigma, St. Louis, MO, U.S.A.) used in this study was pre-aggregated prior to use. Briefly, 1 mg of Aβ_25-35 was dissolved in about 940 µL of double distilled (d.d.) water and incubated in a 37°C incubator for 7 d to induce aggregation. The aggregated Aβ_25-35 was then diluted to 500 µM and stored at −80°C before use.²²⁾

Cell Viability Assay

The MTT assay was carried out as previously described.²³⁾ Cells were plated in 96-well plates, and cell viability was determined using a conventional cytosine arabinoside, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay. After various incubations, cells were treated with the MTT solution (5 mg/mL) at 37°C for 4 h in the dark. The culture medium was replaced with 150 µL of DMSO, and optical density of formazan was measured at 495 nm using a microplate reader (MR-96A, mindray, Shenzhen, China). Wells containing the medium plus water alone were used to provide blanks for absorbance readings. The following formula was used: OD value=OD_experiment/OD_control

Immunofluorescent Staining

Cells in 96-wells plates were fixed in 4% Para formaldehyde for 30 min, followed by washing with phosphate buffered saline (PBS), and blocked in PBS containing 0.1% (w/v) Triton X-100 for 30 min at room temperature.²⁴⁾ The fixed and permeabilized cells were overlaid with anti-NF-M antibody (1 : 100; StemCell Technologies, Inc., Vancouver, BC, Canada) and incubated at 4°C overnight. After washing off the primary antibody, cells were overlaid with a 1 : 200 dilution of Cy™ 3-conjugated goat anti-mouse immunoglobulin G (IgG) and incubated at room temperature for 1 h. Incubation with primary rabbit polyclonal anti-synapsin-1 (1 : 150), anti-Aβ (1 : 150), and anti-p-CREB (1 : 150) antibodies (all from Abcam, Cambridge, MA, U.K.) was followed by Cy™3 (1 : 200) conjugated goat anti-rabbit IgG secondary antibody incubation. Incubation with primary chicken polyclonal anti-BDNF antibody (1 : 100) was followed by incubation with Cy™3 (1 : 200) (all secondary antibodies from Jackson, West Grove, PA, U.S.A.) conjugated goat anti-chicken IgG secondary antibody. All of the above were supplemented with 4′,6-diamidino-2-phenylindole (DAPI) nuclear dye (1%; Sigma), cover-slipped with anti-fade aqueous mounting media (Southern Biotech, Birmingham, AL, U.S.A.) and viewed using an inverted fluorescence microscope (Nikon Eclipse E600). ImageJ (NIH; Bethesda; MD, U.S.A.) was used for quantitative analysis.

Quantification of Cell Apoptosis by Hoechst 33258 Nuclear Staining

The cells were fixed with 4% paraformaldehyde for 1 h at room temperature. After several washes with PBS, the cells were incubated with Hoechst 33258 (0.5 µg/mL in PBS; Sigma) for 30 min, then examined by inverted fluorescence microscope. Apoptotic cells were identified as distinctive condensed or fragmented nuclear structure.²⁵⁾ Percentage of apoptotic cells in total number of cells was determined. All experiments were conducted in triplicate.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) Quantification of mRNA

RT-PCR was carried out as previously described.²⁴⁾ Total RNA was extracted with TriZoland converted to cDNA by reverse transcriptase using a RevertAid™ First Strand cDNA Synthesis Kit (Thermo, Vilnius, Lithuania). The RT-PCR primers used in this study were as follows: CREB sense primer, 5′-ATA AAG CCT GCA ACA GCC AAC T-3′; CREB antisense primer, 5′-CAA AGA CCT GCT AAT CCT CAC G-3′; Bcl-2 sense primer, 5′-GCG GCG TTC TCA GTG GTG TT-3′; Bcl-2 antisense primer, 5′-GCC TTG TGA ATC CTC GTT ATG GTC-3′; Bax sense primer, 5′-AGC AAC CAC GAA ATC TAC CAA A-3′; Bax antisense primer, 5′-ATG TTG AGC CCG TTC CAG AG-3′; β-actin sense primer, 5′-TGC TGT CCC TGT ATG CCT CT-3′; and β-actin antisense primer, 5′-TTT GAT GTC ACG CAC GAT TT-3′ The PCR reaction was for 35 cycles using a DreamTaq™ Green PCR Master Mix Kit (Thermo). β-Actin served as the control. RT-PCR products were resolved in 1.5% agarose gel stained with ethidium bromide. Optical density analysis was performed using ImageJ.

PS-1 and β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 (BACE1) Production by Enzyme-Linked Immunosorbent Assay (ELISA)

Neurons (1.0×10⁶/mL) were cultured in DMEM with 10% FBS in 96-well plates. Media were collected and assayed for PS-1 and BACE1 using an ELISA Kit (R&D, Minneapolis, MN, U.S.A.). The reaction mixtures were quenched and absorbed at 450 nm by a fluorescent plate reader.²⁶⁾

Statistical Analysis

All data are expressed as mean±S.D. of at least three independent experiments. One-way ANOVA (post hoc test Bonferroni’s) has been used to evaluate differences between multiple groups in Figs. 4, 5, 6 and 7; unpaired, two-tailed, Student’s t-tests has been used to compare differences between two groups in Fig. 3. IBM SPSS Statistics 13.0 was used, and the level of significance was set at a p<0.05.

RESULTS

Osthole Reduced Intracellular Aβ Levels in Neural Cells

Figure 2 showed that both primary neurons (A) and SH-SY5Y cells (B) were positive to NF-M. To access the influence of osthole on Aβ level in these cells, neurons and SH-SY5Y cells were incubated with osthole (50 µM) for 24 h,²⁷⁾ then endogenous Aβ was determined by immunofluorescence and quantitative analysis. Results showed that osthole reduced intensity of Aβ_1-42 in these cells obviously (72.1%, 67.1% in neurons and SH-SY5Y cells with osthole vs. 100% in the two controls, Figs. 3A and B, p<0.05), implicating the neuroprotective effects of osthole on the cells, which was consistent with others reports.^1–4)

We further investigated PS-1 and BACE1 production by these cells using ELISA assay. It was found that osthole challenge decreased BACE1 (2363.4 pg/mL, 2185.1 pg/mL in neurons and SH-SY5Y cells with osthole vs. 2678.6 pg/mL, 2460.3 pg/mL in the two controls, p<0.05; Fig. 3C); while no obvious change of PS-1 was found in the presence or absence of osthole (Fig. 3C, p>0.05). These data indicated that osthole decreased intracellular Aβ levels in neural cells, which may be related to the reduction of BACE1 protein.

Fig. 2. Characterization of Neurons and SH-SY5Y Cells by Immunostaining

Neurons (A) and SH-SY5Y cells (B) were immunostained with anti-NF-M antibody (red), their nuclei were counterstained with DAPI (blue). Scale bar: 50 µm in phase-contrast images, 25 µm in immunofluorescence images. (Color images were converted into gray scale.)

Fig. 3. Osthole Reduced Intracellular Aβ_1-42 Levels in Neural Cells

(A) Immunofluorescence staining for Aβ_1-42 (red) in neurons and SH-SY5Y cells. Nuclei were stained with DAPI (blue). Scale bar: 20 µm; (B) Quantitative analysis of Aβ_1-42 immunofluorescence intensity using Image J software. (C) PS-1 and BACE1 concentrations measured by ELISA assay. ^# p<0.05 vs. control. Values are expressed as the mean±S.D. and represent three independent experiments. (Color images were converted into gray scale.)

Osthole Attenuated Aβ-Induced Loss of Cell Viability

We firstly did dose-response study of exogenous Aβ_25-35 on cell viability. After 10 d’s culture, neurons were exposed to Aβ_25-35 at different concentrations (10, 20, 30, 40 and 50 µM) for 24 h, then MTT assay was used to determine the cell viability. Results showed that Aβ_25-35 insult decreased cell survival in a concentration dependent manner (Fig. 4A), and 30 µM of Aβ_25-35 was the sublethal level, thus was used in the following experiments.

To study the neuroprotective effect of osthole, neurons were pre-treated with osthole in different concentrations for 24 h before Aβ_25-35 insult. Results showed that osthole (0–100 µM) did not affect cell viability in the absence of Aβ (Fig. 4B); while in the presence of Aβ, osthole rescued the neural viability in a dose-dependent manner, 50 µM osthole restored cell survival to 91.9% (vs. 68.6% in Aβ control, p<0.01, Fig. 4B).

Fig. 4. Osthole Attenuated Aβ-Induced Loss of Cell Viability

MTT assay was used to determined neurons viability. (A) Dose-dependent cytotoxicity of Aβ_25-35 on neurons. (B) Osthole protected neurons against Aβ-induced viability loss. Osthole alone (0 to 100 µM for 24 h) did not affect neurons viability in the absence of Aβ; pretreatment with osthole before Aβ_25-35 insult (30 µM, 24 h) rescued neurons viability in a dose-dependent manner. ^# p<0.05, ^## p<0.01 vs. control, ** p<0.01 vs. Aβ alone. Values are expressed as mean±S.D. and represent three independent experiments.

Osthole Inhibited Aβ-Induced Synapsin-1 Reduction

To further investigate the effect of osthole on synaptic proteins, neurons were pre-treated with osthole (50 µM, 24 h) before Aβ_25-35 exposure, and synapsin-1 expression was measured by immunofluorescence and quantitative analysis. As shown in Figs. 5A, B, synapsin-1 protein was strong expressed in control neurons, while Aβ_25-35 reduced intensity of synapsin-1 (49.5% vs. 100% in control, p<0.01); pre-treatment with osthole inhibited the reduction of synapsin-1 and restored the protein obviously (68.2% vs. 49.5% in Aβ control, p<0.01).

Fig. 5. Osthole Inhibited Aβ-Induced Reduction of Synapsin-1

(A) Immunostaining with anti-synapsin-1 antibody (red) in neurons. Scale bar: 25 µm; (B) Quantitative analysis of synapsin-1 immunofluorescence intensity. ^## p<0.01 vs. control, ** p<0.01 vs. Aβ alone. Values are expressed as mean±S.D. and represent three independent experiments. (Color images were converted into gray scale.)

Osthole Protected Neural Cells against Aβ-Induced Apoptosis

Figure 6A showed that control SH-SY5Y cells displayed a normal morphology with 1–2 long neuritis which contacted adjacent cells; after co-culture with Aβ_25-35 (30 µM, 24 h), the cells appeared shrunken with retracted neuritis; while pre-treatment with osthole (50 µM, 24 h) restored the cell morphology nearly to normal.

To investigate the role of osthole on Aβ-induced apoptosis, SH-SY5Y cells and primary cortical neurons were stained with Hoechst 33258, and their nuclei morphology were assessed.²⁵⁾ As shown in Figs. 6B and C, apoptotic cells had blue-stained nuclei with multiple bright specks of fragmented or condensed chromatin. Aβ_25-35 induced an increase in apoptosis (24.1%, 18.0% in neurons and SH-SY5Y cells respectively vs. 4.7%, 6.0% in normal controls, p<0.01); while osthole reduced the number of condensed and fragmented nuclei in these cells obviously (11.6% in neurons and 10.3% in SH-SY5Y cells respectively), demonstrating an anti-apoptotic effect against Aβ_25-35 injury.

To further elucidate potential mechanism of anti-apoptosis of osthole, Bcl-2 and Bax mRNA expression were examed using RT-PCR analysis. Bcl-2 is anti-apoptotic while Bax is pro-apoptotic. Balance between the two gene expression is critical to turning the cellular apoptotic machinery on and off.²⁸⁾ Results in this study showed that Aβ induced low Bcl-2 expression and opposite Bax expression, which is consistent with other’s report²²⁾; osthole markedly increased Bcl-2 mRNA in Aβ-insulted neurons, but attenuated Bax mRNA, thus lessened the ratio of Bax/Bcl-2 mRNA (1.18 in group of Aβ plus osthole vs. 1.82 in Aβ control, p<0.05, Figs. 6D, E, F), which may be part of the reason of anti-apoptotic effect of osthole.

Fig. 6. Osthole Protected Neural Cells against Aβ-Induced Apoptosis

(A) SH-SY5Y cells morphology identified by NF-M immunostaining (red). Scale bar: 20 µm; (B) Apoptotic neurons identified by Hoechst 33258 nuclei stain (blue). Scale bar: 50 µm; (C) Quantification of percentages of apoptotic neurons and SH-SY5Y cells. (D) Expression of Bcl-2 and Bax mRNA in neurons examined by RT-PCR. (E) Quantification of Bax and Bcl-2 mRNA expression. (F) Quantification of the ratio of Bax/Bcl-2 mRNA. ^## p<0.01 vs. control, ** p<0.01 vs. Aβ alone. Values are expressed as mean±S.D. and represent three independent experiments. (Color images were converted into gray scale.)

Osthole Reestablished Aβ-Impaired CREB Phosphorylation

We then study the correlation between CREB phosphorylation activity and neuroprotective potential of osthole in neurons. We found that CREB mRNA expression was not changed by exposure to Aβ_25-35 (30 µM, 24 h) in the presence or absence of osthole (50 µM) (p﹥0.05, Figs. 7A, B). While phosphorylation of CREB was inhibited by Aβ_25-35 challenge (111.7%, vs. 137.1% in normal control, p<0.05, Figs. 7C, D), pre-treatment with osthole effectively blocked the decline of CREB phosphorylation (134.7%, vs. 111.7% in Aβ control, p<0.05, Figs. 7C, D). These data suggest that target of osthole is CREB phosphorylation, primarily at a post-transcriptional level, not CREB mRNA.

To further confirm that enhancement of CREB phosphorylation contributed to neuroprotection of osthole, H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate, Cell Signaling, Danvers, MA, U.S.A.), an inhibitor of PKA was used to block CREB phosphorylation.²⁹⁾ Our data indicated that H89 itself (20 µM, 1 h) reduced p-CREB intensity, accompanied by reduction of BDNF expression and cell viability loss both in the normal neurons and Aβ-insulted neurons (Figs. 7C–E); osthole itself did not affect CREB phosphorylation, BDNF expression and viability of the normal cells, while partially reversed H89- or H89+Aβ-induced decline of p-CREB and BDNF intensity and loss of cell viability (Figs. 7C–E). These findings implicated that osthole may exert neuroprotective effect by relieving the block of H89 on CREB phosphorylation, and consequently enhancing CREB phosphorylation in neural cells.

Fig. 7. Osthole Re-established Aβ-Impaired CREB Phosphorylation

(A) CREB mRNA expression in neurons examined by RT-PCR. (B) Quantification of CREB mRNA expression. (C) Immunostaining with anti-p-CREB (upper row) and anti-BDNF (lower row) (red), scale bar: 20 µm; (D) Quantitative analysis of p-CREB and BDNF immunofluorescence intensity. Phosphorylation of CREB in neurons is briefly stimulated by treatment with 100 µM glutamate for 15 min. (E) Cell viability determined by MTT assay. ^## p<0.01 vs. control, ^△△ p<0.01 vs. H89 alone, ** p<0.01 vs. Aβ alone, ^^ p<0.01 vs. Aβ plus H89. Values are expressed as mean±S.D. and represent three independent experiments. (Color images were converted into gray scale.)

DISCUSSION

AD is defined by an accumulation of extracellular senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs). Aβ is the primary cause of SPs in AD. Increasing evidence has shown that the production and accumulation of Aβ is central to the pathogenesis of AD. In brains of AD patients, there is recognized neural death and then loss of synaptic connections within selective regions caused by Aβ neurotoxicity. Aβ peptides are derived from sequential proteolytic cleavages of the amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1), a membrane-bound aspartyl protease identified as β-secretase^30,31) and another protease, γ-secretase.³²⁾ Results in this study demonstrated that osthole markedly reduced intracellular Aβ_1-42 level in neurons and SH-SY5Y cells as determined by immunofluorescence analysis (Figs. 3A, B), which implicated the neuroprotective effects of osthole on the cells.^1–4) A reduction of BACE1 protein in these cells was also revealed by ELISA assays (Fig. 3C). These data indicated that osthole may have potential inhibition on Aβ formation by reducing BACE1 level.

The toxicity of Aβ_25-35 is very similar to that of Aβ_1-42, both of the peptides are easily amyloid genic.^33,34) It has been confirmed that Aβ_25-35 can cause neural cell death, induced suppression of extracellular signal-regulated kinase (ERK) or its downstream intermediate CREB in neuroblastoma cells and in rats after injection of Aβ_25-35.^35,36) In the present study, we thus used Aβ_25-35 to induce cytotoxicity in neurons and SH-SY5Y cells. It was found that Aβ_25-35 insult markedly decreased cell viability and synapsin-1 protein level (Figs. 4, 5), but increased apoptosis of these cells (Fig. 6); pre-treatment with osthole at a dose of 50 µM rescued the decline of cell survival and synapsin-1 expression (Figs. 4, 5), while decreased the cells apoptosis obviously, which may be associated with the reduction of ratio of Bax/Bcl-2 mRNA (Fig. 6). These results suggested that osthole may effectively protect neural cells from Aβ-induced cytotoxicity by recovering the synaptic functional protein and inhibiting apoptosis of the cells.

To elucidate the underlying mechanism of neuroprotective effects of osthole, we assessed the influence of osthole on CREB phosphorylation in neurons. The nuclear transcription factor CREB is an important determinant of cellular processes involved in the growth, survival, and function of hippocampal neurons.³⁷⁾ Many studies have demonstrated that activation of CREB signaling is important for neural survival.^38,39) cAMP/PKA is the classic signaling pathway of CREB phosphorylation, and PKA inhibitor H89 is commonly used to inhibit CREB phosphorylation.⁴⁰⁾ We in the present study demonstrated that Aβ_25-35 challenge inhibited CREB phosphorylation, but did not affect CREB mRNA expression; pre-treatment with osthole effectively blocked the decline of CREB phosphorylation, while did not influence CREB mRNA. These data suggested that promotion of CREB phosphorylation is the main objective of osthole. However, further experiments should be performed to detect the total protein level of CREB and the ratio of pCREB/CREB using immunoblot, which will be helpful to deep comprehend the effects of osthole on the activation of CREB.

To further study whether or not the enhancement of CREB phosphorylation contributed to neuroprotection of osthole, H89 was used to block CREB phosphorylation.²⁹⁾ It was found that H89 reduced p-CREB intensity in neuron, accompanied by reduction of BDNF expression and loss of cell viability both in the normal neurons and Aβ-insulted neurons; osthole itself did not affect CREB phosphorylation, BDNF expression and viability of the normal cells, while partially reversed H89- or H89+Aβ-induced decline of p-CREB and BDNF and loss of cell viability (Fig. 7), implicating the neuroprotective effect of osthole by relieving the block of H89 on CREB phosphorylation and consequently enhancing CREB phosphorylation in neural cells. Both inhibition of Aβ formation by reducing the BACE level and alleviation of Aβ-induced cytotoxicity by activation of CREB phosphorylation account for the neuroprotection of osthole against AD, the correlation between them is not very clear and needs to be further explored.

CONCLUSION

These observations indicated that osthole reduced intracellular Aβ levels in neural cells, which was associated with decreased BACE1 protein; osthole reversed exogenous Aβ_25-35-induced cell viability loss, apoptosis, and synapsin-1 reduction, which might be related to the reestablishment of CREB phosphorylation. The collective evidence suggests that osthole possesses the ability to protect neural cells against Aβ injury, and the underlying mechanism may be attributed to the enhancement of CREB phosphorylation.

Acknowledgment

This work was financially supported by the National Nature Science Foundation of China (No. 81173580), Liaoning Province Nature Science Foundation (No. 201102144), Liaoning Province Excellent Talents Project, and Special Fund Project for Technology Innovation of Shenyang City (No. F11-264-1-42).

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