Accepted Manuscript Silibinin Inhibits Acetylcholinesterase Activity and Aβ Peptide Aggregation: A DualTarget Drug for Alzheimer's Disease Treatment Songwei Duan, Xiaoyin Guan, Runxuan Lin, Xincheng Liu, Ying Yan, Ruibang Lin, Tianqi Zhang, Xueman Chen, Jiaqi Huang, Xicui Sun, Qingqing Li, Shaoliang Fang, Jun Xu, Zhibin Yao, Huaiyu Gu PII:
S0197-4580(15)00102-5
DOI:
10.1016/j.neurobiolaging.2015.02.002
Reference:
NBA 9204
To appear in:
Neurobiology of Aging
Received Date: 29 April 2014 Revised Date:
3 February 2015
Accepted Date: 4 February 2015
Please cite this article as: Duan, S., Guan, X., Lin, R., Liu, X., Yan, Y., Lin, R., Zhang, T., Chen, X., Huang, J., Sun, X., Li, Q., Fang, S., Xu, J., Yao, Z., Gu, H., Silibinin Inhibits Acetylcholinesterase Activity and Aβ Peptide Aggregation: A Dual-Target Drug for Alzheimer's Disease Treatment, Neurobiology of Aging (2015), doi: 10.1016/j.neurobiolaging.2015.02.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Silibinin Inhibits Acetylcholinesterase Activity and Aβ Peptide
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Aggregation: A Dual-Target Drug for Alzheimer's Disease Treatment
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Songwei Duan1, 4, Xiaoyin Guan1, Runxuan Lin1, Xincheng Liu1, Ying Yan1, Ruibang Lin1,4,
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Tianqi Zhang1, Xueman Chen1, Jiaqi Huang1, Xicui Sun5, Qingqing Li1, Shaoliang Fang2,
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Jun Xu3, Zhibin Yao1, Huaiyu Gu1*
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Department of Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China 510080; 2Key Lab of High Performance Computing of Guangdong Province,
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Guangzhou, China 510033; 3 Research Center for Drug Discovery and Institute of Human
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Virology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China
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510006; 4Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong,
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China 510080; 5Guangzhou Brain and Psychiatric Hospital, Guangzhou, China,510450.
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*Corresponding author: Huaiyu Gu: 1Department of Anatomy, Zhongshan School of
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Medicine of Sun Yat-sen University, Guangzhou, Guangdong, China, 510080. Tel: (8620)
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8733 2086; Fax: (8620) 8733 2086; E-mail: [email protected]
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Number of pages: (43)
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Number of figures, tables: figures (9), tables (1).
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Number of words for Abstract, Introduction, and Discussion: Abstract (203), Introduction
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(578), and Discussion (1842).
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Abstract Alzheimer’s disease (AD) is characterized by amyloid-β (Aβ) peptide aggregation and
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cholinergic neurodegeneration. Therefore, in this paper, we examined silibinin, a flavonoid
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extracted from Silybum marianum, to determine its potential as a dual inhibitor of
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acetylcholinesterase (AChE) and Aβ peptide aggregation for AD treatment. To achieve this,
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we used molecular docking and molecular dynamics (MD) simulations to examine the affinity
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of silibinin with Aβ and AChE in silico. Next, we used circular dichroism (CD) and
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transmission electron microscopy (TEM) to study the anti-Aβ aggregation capability of
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silibinin
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immunohistochemistry, BrdU double labeling and a gene gun experiment were performed on
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silibinin-treated APP/PS1 transgenic mice. In MD simulations, silibinin interacted with Aβ
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and AChE to form different stable complexes. After the administration of silibinin, AChE
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activity and Aβ aggregations were down-regulated, and the quantity of AChE also decreased.
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In addition, silibinin-treated APP/PS1 transgenic mice had higher scores in the Morris Water
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Maze. Moreover, silibinin could increase the number of newly generated microglia,
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astrocytes, neurons and neuronal precursor cells. Taken together, the data suggested that
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silibinin could act as a dual inhibitor of AChE and Aβ peptide aggregation, therefore
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suggesting a therapeutic strategy for AD treatment.
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Keywords: Alzheimer’s disease; amyloid β protein; MD simulation; silibinin; memory
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deficits; acetylcholinesterase; APP/PS1 transgenic mice
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1. Introduction Alzheimer’s disease (AD) is a common and severe neurodegenerative disorder among the
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elderly, which is characterized by a cascade of pathological changes. These changes include
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abnormal Amyloid β (Aβ) peptide aggregation with the consequent formation of senile
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plaques in the cerebrocortical and limbic regions and a reduction in the levels of acetylcholine
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(ACh) (Isacson et al., 2002), together with progressive neuron loss (Gómez Isla et al.,
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1997). Although considerable studies have been conducted on AD in the past years, the
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etiology of it remains unclear. Based on the cholinergic hypothesis of AD (Terry and
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Buccafusco, 2003) (Perry et al., 1999) (Deutsch, 1971), the current strategy of AD
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intervention is mainly to ameliorate the cognitive symptoms related to ACh depletion, thereby
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enhancing the central cholinergic neurotransmission by reversing acetylcholinesterase
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(AChE) inhibition. This hypothesis is based on several findings that cholinergic
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neurodegeneration can be a major pathological feature in the brains of AD patients, and
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experimental studies suggest ACh plays a vital role in learning and memory (Bloem et al.,
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2014).
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Aβ aggregation is thought to be responsible for initiating the pathogenic cascade that
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results in neuronal loss and dementia (Estrada and Soto, 2007). Aβ aggregation has been
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shown to have crucial neurotoxic effects, which significantly supports the amyloid hypothesis
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(Skovronsky et al., 1998, Haass and Selkoe, 2007). Hence, the inhibition of Aβ aggregation
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and toxicity may carry therapeutic value by hindering the pathogenesis of AD (Hardy and
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Selkoe, 2002).
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vivo (Laursen et al., 2012). Additionally, it has been reported that AChE inhibitors can inhibit
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AChE-induced Aβ polymerization (Bartolini et al., 2003). One non-cholinergic role of AChE
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in AD pathogenesis is that AChE may play a substantial part in the development of the senile
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plaques by accelerating Aβ polymerization, which will induce greater neurotoxicity
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depending on the amount of AChE bound to the complexes (Muñoz and Inestrosa, 1999).
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Dual inhibitors of both Aβ aggregation and AChE activity are considered to be potential
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therapeutic approaches that can slow or mitigate the progression of AD (Yan and Feng, 2004,
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Inestrosa et al., 2008).
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Silibinin is a flavonoid extracted from the medicinal plant Silybum marianum (milk
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thistle) and has traditionally been used to treat liver diseases on account of its
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hepatoprotective properties (Křen and Walterova, 2005). Additionally, silibinin can act as an
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antioxidant against oxidative stress-related neuropathy (Di Cesare Mannelli et al., 2012).
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Another study shows that silibinin can prevent Aβ-induced memory impairment and oxidative
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stress in male ICR mice (Lu et al., 2009, Yin et al., 2011). Furthermore, silibinin can attenuate
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streptozocin-induced memory impairment by reducing oxidative and nitrosative stress and
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synaptosomal calcium levels, thereby restoring the activity and mRNA expression of AChE
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(Tota et al., 2011). We assume that silibinin can inhibit Aβ aggregation and AChE activity
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simultaneously and impede the interaction between Aβ and AChE during AD pathogenesis.
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This effect may be attributed to the inhibitory effects of silibinin directly on the Aβ peptides,
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which differs from epigallocatechin gallate (EGCG), another flavonoid that reduces Aβ
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aggregation through modulating amyloid precursor protein cleavage (Rezai-Zadeh et al.,
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2005) In the present study, we attempt to explain the potential molecular mechanism of the
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inhibitory features using a molecular dynamics simulation in silico, along with a series of
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assays that include circular dichroism (CD), transmission electron microscopy (TEM) and
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whole-cell patch-clamp in vitro and, ultimately studying the inhibition of Aβ aggregation and
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AChE activity in APP/PS1 transgenic mice in vivo.
2. Materials and Methods
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2.1 Animals
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APPswe/PS1dE9 double transgenic mice were obtained from the Model Animal
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Research Center of Nanjing University (Nanjing, China) (strain type B6C3-Tg [APPswe,
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PSEN1dE9] 85Dbo/J; stock number 004462). Age-matched wild-type (Wt) littermates were
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used as controls. The Sprague-Dawley Rats were obtained from the Guangdong Medical
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Laboratory Animal Center (Foshan, China). All experimental procedures involving the
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animals were performed according to the regulations of the Institutional Animal Care and Use
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Committee (IACUC) of Sun Yat-sen University, Guangzhou, China. The mice and rats were
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housed at 25 °C and 60% relative humidity under a 12 h light-dark cycle (light turned on at 7
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a.m.) with ad libitum access to food and water. For this study, 50 SPF 8-month-old Wt and
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APP/PS1 double transgenic (Tg) mice were used (weight = 27.70 g± 3.47 g). And 12 rats
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were assigned for these experiments. The number of female and male mice used for the study
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was equalized to prevent sex differences from influencing the results.
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Drosophila melanogaster stocks were reared on standard cornmeal agar medium
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method (Gu and O'Dowd, 2006, Gu and O'Dowd, 2007) was used to examine the cholinergic
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miniature excitatory postsynaptic currents (mEPSCs) of the projection neurons (PNs) in the
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whole brain. Drosophila melanogaster matured into adults at approximately every 14 days. To
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record the drosophila at accurate time points, all of the experiments were performed two days
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before eclosion on the wild-type Canton-S female flies, which were identified by their red
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eyes and transparent wings in the puparium.
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2.2 Drug administration
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Due to its low solubility in water (80 µmol/l), silibinin was suspended in a 0.5% (w/v)
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carboxymethylcellulose sodium (CMC) solution to enhance the solute concentration
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immediately before intraperitoneal injection with a constant volume of 10 ml/kg b.w.; this
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method ensured better drug absorption by the mice, compared to oral administration. Mice
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were administered silibinin (2, 20 and 200 mg·kg-1) or 0.5% CMC solution every day for 4
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weeks. The animals were randomly divided into five groups of ten mice each. The groups of
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Tg mice treated with 200, 20 and 2 mg•kg-1 of silibinin were named 200 mg, 20 mg and 2 mg,
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respectively; the groups of transgenic control and wide-type control mice without silibinin
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treatment were named Tg Ctrl and Wt Ctrl, respectively. All of the mice were administered 50
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mg/kg b.w. 5-Bromo-2-deoxyUridine (BrdU) each day for the first 5 days of the silibinin
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administration. For the following 23 days, the mice were administered only with silibinin.
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Each group contained 5 male and 5 female mice to control eventual sex differences from
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confounding the results.
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In the pharmacokinetics (PK) study, silibinin and PBS were administered intravenously
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The groups were administered with 1 mL PBS and 0.01% silibinin, and cerebrospinal fluid
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(CSF) samples were collected 2 min after the injections were named Ctrl (n = 6) and 2 min (n
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= 6), respectively.
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2.3 Molecular docking and molecular dynamics simulations
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All simulations were based on the solution structures of the Human Aβ1-42 (HuAβ42)
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(PDB code 1IYT)(Crescenzi et al., 2002) and the Torpedo californica AChE (TcAChE) (PDB
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code 2 WFZ)(Sanson et al., 2009), which were obtained from the Protein Data Bank (PDB).
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We clearly elucidated the reason why we used 1IYT in silico simulations in our supporting
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information (SI). The missing and truncated residues of the HuAβ42 and TcAChE were
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properly repaired with the Biopolymer module of SYBYL 1.1 (Tripos Inc., St. Louis, MO).
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Program Autodock 4.0 (Morris et al., 1998) was used to identify the docking positions of
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silibinin for HuAβ42, HuAβ40 and the catalytic active site (CAS) of TcAChE with the
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Lamarckian genetic algorithm. The grid map with 80*80*80 points spaced equally at 0.375 Å
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was generated with an AutoGrid program to calculate the binding energies between the ligand
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and the receptors. The docking parameters were set to the default values, except for the
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number of GA runs (200) and energy evaluations (25000000). All docked conformations were
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clustered under a tolerance of 2 Å for the root mean square deviation (RMSD) and ranked
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according to the docking energies at the end of each run.
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The AMBER11 simulation suite (Case et al., 2010) was applied for molecular dynamic
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simulations and data analysis. The all-atom point-charge force field (AMBER ff03), which
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had a good balance between the helix and sheet, was used to represent the peptides (Duan et
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ACCEPTED MANUSCRIPT al., 2003). The water solvent was explicitly represented by the TIP3P model (Jorgensen et al.,
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1983). The parameters for silibinin were generated as follows: the electrostatic potential of
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silibinin was acquired at the HF/6-31G** level after a geometry optimization using the
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Gaussian03 program (Kudin et al., 2004); the partial charges were derived by fitting the
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gas-phase electrostatic potential using the restrained electrostatic potential (RESP) method
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(Bayly et al., 1993), and the other force parameters of the silibinin molecule were taken from
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the AMBER GAFF (Wang et al., 2004) parameter set; the missing interaction parameters in
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the ligand were generated using antechamber tools in AMBER11. The system was first
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minimized using the steepest descent algorithms for 2000 steps. Then, we performed 30 ns
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MD simulations for HuAβ42 and 5 ns simulations for TcAChE with a normal pressure and
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temperature (NPT) ensemble. The pressure was coupled to 1 bar with an anisotropic coupling
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time of 1.0 ps, and the temperature was kept at 300 K during the simulation with a coupling
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time of 0.1 ps. The long-range electrostatic interaction was computed using the Particle-Mesh
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Ewald (PEM) method (Darden et al., 1993). SHAKE (Ryckaert et al., 1977) was used to
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constrain all bonds connected to hydrogen atoms, which enabled a 2.0 fs step in the
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simulation. Cut-offs of 1.2 nm were used for HuAβ42 and TcAChE. There were 2 additional
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sodium ions in the case of the negatively charged Aβ and 9 for AChE. The binding free
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energies (∆Gbinding) were computed with the generalized molecular mechanics. The Born
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surface area (MM-GBSA) approach (Fogolari et al., 2003) at 300 K was built in the
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AMBER11 program. In total, 50 snapshots were taken from the last 2 ns trajectory of
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HuAβ42 and TcAChE with intervals of 40 ps. The vibrational entropy contributions were then
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calculated with NMODE analysis (Chong et al., 1999), and 50 snapshots were accepted for
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the NMODE analysis. The above computation was performed using 192 AMD Opteron (tm)
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Processor CPUs (2.1GHz).
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2.4 Whole-cell patch-clamp from the projection neuron of Drosophila melanogaster Silibinin, dimethyl sulfoxide (DMSO), TTX, PTX and the chemical products used to
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prepare the external and internal solutions were all purchased from Sigma-Aldrich Inc.
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(Shanghai, China). The silibinin was prepared as stock solutions in DMSO and diluted in
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external solutions to the appropriate concentration (100µM) with a final DMSO concentration
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of less than 0.1%, which had no significant effects on the mEPSCs of PNs. The delivery and
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changes to the solution were performed using a gravity perfusion system, and the effects of
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the drugs were observed 5 min after drug application.
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All of the brains were obtained from flies 2 days before eclosion. The entire brain,
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including the optic lobes was obtained and prepared for recordings in standard external
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solution containing (in mM) 101 NaCl, 1 CaCl2, 4 MgCl2, 3 KCl, 5 glucose, 1.25 NaH2PO4,
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and 20.7 NaHCO23, pH 7.2, 250 mOsm as previously described (Gu and O'Dowd, 2006, Gu
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and O'Dowd, 2007). For the cholinergic miniature excitatory postsynaptic currents (mEPSCs)
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recordings, tetrodotoxin (TTX, 1 µM) was added to the external solution to block
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voltage-gated sodium currents, and GABAergic synaptic currents were blocked by picrotoxin
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(PTX, 10 µM). Voltage-clamp recordings were performed using patch-clamp electrodes
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(10-13 MΩ) filled with an internal solution containing (in mM) 102 K-gluconate, 0.085
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CaCl2, 1.7 MgCl2, 17 NaCl, 0.94 EGTA, and 8.5 HEPES, pH of 7.2 and 235 mOsm. All
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recordings were made at room temperature, and only a single projection neuron (PN) was
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examined in each brain. Electrophysiological signals were acquired with an EPC10 amplifier
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digitized at 5 kHz. The miniature excitatory postsynaptic currents (mEPSCs) were detected
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using the Mini Analysis software (Synaptosoft, Decatur, GA). The events were accepted for
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analysis only if they were asymmetrical, with a rising phase faster than 3 ms and a slower
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decaying phase. In addition, the threshold criterion for inclusion was 3 pA. The comparison of
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the frequency and amplitude of mEPSCs before and after the application of silibinin was
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performed with a paired t-test. All of the data were compared with a paired t-test.
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The morphology of the recorded PNs was confirmed using post-hoc staining with
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biocytin (0.4%, Sigma-Aldrich Inc). Briefly, after fixation for 3 h on ice, the brains were
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rinsed with PBS (0.01 M, pH 7.4) containing 0.1% TritonX-100 (PBST). After being blocked
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with 10% goat serum in PBST, the brains were incubated in 1:10 mouse nc82 monoclonal
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antibody (The Developmental Studies Hybridoma Bank, DSHB) and then incubated in 1:200
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goat anti-mouse Alexa Fluor 488 over night (A11001, Invitrogen, USA) and 1:200
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streptavidin-CY3 (Molecular Devices, USA). A Zeiss LSM 710 confocal microscope with a
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40x water-immersion objective and z-stack was used to acquire the photos of dendritic
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arborization of the visual PNs. The 3D post-recording image processing was reconstructed
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using Imaris 6.4.2 software (Bitplane, Zurich, Switzerland).
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2.5 AChE activity assays
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AChE activity assays were carried out based on the colorimetric method (Ellman et al.,
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1961). Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), butyrylcholinesterase
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(BChE, E.C. 3.1.1.8, from equine serum), 5,50-dithiobis- (2-nitrobenzoic acid) (Ellman’s
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reagent, DTNB), butylthiocholine chloride (BTC) and acetylthiocholine chloride (ATC) were
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inhibitor was incubated for 15 min before the substrate was added to initiate the reaction.
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Readings were taken at 10 s intervals for 2 min. Additionally, we used BChE as a Ctrl group
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to illustrate the specificity of silibinin to AChE. The BChE assay utilized the method
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described above.
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2.6 Circular dichroism (CD) and transmission electron microscopy (TEM)
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Aβ1-42 (Sigma-Aldrich Inc., Shanghai, China) was freshly dissolved in 0.01 M
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phosphate buffer (PB, pH 7.4) at a concentration of 1 mg/ml. Then, 50 µM Aβ1-42 was
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incubated in the presence or absence of 50 µM silibinin for 3 days at 37°C. CD spectra were
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recorded from 190 to 260 nm at the indicated time intervals; the secondary structures of
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treated and untreated Aβ1-42 samples were recorded at 25°C with a Chirascan circular
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dichroism spectrometer (Applied PhotoPhysics, Britain) and repeated twice; the data were
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converted to mean residue ellipticity [h] as previously described (Sreerama and Woody,
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2004). These Aβ1-42 structures were confirmed using TEM. An aliquot (5 µL) of each sample
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was spotted onto a glow-discharged, carbon-coated formvar copper grid and stained with 5 µL
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2% phosphotungstic acid (pH 7.0) for 1 min; the samples were then air dried and observed
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under a transmission electron microscope (FEI Inc., USA). All images were captured at a
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voltage of 80 kV.
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2.7 High-Performance Liquid Chromatography (HPLC)
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Silibinin was detected with a high-performance liquid chromatography (HPLC) method
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using a ÄKTA Purifier 10 (GE Healthcare, USA). This system is designed for fast protein
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liquid chromatography (FPLC) and is compatible for HPLC assay. It was equipped with 288
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tests. The mobile phase was methanol-water containing 0.6% glacial acetic acid (50:50, v/v).
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The flow rate was 1.0 mL/min and the monitoring wavelength was 288 nm. This system was
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controlled and the data were collected by UNICORN Control system.
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The cerebrospinal fluid (CSF) samples are collected from the Cisterna Magna of the rats (Liu and Duff, 2008). The capillary tubes (Sutter Instrument, USA, O.D. 1.5mm, I.D. 0.86mm)
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were connected to 1 mL syringes by PE25 tubing (RWD Lifescience, China). After making
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the sagittal incision on the neck, the subcutaneous tissues and muscles were bluntly dissected.
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We used a capillary tube to penetrate through the dura mater immediately after drying the
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dura mater with a cotton swab. And the CSF was collected into the capillary tube with a
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syringe. About 200 µL of CSF sample was collected from each rat. And samples
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contaminated with blood were abandoned. All CSF samples were frozen in -80
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The CSF samples were thawed in room temperature and 500µL of ice-cold methanol was added to precipitate the protein. After 30 min of precipitation in -20
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centrifuged at 6000 g for 10 min. The supernatant was then removed and dried under nitrogen.
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The samples were reconstituted in 200 µL of the mobile phase and filtrated by 0.45 µm
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membranes before injecting into the HPLC system. Besides, the standard solutions of silibinin
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(resolved in mobile phase) were prepared at low level (0.01%) and high level (0.1%). 200 µL
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of each quality-controlled solution was also injected into the HPLC system.
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2.8 Morris Water Maze
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, the samples were
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The Morris Water Maze (MWM) (MT-200, Chengdu, China) was used to evaluate the
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ACCEPTED MANUSCRIPT spatial learning of APP/PS1 transgenic mice after silibinin treatment. According to Charles V
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Vorhees’ procedure (Vorhees and Williams, 2006), the MWM test consists of a consecutive 5
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day learning trial and a 1 day probe trial. Altogether, there were 4 learning trials per day, each
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from a different starting position, and 24 hours after the end of the learning trials, the probe
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trials were immediately carried out for one day. The timer was set to 60 s and automatically
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stopped once the mouse reached the platform within the 60 s and remained on the platform for
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5 s. The time required to locate and get onto the platform was recorded as the escape latency.
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The mice were manually moved to the platform and allowed to stay on the platform for 20 s if
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they could not find the platform. Only mice swimming faster than 8 cm/s were recorded for
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the analysis, and the data from floating mice were discarded. After each trial, mice were dried
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off in their housing facilities next to an electric heater for 30 min. The trajectory and escape
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latency of the mice were recorded, and the average escape latency was analyzed each day.
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Then, the probe trial was performed 24 h after the completion of the learning trial. The
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platform was removed, and the probe trial was set to 60 s. In our experiment, we recorded the
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platform-site crossovers, the path length of each group in each quadrant and the duration of
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each group in each quadrant in the probe trials for 60 s.
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2.9 Tissue Preparation
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After the behavioral tests, all of the mice were sacrificed under deep anesthesia
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(pentobarbital sodium, 40 mg/kg). In each group, 6 mice were randomly chosen and used for
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the immunohistochemistry assay and ELISA; the other 4 mice were used for the synaptic
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morphological analysis after DiI-labeling with a Gene Gun, each analysis had the same
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amount of male and female mice. The mice were anesthetized, and venous blood was
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saline for 5 min, which included protease and phosphatase inhibitors. Then, the blood samples
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were allowed to precipitate for two hours at room temperature before being centrifuged for 15
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min at 1000 ×g. The serum was removed, assayed immediately afterwards and stored at -80
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°C. The brains of the 6 mice were removed and bisected sagittally. The right hemisphere was
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immediately frozen at -80
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stored in 4% paraformaldehyde overnight, and dehydrated in 30% sucrose in 0.1 M phosphate
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buffer for 48 h for immunohistochemical analysis, for which 40 µm thick free-floating
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coronal sections of fixed hemi-brains were collected using a freezing microtome (Leica,
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Germany). And we randomly collected six intact sections of each brain for the analysis. The
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other 4 mice were perfused with 2% paraformaldehyde for 24 hours; then the coronal sections
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(200 µm) were cut on a vibratome (Leica VT 1000 S, Germany), and one intact section was
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stored in 0.01 M PBS. The dentate gyrus and adjacent cortex -2.2 mm to -2.4 mm relative to
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the bregma were taken as the region of interest (ROI) in both analyses.
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2.10 Immunohistochemistry and Immunofluorescence staining
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for biochemical analysis, and the left hemisphere was fixed and
Amyloid pathology was assessed using a primary antibody to Aβ1-42 (dilution, 1:2000,
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Invitrogen), labeled using a biotinylated secondary antibody (Invitrogen) for 1 h at room
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temperature and visualized using DAB development. The surface area of the senile plaques
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were measured and compared as percentage of the dentate gyrus with Image-Pro Plus 6.0
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(Media Cybernetics, Inc. Maryland, USA). BrdU was used to stain newly generated cells. The
307
specimens were incubated with formamide, added to 2× SSC (saline sodium citrate, volume
308
ratio was 1:1) at 65 °C for 2 h and incubated in 2 M HCl at 30 ºC for 30 min to unfold the
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times, and blocked with blocking buffer (5% BSA and 0.03% Triton X-100 in PBS) for 30
311
min at 37 °C. The next day, the primary antibody rat anti-BrdU (1:400; Oxford
312
Biotechnology) together with either mouse anti-NeuN (neuronal nuclear antigen, 1:400;
313
Sigma-Aldrich Inc; to stain mature neurons), mouse anti-GFAP (glial fibrillary acidic protein,
314
1:10000; Sigma-Aldrich Inc; to stain astrocytes), rabbit anti-Iba1 (ionized calcium binding
315
adapter molecule, 1:500; Sigma-Aldrich Inc; to stain microglia) or goat anti-DCX
316
(doublecortin, 1:40; Sigma-Aldrich Inc; to stain neonatal neurons) was added and incubated
317
for 2 h at 37 °C, then overnight at 4 °C. Afterward, a second corresponding fluorescent
318
antibody was chosen to display the positive cells. To quantitate the labeled cells, the optical
319
fractionator method using Stereo Investigator (MBF Bioscience) was used to count the
320
number of all of the labeled cells. The cells double-labeled for BrdU+ and DCX+, BrdU+ and
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NeuN+, BrdU+ and Iba1+, and BrdU+ and GFAP+ were quantitated unilaterally in the
322
subgranular zone (SGZ) of the hippocampus. The cell density was calculated from the sum of
323
all cells counted, divided by the counting volume. We counted the number of labeled cells in
324
twelve coronal sections per mouse brain that was stained and mounted on coded slides. The
325
absolute numbers and ratios of double-labeled cells versus BrdU-positive cells were analyzed.
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Meanwhile, a Zeiss LSM 710 laser confocal scanning microscope (Zeiss, Germany) was used
327
to record the immunohistochemical staining.
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2.11 Enzyme-linked immunosorbent assay (ELISA) and AChE activity determination
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In the ELISA, frozen brains were thawed and minced. Then, the cerebral tissues of the
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brain were weighed and homogenized in 50 mM Tris-HCl buffer, pH 8.0, containing a
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332
and the supernatants were stored at -70 °C for additional analysis of soluble Aβ, BDNF and
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AChE. Then, we sonicated the pellet in 5 M guanidine Tris buffer; the samples were
334
incubated for 30 min at room temperature, and then centrifuged (100,000 × g, 1 hr, 4 °C), and
335
the supernatants were stored for analysis of insoluble Aβ.
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The levels of Aβ1-40 and Aβ1-42 in mice brains were measured using ELISA. The
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concentrations of insoluble and soluble Aβ40 and Aβ42 were analyzed using Aβ ELISA kits
338
(Invitrogen, USA), following the protocol of the manufacturer. The levels of brain derived
339
neurotrophic factor (BDNF) in the mouse brain and serum were measured with a Mouse
340
BDNF ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's protocol. And
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the AChE quantities were measured with a Mouse AChE ELISA Kit (CUSABIO, Wuhan,
342
China) following the manufacturer's protocol. Finally, the AChE activities were determined
343
using Ellman’s method (Ellman et al., 1961). We used an Acetylcholinesterase Fluorescent
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Activity kit (ARBOR Assays, USA) to measure it according to the protocol of the
345
manufacturer.
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2.12 DiOlistic labeling and morphological analysis
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The gene gun bullets were prepared as described (Staffend and Meisel, 2011): briefly, 4
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mg of gold particles (1.6 µm diameter) were mixed with 2.5 mg DiI (Sigma-Aldrich Inc) and
349
dissolved in 250 µl methylene chloride. After drying, the coated particles were collected in 1.5
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ml water and placed into a sonicator for 5 min. The solution was vortexed for 15 s and
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immediately transferred to 1 mm diameter gene gun tubing (Bio-Rad). The labeled sections
352
were rinsed in 0.01 M PBS three times and resuspended in PBS at 4 °C overnight for the dye
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ACCEPTED MANUSCRIPT to diffuse through the neuronal membranes. The images of DiI-impregnated cells were taken
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using a Zeiss LSM 710 confocal microscope (Zeiss, Germany). The neuron was scanned at 1
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µm increments along the Z-axis and reconstructed using LAS AF software to analyze the
356
dendrite segments. The density of the dendritic spines was measured on 10 to 30 randomly
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chosen dendrites from 3 to 6 neurons, calculated by quantifying the number of spines per unit
358
length of dendrite and normalized per 10 µm of dendrite length. 3D reconstructions of
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confocal images were performed with an Imaris 6.4.2 (Bitplane, Zurich, Switzerland).
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2.13 Statistical analysis
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The data represented as the mean ± S.E.M. p
S0197-4580(15)00102-5
DOI:
10.1016/j.neurobiolaging.2015.02.002
Reference:
NBA 9204
To appear in:
Neurobiology of Aging
Received Date: 29 April 2014 Revised Date:
3 February 2015
Accepted Date: 4 February 2015
Please cite this article as: Duan, S., Guan, X., Lin, R., Liu, X., Yan, Y., Lin, R., Zhang, T., Chen, X., Huang, J., Sun, X., Li, Q., Fang, S., Xu, J., Yao, Z., Gu, H., Silibinin Inhibits Acetylcholinesterase Activity and Aβ Peptide Aggregation: A Dual-Target Drug for Alzheimer's Disease Treatment, Neurobiology of Aging (2015), doi: 10.1016/j.neurobiolaging.2015.02.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Silibinin Inhibits Acetylcholinesterase Activity and Aβ Peptide
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Aggregation: A Dual-Target Drug for Alzheimer's Disease Treatment
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Songwei Duan1, 4, Xiaoyin Guan1, Runxuan Lin1, Xincheng Liu1, Ying Yan1, Ruibang Lin1,4,
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Tianqi Zhang1, Xueman Chen1, Jiaqi Huang1, Xicui Sun5, Qingqing Li1, Shaoliang Fang2,
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Jun Xu3, Zhibin Yao1, Huaiyu Gu1*
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Department of Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China 510080; 2Key Lab of High Performance Computing of Guangdong Province,
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Guangzhou, China 510033; 3 Research Center for Drug Discovery and Institute of Human
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Virology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China
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510006; 4Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong,
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China 510080; 5Guangzhou Brain and Psychiatric Hospital, Guangzhou, China,510450.
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*Corresponding author: Huaiyu Gu: 1Department of Anatomy, Zhongshan School of
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Medicine of Sun Yat-sen University, Guangzhou, Guangdong, China, 510080. Tel: (8620)
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8733 2086; Fax: (8620) 8733 2086; E-mail: [email protected]
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Number of pages: (43)
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Number of figures, tables: figures (9), tables (1).
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Number of words for Abstract, Introduction, and Discussion: Abstract (203), Introduction
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(578), and Discussion (1842).
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Abstract Alzheimer’s disease (AD) is characterized by amyloid-β (Aβ) peptide aggregation and
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cholinergic neurodegeneration. Therefore, in this paper, we examined silibinin, a flavonoid
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extracted from Silybum marianum, to determine its potential as a dual inhibitor of
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acetylcholinesterase (AChE) and Aβ peptide aggregation for AD treatment. To achieve this,
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we used molecular docking and molecular dynamics (MD) simulations to examine the affinity
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of silibinin with Aβ and AChE in silico. Next, we used circular dichroism (CD) and
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transmission electron microscopy (TEM) to study the anti-Aβ aggregation capability of
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silibinin
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immunohistochemistry, BrdU double labeling and a gene gun experiment were performed on
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silibinin-treated APP/PS1 transgenic mice. In MD simulations, silibinin interacted with Aβ
34
and AChE to form different stable complexes. After the administration of silibinin, AChE
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activity and Aβ aggregations were down-regulated, and the quantity of AChE also decreased.
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In addition, silibinin-treated APP/PS1 transgenic mice had higher scores in the Morris Water
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Maze. Moreover, silibinin could increase the number of newly generated microglia,
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astrocytes, neurons and neuronal precursor cells. Taken together, the data suggested that
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silibinin could act as a dual inhibitor of AChE and Aβ peptide aggregation, therefore
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suggesting a therapeutic strategy for AD treatment.
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Keywords: Alzheimer’s disease; amyloid β protein; MD simulation; silibinin; memory
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deficits; acetylcholinesterase; APP/PS1 transgenic mice
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1. Introduction Alzheimer’s disease (AD) is a common and severe neurodegenerative disorder among the
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elderly, which is characterized by a cascade of pathological changes. These changes include
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abnormal Amyloid β (Aβ) peptide aggregation with the consequent formation of senile
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plaques in the cerebrocortical and limbic regions and a reduction in the levels of acetylcholine
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(ACh) (Isacson et al., 2002), together with progressive neuron loss (Gómez Isla et al.,
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1997). Although considerable studies have been conducted on AD in the past years, the
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etiology of it remains unclear. Based on the cholinergic hypothesis of AD (Terry and
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Buccafusco, 2003) (Perry et al., 1999) (Deutsch, 1971), the current strategy of AD
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intervention is mainly to ameliorate the cognitive symptoms related to ACh depletion, thereby
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enhancing the central cholinergic neurotransmission by reversing acetylcholinesterase
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(AChE) inhibition. This hypothesis is based on several findings that cholinergic
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neurodegeneration can be a major pathological feature in the brains of AD patients, and
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experimental studies suggest ACh plays a vital role in learning and memory (Bloem et al.,
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2014).
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Aβ aggregation is thought to be responsible for initiating the pathogenic cascade that
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results in neuronal loss and dementia (Estrada and Soto, 2007). Aβ aggregation has been
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shown to have crucial neurotoxic effects, which significantly supports the amyloid hypothesis
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(Skovronsky et al., 1998, Haass and Selkoe, 2007). Hence, the inhibition of Aβ aggregation
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and toxicity may carry therapeutic value by hindering the pathogenesis of AD (Hardy and
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Selkoe, 2002).
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ACCEPTED MANUSCRIPT A previous study reports that cholinergic degeneration can accelerate Aβ plaque burden in
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vivo (Laursen et al., 2012). Additionally, it has been reported that AChE inhibitors can inhibit
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AChE-induced Aβ polymerization (Bartolini et al., 2003). One non-cholinergic role of AChE
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in AD pathogenesis is that AChE may play a substantial part in the development of the senile
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plaques by accelerating Aβ polymerization, which will induce greater neurotoxicity
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depending on the amount of AChE bound to the complexes (Muñoz and Inestrosa, 1999).
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Dual inhibitors of both Aβ aggregation and AChE activity are considered to be potential
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therapeutic approaches that can slow or mitigate the progression of AD (Yan and Feng, 2004,
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Inestrosa et al., 2008).
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Silibinin is a flavonoid extracted from the medicinal plant Silybum marianum (milk
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thistle) and has traditionally been used to treat liver diseases on account of its
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hepatoprotective properties (Křen and Walterova, 2005). Additionally, silibinin can act as an
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antioxidant against oxidative stress-related neuropathy (Di Cesare Mannelli et al., 2012).
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Another study shows that silibinin can prevent Aβ-induced memory impairment and oxidative
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stress in male ICR mice (Lu et al., 2009, Yin et al., 2011). Furthermore, silibinin can attenuate
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streptozocin-induced memory impairment by reducing oxidative and nitrosative stress and
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synaptosomal calcium levels, thereby restoring the activity and mRNA expression of AChE
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(Tota et al., 2011). We assume that silibinin can inhibit Aβ aggregation and AChE activity
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simultaneously and impede the interaction between Aβ and AChE during AD pathogenesis.
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This effect may be attributed to the inhibitory effects of silibinin directly on the Aβ peptides,
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which differs from epigallocatechin gallate (EGCG), another flavonoid that reduces Aβ
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aggregation through modulating amyloid precursor protein cleavage (Rezai-Zadeh et al.,
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2005) In the present study, we attempt to explain the potential molecular mechanism of the
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inhibitory features using a molecular dynamics simulation in silico, along with a series of
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assays that include circular dichroism (CD), transmission electron microscopy (TEM) and
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whole-cell patch-clamp in vitro and, ultimately studying the inhibition of Aβ aggregation and
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AChE activity in APP/PS1 transgenic mice in vivo.
2. Materials and Methods
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2.1 Animals
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APPswe/PS1dE9 double transgenic mice were obtained from the Model Animal
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Research Center of Nanjing University (Nanjing, China) (strain type B6C3-Tg [APPswe,
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PSEN1dE9] 85Dbo/J; stock number 004462). Age-matched wild-type (Wt) littermates were
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used as controls. The Sprague-Dawley Rats were obtained from the Guangdong Medical
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Laboratory Animal Center (Foshan, China). All experimental procedures involving the
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animals were performed according to the regulations of the Institutional Animal Care and Use
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Committee (IACUC) of Sun Yat-sen University, Guangzhou, China. The mice and rats were
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housed at 25 °C and 60% relative humidity under a 12 h light-dark cycle (light turned on at 7
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a.m.) with ad libitum access to food and water. For this study, 50 SPF 8-month-old Wt and
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APP/PS1 double transgenic (Tg) mice were used (weight = 27.70 g± 3.47 g). And 12 rats
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were assigned for these experiments. The number of female and male mice used for the study
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was equalized to prevent sex differences from influencing the results.
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Drosophila melanogaster stocks were reared on standard cornmeal agar medium
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method (Gu and O'Dowd, 2006, Gu and O'Dowd, 2007) was used to examine the cholinergic
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miniature excitatory postsynaptic currents (mEPSCs) of the projection neurons (PNs) in the
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whole brain. Drosophila melanogaster matured into adults at approximately every 14 days. To
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record the drosophila at accurate time points, all of the experiments were performed two days
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before eclosion on the wild-type Canton-S female flies, which were identified by their red
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eyes and transparent wings in the puparium.
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2.2 Drug administration
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Due to its low solubility in water (80 µmol/l), silibinin was suspended in a 0.5% (w/v)
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carboxymethylcellulose sodium (CMC) solution to enhance the solute concentration
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immediately before intraperitoneal injection with a constant volume of 10 ml/kg b.w.; this
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method ensured better drug absorption by the mice, compared to oral administration. Mice
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were administered silibinin (2, 20 and 200 mg·kg-1) or 0.5% CMC solution every day for 4
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weeks. The animals were randomly divided into five groups of ten mice each. The groups of
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Tg mice treated with 200, 20 and 2 mg•kg-1 of silibinin were named 200 mg, 20 mg and 2 mg,
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respectively; the groups of transgenic control and wide-type control mice without silibinin
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treatment were named Tg Ctrl and Wt Ctrl, respectively. All of the mice were administered 50
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mg/kg b.w. 5-Bromo-2-deoxyUridine (BrdU) each day for the first 5 days of the silibinin
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administration. For the following 23 days, the mice were administered only with silibinin.
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Each group contained 5 male and 5 female mice to control eventual sex differences from
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confounding the results.
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In the pharmacokinetics (PK) study, silibinin and PBS were administered intravenously
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The groups were administered with 1 mL PBS and 0.01% silibinin, and cerebrospinal fluid
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(CSF) samples were collected 2 min after the injections were named Ctrl (n = 6) and 2 min (n
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= 6), respectively.
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2.3 Molecular docking and molecular dynamics simulations
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All simulations were based on the solution structures of the Human Aβ1-42 (HuAβ42)
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(PDB code 1IYT)(Crescenzi et al., 2002) and the Torpedo californica AChE (TcAChE) (PDB
140
code 2 WFZ)(Sanson et al., 2009), which were obtained from the Protein Data Bank (PDB).
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We clearly elucidated the reason why we used 1IYT in silico simulations in our supporting
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information (SI). The missing and truncated residues of the HuAβ42 and TcAChE were
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properly repaired with the Biopolymer module of SYBYL 1.1 (Tripos Inc., St. Louis, MO).
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Program Autodock 4.0 (Morris et al., 1998) was used to identify the docking positions of
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silibinin for HuAβ42, HuAβ40 and the catalytic active site (CAS) of TcAChE with the
146
Lamarckian genetic algorithm. The grid map with 80*80*80 points spaced equally at 0.375 Å
147
was generated with an AutoGrid program to calculate the binding energies between the ligand
148
and the receptors. The docking parameters were set to the default values, except for the
149
number of GA runs (200) and energy evaluations (25000000). All docked conformations were
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clustered under a tolerance of 2 Å for the root mean square deviation (RMSD) and ranked
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according to the docking energies at the end of each run.
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The AMBER11 simulation suite (Case et al., 2010) was applied for molecular dynamic
153
simulations and data analysis. The all-atom point-charge force field (AMBER ff03), which
154
had a good balance between the helix and sheet, was used to represent the peptides (Duan et
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ACCEPTED MANUSCRIPT al., 2003). The water solvent was explicitly represented by the TIP3P model (Jorgensen et al.,
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1983). The parameters for silibinin were generated as follows: the electrostatic potential of
157
silibinin was acquired at the HF/6-31G** level after a geometry optimization using the
158
Gaussian03 program (Kudin et al., 2004); the partial charges were derived by fitting the
159
gas-phase electrostatic potential using the restrained electrostatic potential (RESP) method
160
(Bayly et al., 1993), and the other force parameters of the silibinin molecule were taken from
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the AMBER GAFF (Wang et al., 2004) parameter set; the missing interaction parameters in
162
the ligand were generated using antechamber tools in AMBER11. The system was first
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minimized using the steepest descent algorithms for 2000 steps. Then, we performed 30 ns
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MD simulations for HuAβ42 and 5 ns simulations for TcAChE with a normal pressure and
165
temperature (NPT) ensemble. The pressure was coupled to 1 bar with an anisotropic coupling
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time of 1.0 ps, and the temperature was kept at 300 K during the simulation with a coupling
167
time of 0.1 ps. The long-range electrostatic interaction was computed using the Particle-Mesh
168
Ewald (PEM) method (Darden et al., 1993). SHAKE (Ryckaert et al., 1977) was used to
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constrain all bonds connected to hydrogen atoms, which enabled a 2.0 fs step in the
170
simulation. Cut-offs of 1.2 nm were used for HuAβ42 and TcAChE. There were 2 additional
171
sodium ions in the case of the negatively charged Aβ and 9 for AChE. The binding free
172
energies (∆Gbinding) were computed with the generalized molecular mechanics. The Born
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surface area (MM-GBSA) approach (Fogolari et al., 2003) at 300 K was built in the
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AMBER11 program. In total, 50 snapshots were taken from the last 2 ns trajectory of
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HuAβ42 and TcAChE with intervals of 40 ps. The vibrational entropy contributions were then
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calculated with NMODE analysis (Chong et al., 1999), and 50 snapshots were accepted for
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the NMODE analysis. The above computation was performed using 192 AMD Opteron (tm)
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Processor CPUs (2.1GHz).
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2.4 Whole-cell patch-clamp from the projection neuron of Drosophila melanogaster Silibinin, dimethyl sulfoxide (DMSO), TTX, PTX and the chemical products used to
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prepare the external and internal solutions were all purchased from Sigma-Aldrich Inc.
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(Shanghai, China). The silibinin was prepared as stock solutions in DMSO and diluted in
183
external solutions to the appropriate concentration (100µM) with a final DMSO concentration
184
of less than 0.1%, which had no significant effects on the mEPSCs of PNs. The delivery and
185
changes to the solution were performed using a gravity perfusion system, and the effects of
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the drugs were observed 5 min after drug application.
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All of the brains were obtained from flies 2 days before eclosion. The entire brain,
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including the optic lobes was obtained and prepared for recordings in standard external
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solution containing (in mM) 101 NaCl, 1 CaCl2, 4 MgCl2, 3 KCl, 5 glucose, 1.25 NaH2PO4,
190
and 20.7 NaHCO23, pH 7.2, 250 mOsm as previously described (Gu and O'Dowd, 2006, Gu
191
and O'Dowd, 2007). For the cholinergic miniature excitatory postsynaptic currents (mEPSCs)
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recordings, tetrodotoxin (TTX, 1 µM) was added to the external solution to block
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voltage-gated sodium currents, and GABAergic synaptic currents were blocked by picrotoxin
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(PTX, 10 µM). Voltage-clamp recordings were performed using patch-clamp electrodes
195
(10-13 MΩ) filled with an internal solution containing (in mM) 102 K-gluconate, 0.085
196
CaCl2, 1.7 MgCl2, 17 NaCl, 0.94 EGTA, and 8.5 HEPES, pH of 7.2 and 235 mOsm. All
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recordings were made at room temperature, and only a single projection neuron (PN) was
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examined in each brain. Electrophysiological signals were acquired with an EPC10 amplifier
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digitized at 5 kHz. The miniature excitatory postsynaptic currents (mEPSCs) were detected
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using the Mini Analysis software (Synaptosoft, Decatur, GA). The events were accepted for
202
analysis only if they were asymmetrical, with a rising phase faster than 3 ms and a slower
203
decaying phase. In addition, the threshold criterion for inclusion was 3 pA. The comparison of
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the frequency and amplitude of mEPSCs before and after the application of silibinin was
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performed with a paired t-test. All of the data were compared with a paired t-test.
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The morphology of the recorded PNs was confirmed using post-hoc staining with
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biocytin (0.4%, Sigma-Aldrich Inc). Briefly, after fixation for 3 h on ice, the brains were
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rinsed with PBS (0.01 M, pH 7.4) containing 0.1% TritonX-100 (PBST). After being blocked
209
with 10% goat serum in PBST, the brains were incubated in 1:10 mouse nc82 monoclonal
210
antibody (The Developmental Studies Hybridoma Bank, DSHB) and then incubated in 1:200
211
goat anti-mouse Alexa Fluor 488 over night (A11001, Invitrogen, USA) and 1:200
212
streptavidin-CY3 (Molecular Devices, USA). A Zeiss LSM 710 confocal microscope with a
213
40x water-immersion objective and z-stack was used to acquire the photos of dendritic
214
arborization of the visual PNs. The 3D post-recording image processing was reconstructed
215
using Imaris 6.4.2 software (Bitplane, Zurich, Switzerland).
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2.5 AChE activity assays
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AChE activity assays were carried out based on the colorimetric method (Ellman et al.,
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1961). Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), butyrylcholinesterase
219
(BChE, E.C. 3.1.1.8, from equine serum), 5,50-dithiobis- (2-nitrobenzoic acid) (Ellman’s
220
reagent, DTNB), butylthiocholine chloride (BTC) and acetylthiocholine chloride (ATC) were
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inhibitor was incubated for 15 min before the substrate was added to initiate the reaction.
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Readings were taken at 10 s intervals for 2 min. Additionally, we used BChE as a Ctrl group
224
to illustrate the specificity of silibinin to AChE. The BChE assay utilized the method
225
described above.
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2.6 Circular dichroism (CD) and transmission electron microscopy (TEM)
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Aβ1-42 (Sigma-Aldrich Inc., Shanghai, China) was freshly dissolved in 0.01 M
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phosphate buffer (PB, pH 7.4) at a concentration of 1 mg/ml. Then, 50 µM Aβ1-42 was
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incubated in the presence or absence of 50 µM silibinin for 3 days at 37°C. CD spectra were
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recorded from 190 to 260 nm at the indicated time intervals; the secondary structures of
231
treated and untreated Aβ1-42 samples were recorded at 25°C with a Chirascan circular
232
dichroism spectrometer (Applied PhotoPhysics, Britain) and repeated twice; the data were
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converted to mean residue ellipticity [h] as previously described (Sreerama and Woody,
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2004). These Aβ1-42 structures were confirmed using TEM. An aliquot (5 µL) of each sample
235
was spotted onto a glow-discharged, carbon-coated formvar copper grid and stained with 5 µL
236
2% phosphotungstic acid (pH 7.0) for 1 min; the samples were then air dried and observed
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under a transmission electron microscope (FEI Inc., USA). All images were captured at a
238
voltage of 80 kV.
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2.7 High-Performance Liquid Chromatography (HPLC)
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Silibinin was detected with a high-performance liquid chromatography (HPLC) method
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using a ÄKTA Purifier 10 (GE Healthcare, USA). This system is designed for fast protein
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liquid chromatography (FPLC) and is compatible for HPLC assay. It was equipped with 288
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tests. The mobile phase was methanol-water containing 0.6% glacial acetic acid (50:50, v/v).
245
The flow rate was 1.0 mL/min and the monitoring wavelength was 288 nm. This system was
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controlled and the data were collected by UNICORN Control system.
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The cerebrospinal fluid (CSF) samples are collected from the Cisterna Magna of the rats (Liu and Duff, 2008). The capillary tubes (Sutter Instrument, USA, O.D. 1.5mm, I.D. 0.86mm)
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were connected to 1 mL syringes by PE25 tubing (RWD Lifescience, China). After making
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the sagittal incision on the neck, the subcutaneous tissues and muscles were bluntly dissected.
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We used a capillary tube to penetrate through the dura mater immediately after drying the
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dura mater with a cotton swab. And the CSF was collected into the capillary tube with a
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syringe. About 200 µL of CSF sample was collected from each rat. And samples
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contaminated with blood were abandoned. All CSF samples were frozen in -80
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were used.
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The CSF samples were thawed in room temperature and 500µL of ice-cold methanol was added to precipitate the protein. After 30 min of precipitation in -20
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centrifuged at 6000 g for 10 min. The supernatant was then removed and dried under nitrogen.
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The samples were reconstituted in 200 µL of the mobile phase and filtrated by 0.45 µm
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membranes before injecting into the HPLC system. Besides, the standard solutions of silibinin
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(resolved in mobile phase) were prepared at low level (0.01%) and high level (0.1%). 200 µL
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of each quality-controlled solution was also injected into the HPLC system.
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2.8 Morris Water Maze
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, the samples were
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The Morris Water Maze (MWM) (MT-200, Chengdu, China) was used to evaluate the
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ACCEPTED MANUSCRIPT spatial learning of APP/PS1 transgenic mice after silibinin treatment. According to Charles V
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Vorhees’ procedure (Vorhees and Williams, 2006), the MWM test consists of a consecutive 5
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day learning trial and a 1 day probe trial. Altogether, there were 4 learning trials per day, each
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from a different starting position, and 24 hours after the end of the learning trials, the probe
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trials were immediately carried out for one day. The timer was set to 60 s and automatically
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stopped once the mouse reached the platform within the 60 s and remained on the platform for
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5 s. The time required to locate and get onto the platform was recorded as the escape latency.
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The mice were manually moved to the platform and allowed to stay on the platform for 20 s if
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they could not find the platform. Only mice swimming faster than 8 cm/s were recorded for
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the analysis, and the data from floating mice were discarded. After each trial, mice were dried
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off in their housing facilities next to an electric heater for 30 min. The trajectory and escape
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latency of the mice were recorded, and the average escape latency was analyzed each day.
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Then, the probe trial was performed 24 h after the completion of the learning trial. The
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platform was removed, and the probe trial was set to 60 s. In our experiment, we recorded the
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platform-site crossovers, the path length of each group in each quadrant and the duration of
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each group in each quadrant in the probe trials for 60 s.
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2.9 Tissue Preparation
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After the behavioral tests, all of the mice were sacrificed under deep anesthesia
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(pentobarbital sodium, 40 mg/kg). In each group, 6 mice were randomly chosen and used for
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the immunohistochemistry assay and ELISA; the other 4 mice were used for the synaptic
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morphological analysis after DiI-labeling with a Gene Gun, each analysis had the same
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amount of male and female mice. The mice were anesthetized, and venous blood was
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ACCEPTED MANUSCRIPT collected with serum separator tube (SST), followed by transcardial perfusion with normal
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saline for 5 min, which included protease and phosphatase inhibitors. Then, the blood samples
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were allowed to precipitate for two hours at room temperature before being centrifuged for 15
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min at 1000 ×g. The serum was removed, assayed immediately afterwards and stored at -80
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°C. The brains of the 6 mice were removed and bisected sagittally. The right hemisphere was
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immediately frozen at -80
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stored in 4% paraformaldehyde overnight, and dehydrated in 30% sucrose in 0.1 M phosphate
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buffer for 48 h for immunohistochemical analysis, for which 40 µm thick free-floating
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coronal sections of fixed hemi-brains were collected using a freezing microtome (Leica,
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Germany). And we randomly collected six intact sections of each brain for the analysis. The
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other 4 mice were perfused with 2% paraformaldehyde for 24 hours; then the coronal sections
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(200 µm) were cut on a vibratome (Leica VT 1000 S, Germany), and one intact section was
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stored in 0.01 M PBS. The dentate gyrus and adjacent cortex -2.2 mm to -2.4 mm relative to
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the bregma were taken as the region of interest (ROI) in both analyses.
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2.10 Immunohistochemistry and Immunofluorescence staining
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for biochemical analysis, and the left hemisphere was fixed and
Amyloid pathology was assessed using a primary antibody to Aβ1-42 (dilution, 1:2000,
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Invitrogen), labeled using a biotinylated secondary antibody (Invitrogen) for 1 h at room
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temperature and visualized using DAB development. The surface area of the senile plaques
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were measured and compared as percentage of the dentate gyrus with Image-Pro Plus 6.0
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(Media Cybernetics, Inc. Maryland, USA). BrdU was used to stain newly generated cells. The
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specimens were incubated with formamide, added to 2× SSC (saline sodium citrate, volume
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ratio was 1:1) at 65 °C for 2 h and incubated in 2 M HCl at 30 ºC for 30 min to unfold the
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ACCEPTED MANUSCRIPT DNA double helix structure. Next, the sections were washed with boric acid and PBS three
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times, and blocked with blocking buffer (5% BSA and 0.03% Triton X-100 in PBS) for 30
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min at 37 °C. The next day, the primary antibody rat anti-BrdU (1:400; Oxford
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Biotechnology) together with either mouse anti-NeuN (neuronal nuclear antigen, 1:400;
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Sigma-Aldrich Inc; to stain mature neurons), mouse anti-GFAP (glial fibrillary acidic protein,
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1:10000; Sigma-Aldrich Inc; to stain astrocytes), rabbit anti-Iba1 (ionized calcium binding
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adapter molecule, 1:500; Sigma-Aldrich Inc; to stain microglia) or goat anti-DCX
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(doublecortin, 1:40; Sigma-Aldrich Inc; to stain neonatal neurons) was added and incubated
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for 2 h at 37 °C, then overnight at 4 °C. Afterward, a second corresponding fluorescent
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antibody was chosen to display the positive cells. To quantitate the labeled cells, the optical
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fractionator method using Stereo Investigator (MBF Bioscience) was used to count the
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number of all of the labeled cells. The cells double-labeled for BrdU+ and DCX+, BrdU+ and
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NeuN+, BrdU+ and Iba1+, and BrdU+ and GFAP+ were quantitated unilaterally in the
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subgranular zone (SGZ) of the hippocampus. The cell density was calculated from the sum of
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all cells counted, divided by the counting volume. We counted the number of labeled cells in
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twelve coronal sections per mouse brain that was stained and mounted on coded slides. The
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absolute numbers and ratios of double-labeled cells versus BrdU-positive cells were analyzed.
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Meanwhile, a Zeiss LSM 710 laser confocal scanning microscope (Zeiss, Germany) was used
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to record the immunohistochemical staining.
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2.11 Enzyme-linked immunosorbent assay (ELISA) and AChE activity determination
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In the ELISA, frozen brains were thawed and minced. Then, the cerebral tissues of the
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brain were weighed and homogenized in 50 mM Tris-HCl buffer, pH 8.0, containing a
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ACCEPTED MANUSCRIPT cocktail of protease inhibitors. The homogenates were centrifuged (100,000 × g, 1 hour, 4°C),
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and the supernatants were stored at -70 °C for additional analysis of soluble Aβ, BDNF and
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AChE. Then, we sonicated the pellet in 5 M guanidine Tris buffer; the samples were
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incubated for 30 min at room temperature, and then centrifuged (100,000 × g, 1 hr, 4 °C), and
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the supernatants were stored for analysis of insoluble Aβ.
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The levels of Aβ1-40 and Aβ1-42 in mice brains were measured using ELISA. The
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concentrations of insoluble and soluble Aβ40 and Aβ42 were analyzed using Aβ ELISA kits
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(Invitrogen, USA), following the protocol of the manufacturer. The levels of brain derived
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neurotrophic factor (BDNF) in the mouse brain and serum were measured with a Mouse
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BDNF ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's protocol. And
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the AChE quantities were measured with a Mouse AChE ELISA Kit (CUSABIO, Wuhan,
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China) following the manufacturer's protocol. Finally, the AChE activities were determined
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using Ellman’s method (Ellman et al., 1961). We used an Acetylcholinesterase Fluorescent
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Activity kit (ARBOR Assays, USA) to measure it according to the protocol of the
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manufacturer.
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2.12 DiOlistic labeling and morphological analysis
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The gene gun bullets were prepared as described (Staffend and Meisel, 2011): briefly, 4
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mg of gold particles (1.6 µm diameter) were mixed with 2.5 mg DiI (Sigma-Aldrich Inc) and
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dissolved in 250 µl methylene chloride. After drying, the coated particles were collected in 1.5
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ml water and placed into a sonicator for 5 min. The solution was vortexed for 15 s and
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immediately transferred to 1 mm diameter gene gun tubing (Bio-Rad). The labeled sections
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were rinsed in 0.01 M PBS three times and resuspended in PBS at 4 °C overnight for the dye
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ACCEPTED MANUSCRIPT to diffuse through the neuronal membranes. The images of DiI-impregnated cells were taken
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using a Zeiss LSM 710 confocal microscope (Zeiss, Germany). The neuron was scanned at 1
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µm increments along the Z-axis and reconstructed using LAS AF software to analyze the
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dendrite segments. The density of the dendritic spines was measured on 10 to 30 randomly
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chosen dendrites from 3 to 6 neurons, calculated by quantifying the number of spines per unit
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length of dendrite and normalized per 10 µm of dendrite length. 3D reconstructions of
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confocal images were performed with an Imaris 6.4.2 (Bitplane, Zurich, Switzerland).
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2.13 Statistical analysis
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The data represented as the mean ± S.E.M. p
