Resveratrol Inhibits β-Amyloid-Induced Neuronal Apoptosis through Regulation of SIRT1-ROCK1 Signaling Pathway

Alzheimer’s disease (AD) is characterized by the accumulation of β-amyloid peptide (Aβ) and loss of neurons. Recently, a growing body of evidences have indicated that as a herbal compound naturally derived from grapes, resveratrol modulates the pathophysiology of AD, however, with a largely unclear mechanism. Therefore, we aimed to investigate the protection of resveratrol against the neurotoxicity of β-amyloid peptide 25–35 (Aβ25–35) and further explore its underlying mechanism in the present study. PC12 cells were injuried by Aβ25–35, and resveratrol at different concentrations was added into the culture medium. We observed that resveratrol increased cell viability through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) colorimetric assays. Flow cytometry indicated the reduction of cell apoptosis by resveratrol. Moreover, resveratrol also stabilized the intercellular Ca2+ homeostasis and attenuated Aβ25–35 neurotoxicity. Additionally, Aβ25–35-suppressed silent information regulator 1 (SIRT1) activity was significantly reversed by resveratrol, resulting in the downregulation of Rho-associated kinase 1 (ROCK1). Our results clearly revealed that resveratrol significantly protected PC12 cells and inhibited the β-amyloid-induced cell apoptosis through the upregulation of SIRT1. Moreover, as a downstream signal molecule, ROCK1 was negatively regulated by SIRT1. Taken together, our study demonstrated that SIRT1-ROCK1 pathway played a critical role in the pathomechanism of AD.


Introduction
Alzheimer's disease (AD) was firstly described by Alois Alzheimer about a century ago [1]. It is the most common neurodegenerative disease in the elderly, which eventually impairs the cognitive function of brain. The prevalence of AD exponentially increases with age. Currently, nearly one-eighth of people older than 65 years present with AD, and worldwide prevalence of the disease is expected to be close to 30 millions by 2050 [2]. The pathological features of AD include extracellular Ab plaques and intracellular neurofibrillary tangles [3]. Ab protein abnormally deposits in the brain, which is the typical hallmark of AD. As a toxic factor, Ab aggregation plays a critical role in the initiation phase of AD pathogenesis [4]. Therefore, a large amount of therapeutic efforts have been focused on reducing the toxicity of Ab protein and preventing the formation of Ab oligomer [5].
As a natural herbal compound, resveratrol is originally found in grapes, peanuts and other plants [6]. It has long been reported that resveratrol possesses a wide range of biological activities, such as anti-oxidant, anti-inflammatory, anti-cancer and anti-aging effects in numerous organisms [7,8]. Recently, resveratrol has also attracted the attention from neuroscientists because of its neuroprotective properties. For example, resveratrol regulates neurological disorders including strokes and Huntington's disease [9]. Recent studies reported that resveratrol protects neurons against peroxide (H 2 O 2 ), 1-methyl-4-phenylpyridine ion (MPP) and Ab injury [10,11,12]. A rat model of AD suggests that resveratrol can prevent the cognitive impairment [13]. However, the neuroprotection of resveratrol against Ab cytotoxicity, especially the underlying mechanism, remains largely unknown due to its wide pharmacological actions. Therefore, this study investigated the protective effect of resveratrol against Ab cytotoxicity and explored the possible underlying mechanisms.
Resveratrol triggers the overexpression of SIRT1, a member of the sirtuin family [14,15], which is a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase and plays an essential role in regulating cellular functions, such as transcriptional silencing of telomeres and life-span extension [16,17]. SIRT1 is also involved in calorie restriction and aging [18]. Two recent studies strongly implied that SIRT1 exerts a role in neuroprotection. Firstly, resveratrol-induced SIRT1 expression rescues the neuronal dysfunction against polyglutamines (polyQ) toxicity in Huntington's disease [19]. Secondly, in a mouse model of slow Wallerian degeneration, resveratrol protects neurons from degeneration due to axotomy [20]. These results suggested that resveratrol is therapeutically valuable against the neurological disorder. More recently, Julien et al. [21] declared that SIRT1 may regulate the aging and metabolic processes in AD, and the loss of SIRT1 is closely associated with the Ab accumulation and disease progression.
ROCK is serine/threonine protein kinase. There are two different diastereomers: ROCK1 and ROCK2. The former is known partly for its role in inhibiting the non-amyloidogenic, asecretase processing of amyloid precursor protein (APP) [22]. A previous study showed that SIRT1 overexpression in primary neurons enhances cell viability and reduces Ab secretion and ROCK1 expression, suggesting that SIRT1 enhances a-secretasemediated non-amyloidogenic APP processing partly via ROCK1 signaling [23]. Additionally, in experiments with squirrel monkeys, calorie restriction attenuates AD type brain amyloidosis, while the protein content of SIRT1 is increased, and ROCK1 is decreased [24]. Based on these data, we hypothesized that resveratrol protected the neurons against Ab neurotoxicity possibly through activating the SIRT1 expression, likely involving the subsequent regulation of ROCK1.
To test the above hypothesis regarding Ab 25-35 neurotoxicity and explore the underlying mechanism, we examined the protective effect of resveratrol on a neurotoxic cell model of Ab 25-35 injury using PC12 cells. MTT and LDH assays were employed to determine the cell viability; intercellular calcium ([Ca 2+ ]i) level was measured using fluorescent Ca 2+ indicator (FLuo-3/AM); and flow cytometry with Annexin V-FITC/PI double staining was used to detect the cell apoptosis. Furthermore, real time quantitative PCR and Western blotting were performed to detect the expressions of SIRT1 and ROCK1 at both the mRNA and protein levels, respectively. Finally, SIRT1 inhibitor nicotinamide and ROCK1 inhibitor Y-27632 were used to further explore the mechanisms of both proteins in the neuroprotection against neurotoxicity by resveratrol.

MTT Assay
Cell viability was assessed by MTT assay according to a previous report [27]. Briefly, cells were added with MTT solution to a final concentration of 0.5 mg/ml and incubated at 37uC for 4 h. Then the medium was gently aspirated, and DMSO was added into each well to dissolve the formazan product by shaking at room temperature for 10 min. The absorbance of each sample at a wavelength of 490 nm (A490) was determined using a microplate reader (Multiskan MK3, Thermo Labsystems, Philadelphia, PA, USA). Cell viability was quantified based on the recorded A490.

LDH Assay
To further assess the cell viability and neurotoxicity of Ab 25-35 , LDH assay was performed with a modified method [28] using LDH kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. The absorbance of each sample was measured at a wavelength of 450 nm (A450) using a microplate reader (Multiskan MK3, Thermo Labsystems, Philadelphia, PA, USA). Cell viability was then quantified based on the recorded A450.

Intracellular Ca 2+ Measurement
Intracellular Ca 2+ level was examined using fluorescent Ca 2+ indicator (FLuo-3/AM) as previously described [28]. Briefly, PC12 cells were cultured in 96-well plates in the presence or absence of Ab [25][26][27][28][29][30][31][32][33][34][35] and resveratrol, respectively. After 24 h of in vitro culture, the culture medium was aspirated, and the cells were washed by D-Hanks buffer. Then the cells were incubated in D-Hanks buffer containing FLuo-3/AM at a final concentration of 5 mM for 45 min at 37uC. A Zeiss LSM 780 Laser Scanning Confocal Microscope (Carl Zeiss SAS, Jena, Germany) was used to detect the fluorescence intensities. The excitation was set at a wavelength of 488 nm, and the emission was recorded at 526 nm. The obtained images were analyzed by the ZEN software package of Zeiss.

Cell Apoptosis Analysis
PC12 cells in different groups were double-stained with Hoechst 33342 and propidium iodide (PI) at a concentration of 10 mg/ml for 15 min. Labeled cells were observed under a LSM 780 Laser Scanning Confocal Microscope (Carl Zeiss SAS, Jena, Germany). In order to further discriminate the early and late cell apoptosis and perform a quantitative analysis, flow cytometry with Annexin V-FITC/PI double staining was employed as previously described [29]. Briefly, PC12 cells were detached with 0.125% trypsin, centrifuged at 1,000 rpm for 5 min, and then washed twice with PBS. Subsequently, 5610 5 cells were resuspended in binding buffer and stained with Annexin V-FITC and PI for 15 min in the dark at room temperature. Finally, the fluorescence of each group was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

Real Time Quantitative PCR
In order to investigate the protective mechanism of resveratrol, the expression of SIRT1 and ROCK1 was examined by real time quantitative PCR. Total RNA was extracted from PC12 cells using Trizol reagent (Sangon Biotech Co., Ltd., Shanghai, China). The RNA integrity was spectrophotometrically examined according to its A260/A280 absorption. Subsequently, cDNA was synthesized through reverse transcription. Real time quantitative PCR was performed on Mastercycler ep realplex (Eppendorf, Hamburg, Germany). Briefly, the amplification reaction was

Western Blotting
PC12 cells were collected and lysed by RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Total proteins were extracted, and the protein concentration was determined using BCA kit (Boster Biological Technology, Wuhan, China). The protein samples were separated on 8% polyacrylamide gels and electro-transferred onto nitrocellulose (NC) membranes in an ice-water environment. Blots were blocked by 5% defatted milk in Tris-buffer containing 0.1% Tween-20 and then incubated with primary anti-SIRT1 (1:1,000, Santa Cruz Biotechnology, Inc., CA, USA) and anti-ROCK1 (1:3,000, Sigma-Aldrich, St. Louis, MO, USA) antibodies at 4uC overnight. Subsequently, the blots were incubated with the anti-rabbit secondary antibody (1:1,000, ZSGB-BIO ORIGENE, Beijing, China) at room temperature for 1 h. Then the blots were

Effect of Nicotinamide on Resveratrol's Protection
The role of SIRT1 in the neuroprotection of resveratrol against Ab 25-35 toxicity was investigated by using SIRT1 inhibitor nicotinamide. Nicotinamide was diluted in deionized distilled water and stored at 4uC. In the resveratrol protection group, nicotinamide and resveratrol were simultaneously added into the culture medium, and the final concentration of nicotinamide was 5 mM as previously reported [30]. The cell growth determined by MTT assay was used to analyze the role of SIRT1 in the protection process. Effects of Nicotinamide and Y-27632 on SIRT1 and ROCK1 Expression Y-27632 is a potent and specific inhibitor of ROCK1. Nicotinamide and Y-27632 were used to further investigate whether SIRT1 regulated the ROCK1 expression. After 24 h of in vitro culture, PC12 cells were treated with 5 mM nicotinamide and 10 mM Y-27632, respectively. The concentration of Y-27632 was referred a previous report [31]. Cells without the addition of either nicotinamide or Y-27632 were served as the normal control group. After 24 h treatment with the inhibitors, the expressions of SIRT1 and ROCK1 were analyzed by real time quantitative PCR and Western blotting.

Statistical Analysis
Statistical analysis was performed using the SPSS 17.0 software. Data of MTT and LDH assays were processed with analysis of variance (ANOVA) followed by Newman-Keuls. Other data were analyzed by Student's t-test. All the data were expressed as mean 6 S.E.M. p , 0.05 was considered as statistically significant.

Resveratrol Upregulates the SIRT1 Expression and Downregulates the ROCK1 Expression in Ab 25-35 -treated Cells
To further explore the protective mechanism of resveratrol on PC12 cells against Ab 25-35 neurotoxicity, we examined the expression of SIRT1 and ROCK1 at the mRNA level using real time quantitative PCR. As shown in Fig. 5B, the SIRT1 expression was significantly decreased for around 8-fold by Ab 25-35 compared with the normal control group (Fig. 5F, p , 0.01). Meanwhile, the expression of SIRT1 was markedly increased when PC12 cells were incubated with resveratrol. However, the ROCK1 expression was increased by Ab [25][26][27][28][29][30][31][32][33][34][35] compared with the normal control group, while such increase was significantly attenuated by resveratrol ( Fig. 5C and G, p , 0.01). We also analyzed the protein expression of SIRT1 and ROCK1 by Western blotting. The SIRT1 expression was increased in the presence of resveratrol compared with that in the Ab 25-35 injury group ( Fig. 5A and D, p , 0.01), though it remained lower than that in the normal control group (Fig. 5D, p , 0.01). The ROCK1 expression was decreased in the presence of resveratrol compared with that in the Ab 25-35 injury group ( Fig. 5A and E, p , 0.01); however, it was still higher than that in the normal control group (Fig. 5E, p , 0.01). These data implied that SIRT1 and ROCK1 partially participated in the neuroprotection of resveratrol against Ab 25-35 injury.

Nicotinamide Partially Attenuates the Neuroprotective Effect of Resveratrol on PC12 Cells
Based on the changes of SIRT1 expression in PC12 cells, we investigated the role of SIRT1 in the neuroprotection of resveratrol by adding nicotinamide, the SIRT1 inhibitor, into the culture medium of the resveratrol protection group. The results showed that although the growth of PC12 cells in the resveratrol protection group (Fig. 6B) was significantly improved compared with that in the Ab 25-35 injury group (Fig. 6A), such protective effect of resveratrol was attenuated by the addition of nicotinamide, evidenced by loss of neurites in more cells (Fig. 6C). Moreover, Hoechst 33342/PI double staining showed that more apoptotic cells were observed with nicotinamide treatment (Fig. 6D-F). MTT assay also confirmed that the cell viability in the nicotinamide group was lower than that in the resveratrol protection group (Fig. 6G, p , 0.01), though it remained higher than that in the Ab 25-35 injury group. Furthermore, cell apoptosis analysis demonstrated that the apoptotic rate in the nicotinamide group was higher than that in the resveratrol protection group (Fig. 6H, p , 0.01). Therefore, resveratrol protected PC12 cells against the Ab 25-35 neurotoxicity partially through regulating the SIRT1 expression.

SIRT1 Downregulates ROCK1 Expression
To further explore the mechanism in the neuroprotective effects of resveratrol against Ab [25][26][27][28][29][30][31][32][33][34][35] and whether SIRT1 regulated the ROCK1 expression, the expression of SIRT1 and ROCK1 were determined at the mRNA level in PC12 cells treated with nicotinamide or Y-27632 (a selective inhibitor of ROCK1). The results showed that nicotinamide significantly inhibited the SIRT1 expression and increased the ROCK1 expression compared with the normal control group (Fig. 7B, C, F, and G, p , 0.01). Y-27632 greatly inhibited the ROCK1 expression compared with the normal control group ( Fig. 7C and G, p , 0.01); however, the expression of SIRT1 was not affected. Furthermore, we analyzed the protein expression of SIRT1 and ROCK1 by Western blotting. The expression of SIRT1 and ROCK1 in groups treated with nicotinamide or Y-27632 was consistent with the results of real time quantitative PCR (Fig. 5A, D, and E, p , 0.01). Therefore, nicotinamide inhibited the SIRT1 expression and activated the ROCK1 expression, whereas Y-27632 only inhibited the ROCK1 expression. These data indicated that ROCK1 was a downstream signal molecule and could be suppressed by SIRT1.
Resveratrol is naturally synthesized when plant suffers from fungal attack and is exposed to ultraviolet light, and it is mainly distributed in the skin and seeds of purple grapes and peanuts [32]. As an active ingredient of polyphenols in red wine and many plants, resveratrol has received increasing attention due to its therapeutic potentials in treating inflammation, cancer and neurologic disorders. Actually, quite a few reports suggested that drinking red wine can attenuate the cognitive degeneration, which is mainly attributed to resveratrol, the polyphenol compound [33,34]. This study showed that resveratrol increased the cell viability against Ab 25-35 toxicity. The growth of PC12 cells in the resveratrol protection group was significantly improved compared with that in the Ab 25-35 injury group. Besides resveratrol, other herbal medicines, such as Ginkgolide B [35] and Bacopa monnieri [36], also demonstrate neuronal protection effects against Ab [25][26][27][28][29][30][31][32][33][34][35] . In recent years, more than 50 different plants or herbs, either in pure molecular form or in specific extracts, have been identified potentially useful for AD treatment [37]. Investigation of the health benefits of these natural compounds including resveratrol poses substantial challenges to modern medicine; especially, herb-derived drugs become popular in recent days because of their good safety profiles and low incidence of side effects [38].
In our study, we found that the protection of 50 mM resveratrol was better than that in other groups. Conte et al. [39] also suggested that 50 mM resveratrol protects PC12 cells from Ab 1-41 injury, and the concentration higher than 50 mM exerts the inhibitory effect, which is consistent with our study. However, Alvira et al. [40] reported that 100 mM resveratrol still renders the protection on cerebellar neurons against MPP, which is slightly better than 50 mM. This is possibly because that Ab [25][26][27][28][29][30][31][32][33][34][35] and Ab 1-41 might insult cells differentially, and the resistance to drugs of different cells also has variations among studies.
A growing body of evidences demonstrate that [Ca 2+ ]i is a universal signaling molecule that regulates many cellular functions, and it is one of the key elements of apoptotic signaling pathways [41,42]. Many toxic factors trigger apoptosis by early transient elevation of intracellular free calcium, thereby resulting in the increase of membrane permeability and mitochondrial membrane disruption [43]. Ferreiro et al. [44] reported that Ab [25][26][27][28][29][30][31][32][33][34][35] changes the [Ca 2+ ]i homeostasis. Therefore, we measured the changes of [Ca 2+ ]i level in the prevention of resveratrol against Ab 25-35 toxicity. Our data indicated that Ab 25-35 obviously increased the [Ca 2+ ]i levels in PC12 cells, while resveratrol restored the Ca 2+ homeostasis. This was consistent with the high signals in MTT and LDH assays. Therefore, the increase of [Ca 2+ ]i was the initial step in the injury of PC12 cells, and resveratrol possibly protected cells from the damage of Ab [25][26][27][28][29][30][31][32][33][34][35] toxicity at the beginning. Further study is necessary to determine the effect of resveratrol on intracellular Ca 2+ homeostasis.
Resveratrol is also a calorie restriction mimetics that triggers the overexpression of sirtuins, of which SIRT1 is closely associated with aging and Ab accumulation [14,21]. To further explore the underlying mechanisms of resveratrol's protection against Ab [25][26][27][28][29][30][31][32][33][34][35] neurotoxicity, we examined the expression of SIRT1, a silent information regulator. Our results showed that Ab 25-35 obviously reduced the expression of SIRT1, which was partially recovered by resveratrol. A current report also suggested the low expression of SIRT1 in cerebral cortex of AD [15].
Recently, Qin et al. [23] reported that a-secretase activity is increased in SIRT1 transgenic mice, which is correlated with a reduction in the ROCK1 expression. Coincidentally, a similar result has been reported in a squirrel monkey model of calorie restriction [24]. In this study, Ab caused the loss of SIRT1 and the increase of ROCK1. Importantly, resveratrol increased the SIRT1 level accompanied by ROCK1 reduction, suggesting that SIRT1 and ROCK1 played key roles in the anti-neurotoxicity of resveratrol. However, the feedback loop between SIRT1 and ROCK1 remains unclear. We revealed the regulation of ROCK1 by SIRT1 using nicotinamide and Y-27632, which are inhibitors of SIRT1 and ROCK1, respectively. As expected, nicotinamide significantly inhibited the SIRT1 expression and simultaneously increased the ROCK1 expression. Y-27632 greatly inhibited the ROCK1 expression, while had no effect on the SIRT1 expression. These results indicated that ROCK1 was a downstream signal molecule and downregulated by SIRT1, which in turn inhibited the Ab 25-35 -induced cell apoptosis.
Although we demonstrated the connection among resveratrol, SIRT1, ROCK1 and Ab in the present study, the downstream events in neuronal protection after the regulation of SIRT1 and ROCK1 expression remain unclear. Recent reports demonstrated that SIRT1 also regulates two apoptosis-associated proteins, P53 and FOXO [45,46]. Additionally, a recent study indicated that SIRT1 inhibits the nuclear factor kappa B signaling and protects the neurons [47]. Moreover, it has also been shown that resveratrol directly binds to Ab 42 and interferes with its aggregation, leading to an attenuated Ab cytotoxicity [48]. More likely, these functions play a synergistic role in the neuronal protection, which warrants further investigations.
Taken together, resveratrol protected PC12 cells from Abinduced neurotoxicity and inhibited the cell apoptosis. It prevented the LDH leakage and maintained the intracellular Ca 2+ homeostasis. Especially, Ab 25-35 suppressed the SIRT1 expression and hence upregulated the expression of downstream ROCK1, which was significantly recovered by resveratrol. Our data further demonstrated that the anti-apoptosis effects of resveratrol were partially attributed to the SIRT1-ROCK1 pathway. This study provided new insights into the pathogenesis and treatment of neurodegenerative disease.