Pinostrobin Exerts Neuroprotective Actions in Neurotoxin-Induced

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Bioactive Constituents, Metabolites, and Functions

Pinostrobin exerts neuroprotective actions in neurotoxininduced Parkinson’s disease models through Nrf2 induction Chuwen Li, Benqin Tang, Yu Feng, Fan Tang, Maggie Pui Man Hoi, Ziren Su, and Simon Ming-Yuen Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02607 • Publication Date (Web): 01 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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Journal of Agricultural and Food Chemistry

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Title Page

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Pinostrobin exerts neuroprotective actions in neurotoxin-induced Parkinson’s

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disease models through Nrf2 induction

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Chuwen Li †, Benqin Tang†, §, Yu Feng†, Fan Tang†, Maggie Pui-Man Hoi†, Ziren Su‡,

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and Simon Ming-Yuen Lee†, *

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†

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Medical Sciences, University of Macau, Macau, China

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§

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‡

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese

Department of Medical Science, Shunde Polytechnic, Shunde, China Guangdong Provincial Key Laboratory of New Drug Development and Research of

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Chinese Medicine, Mathematical Engineering Academy of Chinese Medicine,

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Guangzhou University of Chinese Medicine, Guangzhou, China

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* To whom correspondence should be addressed:

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Simon Ming-Yuen Lee, Ph.D., Professor (Full)

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Institute of Chinese Medical Sciences, University of Macau

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Tel: (+853) 8822-4695; Fax: (+853) 8822-1358

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E-mail: [email protected]

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Abstract

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The aim of the present study was to assess the neuroprotective effects of pinostrobin

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(PSB), a dietary bioflavonoid, and its underlying mechanisms in neurotoxin-induced PD

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models. Firstly, PSB could attenuate MPTP-induced loss of dopaminergic neurons and

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improve behavior deficiency in zebrafish, supporting its potential neuroprotective

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actions in vivo. Next, PSB could decreased apoptosis and death in the MPP+-intoxicated

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SH-SY5Y cells, evidenced by MTT, LDH, Annexin V-FITC/PI and DNA

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fragmentation assay. PSB also blocked MPP+-induced apoptotic cascades, including

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loss of mitochondrial membrane potential, activation of caspase 3, and reduced ratio of

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Bcl-2/Bax. In addition, PSB suppressed MPP+-induced oxidative stress but increased

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antioxidant enzymes, evidenced by decrease of ROS generation and lipid peroxidation

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and up-regulation of GSH-Px, SOD, CAT, GSH/GSSG and NAD/NADH. Further

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investigations showed that PSB significantly enhanced Nrf2 expression and nuclear

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accumulation, improved ARE promoter activity and up-regulated expression of HO-1

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and GCLC. Furthermore, Nrf2 knockdown via specific Nrf2 siRNA abolished PSB-

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induced anti-oxidative and anti-apoptotic effects against MPP+ insults. Interestingly, we

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then found that PSB promoted phosphorylation of PI3K/AKT and ERK, and

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pharmacological inhibition of PI3K/AKT or ERK signaling diminished PSB-induced

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Nrf2/ARE activation and protective actions. In summary, PSB confers neuroprotection

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against MPTP/MPP+-induced neurotoxicity in Parkinson’s disease models. Promoting

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activation of Nrf2/ARE signaling contributes to PSB-mediated anti-oxidative and

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neuroprotective actions, which, in part is mediated by PI3K/AKT and ERK.

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Key words Pinostrobin; Parkinson’s disease; neurotoxin; Nrf2

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Introduction

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Parkinson’s disease (PD) is the second most common neurodegenerative diseases after

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Alzheimer’s disease (AD) and has been considered to be one of global health concerns

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currently.1 PD is usually characterized by a loss of dopaminergic neurons in the

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substantia nigra (SN) with clinical manifestations including bradykinesia, tremor,

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rigidity and postural instability. 2, 3 Nowadays, halting or reversing neuron degeneration

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in PD patients is still difficult. Therefore, it is of great need to discover and develop

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novel anti-parkinsonian drugs for PD treatments.

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Even though the etiology of PD remains unclear, accumulating evidence indicates that

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oxidative stress plays an important role in the pathogenesis of both PD and

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Parkinsonism models.4-6 The abnormal reactive oxygen species (ROS) level,

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imbalanced antioxidant enzymes activity, and other activated oxidase components that

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induce cellular dysfunction and apoptosis in the central nervous system (CNS).4-6

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Insights into PD pathogenesis, the well-known neurotoxin, 1-Methyl-4-phenyl-1,2,3,6-

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tetrahydropyridine (MPTP) and its metabolite, 1-methyl-4-phenylpyridinium (MPP+),

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have long been used to establish experimental PD models.7, 8 MPTP can be selectively

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taken up via dopamine transporters and metabolites into MPP+, which cause

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dopaminergic neurons damage and syndromes closely resembling PD.7, 8 The toxicity of

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MPTP/MPP+ is mainly due to the inhibition of mitochondrial complex I of the electron

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transport chain, which leads to the loss of ATP, collapse of mitochondrial functions,

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oxidative damages, and apoptotic cascades.7-9 Interestingly, the oxidative responses and

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apoptotic signaling can be regulated by the nuclear factor erythroid 2-related factor 2

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(Nrf2) pathway.6, 10 Nrf2 is a kind of transcription factor that modulates endogenous

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antioxidants and antioxidant enzymes.6, 10, 11 In response to stimuli and stress, it can be



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dissociated from Kelch-like ECH-associated protein 1 (Keap1) in the cytosol,

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translocated into the nucleus. In nucleus, Nrf2 binds to the antioxidant response element

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(ARE) and further activates transcription of genes encoding for antioxidants and

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detoxifications like heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase-1

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(NQO-1), and glutathione-synthesizing enzymes, like gutamate-cysteine ligase catalytic

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subunit (GCLC).10

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Pinostrobin (PSB; 5-hydroxy-7-methoxy flavanone, the chemical structure is shown in

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Figure 1A) is a dietary bioflavonoid principally isolated from the heart-wood of pine

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(Pinus strobus L.), also edible food materials such as pigeon pea (Cajanus cajan (L.)

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Millsp.), Thai ginger (Boesenbergia pandurata (Roxb.)), honey and propolis, etc.12-14

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So far, PSB has been applied as functional foods incorporated as multifunctional

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products in the pharmaceutical industry. PSB-mediated biological activities, including

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antioxidant,

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been well reported. In our previous study, we demonstrated that PSB conferred

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protective actions against amyloid-β (Aβ) peptides-induced AD model in PC12 cells,

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via inhibition of oxidative damages and suppression of mitochondria-mediated neural

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apoptosis and death.19 Although PSB exhibited neuroprotective effects in AD models,

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its actions on PD models remained an interesting speculation that needed further

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investigation. Thus, the aim of the present study was therefore to assess the

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neuroprotective effect of PSB against MPP+/MPTP-induced PD models, including SH-

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SY5Y cells and zebrafish.

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Materials and Methods

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Chemicals

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antiviral,

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anti-leukemia,

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and anti-inflammatory activities


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have

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Pinostrobin (PSB, power, purity: UV ≥ 98%) was purchased from Weikeqi Biological

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Technology (Si Chuan, China). MPP+ and MPTP, were purchased from Sigma-Aldrich

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(MO, USA). SP600125, PD98059, SB203580, LY294002, GF109203X, and Compound

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C were purchased from Selleck (Shanghai, China).

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Zebrafish maintenance

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The AB strain of wild type zebrafish was maintained as described in the Zebrafish

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Handbook. Normally developed fertilized eggs were collected for experiments and

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zebrafish were staged by days post fertilization (dpf). All experiments in current study

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were performed according to the Guide to Animal Use and Care of the University of

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Macau (UM) and were approved by the ethics committee of UM.

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Zebrafish anti-tyrosine hydroxylase (TH) whole-mount immunostaining

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Zebrafish embryos at 1dpf were exposed to 360 μM MPTP in the presence or absence

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of indicated concentrations of PSB for 2 days.20 The MPTP -containing buffer was

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renewed per day. Then zebrafish larvae were fixed, permeabilized, and blocked as

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previously described. After that samples were incubated with anti-TH antibody (1:250

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diluted, Millipore, Burlington, MD, USA) overnight at 4 °C and Alexa Fluor 488 goat

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anti-mouse (1:500 dilution, Invitrogen, Carlsbad, CA, USA) for 90 min at room

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temperature, respectively. After that, samples were imaged via a fluorescent microscope

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(Carl Zeiss, Axiovert 200, Oberkochen, Germany). The semi-quantification of TH+

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dopaminergic neurons was performed by an investigator unaware of the drug treatment

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using Image-J software. Results are expressed as integrated intensity of TH+ region.

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Zebrafish locomotion assay



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Zebrafish embryos at 1dpf were exposed to 360 μM MPTP in with or without PSB for 6

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days.20 At 6 dpf, larval zebrafish were transferred to 24-well round microplate (1

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larval/well) and acclimated to testing plate for 24 h.20 The 7dpf larval loading-

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microplate was then transferred to the zebrafish tracking box (ViewPoint Behavior

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Technology, Lyon, France) and acclimated to the testing box for 60 min.

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zebrafish behavior was monitored and recorded using the ViewPoint automated video-

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tracking system (ViewPoint Behavior Technology, Lyon, France). Three 10 min

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sessions were recorded for each zebrafish. The total distance was defined as the distance

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(in mm) that each fish moved during per session.

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Cell culture and treatment

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The human neuroblastoma SH-SY5Y cells were obtained from American type culture

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collection and maintained in DMEM/F12, supplied with 10 % FBS, 100 U/ml penicillin,

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and 100 mg/ml streptomycin (Gibco, Carlsbad, CA, USA) with a humidified

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atmosphere 5 % CO2 at 37 °C. All experiments were carried out about 36 h after cells

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were seeded. PSB, MPP+, and other compounds used were all dissolved in DMSO or

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dd-H2O as stock solutions and added into the culture medium. The DMSO

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concentration was maintained at 0.1 % in all treatments.

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MTT assay and LDH assay

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Cell viability was measured by the MTT assay (Invitrogen, Carlsbad, CA, USA).

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Briefly, SH-SY5Y cells were seeded in 96-well culture plates (2 × 104 cells/well) and

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received indicated treatments. Then, cells were incubated with MTT and finally the

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absorbance at 570 nm was measured. Cytotoxicity was measured by the LDH release

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assay. In brief, the levels of LDH released in the medium following damage of cellular


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After that,

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membranes were measured using the LDH assay kit (Roche, Mannheim, Germany)

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according to the manual. The absorbance at 490 nm was measured.

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Annexin V-FITC/PI apoptosis analysis

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The apoptosis analysis was performed via flow cytometric analysis according to the

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protocol of Annexin V-FITC/PI apoptosis detection kit (BioLegend, San Diego, CA,

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USA). Briefly, after treatments, cells were harvested and then incubated with Annexin

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V-FITC and PI solutions. The quantitative analysis was conducted using a flow

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cytometer (BD Accuri™ C6 Cytometer, BD Biosciences, San Jose, CA, USA).

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Cellular DNA fragmentation assay

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The cellular DNA fragmentation was measured using a Cellular DNA Fragmentation

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ELISA kit (Roche, Mannheim, Germany) according to the protocols. Finally,

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absorbance at 450 nm of each group was measured and represented as the level of DNA

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fragmentation.

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Measurement of mitochondrial membrane potential (Δψm)

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The level of Δψm was analyzed using the commercial fluorescent probe, JC-1

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(Invitrogen, Carlsbad, CA, USA). Cells were incubated with JC-1 (15 μg/ml) for 15

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mins at 37 °C. After that, the intensity of red fluorescence (excitation: 560 nm and

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emission: 595 nm) and green fluorescence (excitation: 485 nm and emission: 535 nm)

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was measured and the level of Δψm was calculated as the JC-1 red/green fluorescence

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intensity ratio. For further observation, staining cells were imaged using the In Cell

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Analyzer 2000 system (GE Healthcare, Milwaukee, WI, USA).

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Measurement of ROS



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After treatments, ROS was analyzed using the fluorescent probe, CM-H2DCFDA

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(Invitrogen, Carlsbad, CA, USA). Then, the level of ROS was measured via

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determining the fluorescence intensity (excitation: 490 nm and emission: 520 nm) of

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each sample. For further observation, staining cells were imaged using the In Cell

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Analyzer 2000 system (GE Healthcare, Milwaukee, WI, USA) .

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Lipid peroxidation assays

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In brief, after treatments, cells were collected and incubated with lysis buffers. The

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supernatant was collected and subject to analysis. The levels lipid peroxidation was

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measured via determination of maleic dialdehyde (MDA) using the lipid peroxidation

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colorimetric assay kit (BioVision, Milpitas, CA, USA) according to the manufacture’s

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protocol.

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Assessment of glutathione (GSH)/oxidized glutathione (GSSG) ratio

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In brief, after treatments, cells were collected and incubated with lysis buffers. The

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supernatant was collected and subject to analysis. The levels of GSH and GSSG were

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measured using the glutathione fluorometric assay kit (BioVision, Milpitas, CA, USA)

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according to the manufacture’s protocol.

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Assessment of NAD+/nicotinamide adenine dinucleotide diaphorase (NADH) ratio

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Briefly, after treatments, cells were collected and incubated with lysis buffers. The

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supernatant was then collected and subject to analysis. The NAD+ and NADH levels

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were measured using the NAD+/NADH quantification kit (BioVision, Milpitas, CA,

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USA) , following the manufacturer’s recommendations.

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Assessment of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and

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catalase (CAT) 8 ACS Paragon Plus Environment

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Briefly, after treatments, cells were collected and incubated with lysis buffers. The

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supernatant was then collected and subject to analysis. The activity of GDH-Px, SOD,

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and CAT were measured using the GDH-Px, SOD, and CAT assay kits (Beyotime,

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Shanghai, China), following manufacturer’s protocols, respectively.

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Luciferase assays

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Briefly, SH-SY5Y cells were transfected with pARE-luc reporter plasmids

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(SABiosciences, Frederick, MD, USA) using the SureFECT transfection reagents

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(SABiosciences, Frederick, MD, USA) according to the manual. 36 hours after

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transfection, cells were used and treated. After that, cell samples were then subjected to

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the Dual-Luciferase® reporter assay system (Promega, Madison, WI, USA) and

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luciferase activities were measured according the manual.

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Nrf2 siRNA transfection

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The Nrf2-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used

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to knock down Nrf2. Briefly, SH-SY5Ycells were seeded in 6-well culture plate, and

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then transfected with Nrf2 siRNA (80 nM) or scrambled siRNA (Santa Cruz

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Biotechnology, Santa Cruz, CA, USA) using Lipofectamine 3000 (Invitrogen, Carlsbad,

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CA, USA) according to the manual.

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Immunofluorescence assay

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After treatments, cells were fixed by 3.7 % PFA for 15 mins, permeabilized by 0.3 %

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Triton X-100-PBS, and then blocked with blocking buffer (0.1% Triton X-100 and 5 %

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BSA in PBS) for another 30 min at room temperature. After that cells were incubated

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with anti-Nrf2 antibody (1:250 diluted, Abcam, Cambridge, MA, USA) overnight at

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4 °C and Alexa Fluor 488 goat anti-rabbit (1:500 dilution, Invitrogen, Carlsbad, CA,



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USA) for 60 min at room temperature, respectively. For the nuclei observation, the cells

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were stained with DAPI for 10 min. Finally, the samples were mounted with prolong

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anti-fade reagent, and imaged by a confocal laser scanning microscope (TCS SP2, Leica,

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Solms, Germany).

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Quantitative PCR (qPCR) assay

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Total RNA was extracted using the High Pure RNA Isolation kit (Roche, Mannheim,

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Germany) according to the manufacturer’s protocol. Isolated RNA was then reverse-

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transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis kit (Roche,

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Mannheim, Germany) following the standard protocol. The qPCR assay was conducted

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using FastStart Universal SYBR Green Master reagents (Roche, Mannheim, Germany)

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with the Applied Biosystems 7900 HT Fast Real-Time PCR System (Applied

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Biosystems, Foster City, CA, USA). Each sample was analyzed in triplicate,

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Comparative Ct method was used to calculate the relative fold changes in gene

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expression normalized against the β-actin. The primer sequences used were listed in the

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Supplementary Table 1.

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Preparation of whole cell, cytoplasmic, and nuclear protein

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For the whole cell protein extraction, cells were collected and incubated with RIPA

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lysis buffer containing 1% PMSF and 1% protease inhibitor cocktail (Invitrogen,

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Carlsbad, CA, USA) for 30 min on ice. Cell lysates were centrifuged and the

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supernatant was collected and stored. For subcellular fractionation preparation, cell

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samples were processed using the nuclear and cytoplasmic Protein extraction kit

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(Beyotime, Shanghai, China). The protein content was assayed using the BCA assay

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(Invitrogen, Carlsbad, CA, USA) .


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Western blotting

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Aliquots protein samples (~30 μg of cell samples and ~40μg of animal samples) were

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resolved by SDS-PAGE (7.5% to 12%) and transferred to PVDF membranes (Bio-Rad,

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San Jose, CA, USA). The blots were then incubated with appropriate primary antibodies:

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Nrf2, HO-1, GCLC, Lamin B, GAPDH and β-actin (1:1000 diluted, Abcam, Cambridge,

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MA, USA), cleaved caspase-3, cleaved PARP, p-AKT, t-AKT, p-ERK, and t-ERK

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(1:1000 diluted, Cell Signaling Technology, Danvers, MA, USA), and peroxidase-

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conjugated secondary antibodies (1:2000, Cell Signaling Technology, Danvers, MA,

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USA). Finally, protein bands were visualized using an ECL plus Western blotting

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detection reagents (GE Healthcare, Milwaukee, WI, USA). The membranes were then

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scanned on a Bio-Rad ChemiDoc XRS Imaging System and the intensity of the protein

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bands were analyzed using Bio-Rad Quantity One Software.

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Graphing and statistical analysis

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Statistical analyses were performed using GraphPad Prism software (ver. 6.0; GraphPad,

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San Diego, CA, USA), and data are represented as means ± standard error of the mean

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(SEM). Statistical analysis of differences between two groups was done using the

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independent-samples t-test and one-way or two-way ANOVA with Bonferroni’s

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correction applied was used for comparison of more than two groups. Pearson’s

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correlation coefficient was used for correlation analyses. P < 0.05 was considered

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significant in all analyses.

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Results

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PSB alleviates MPTP-induced dopaminergic neuron loss and locomotion

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deficiency in zebrafish



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In this study, the anti-TH whole mount immunostaining was used to determine the PSB

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actions on dopaminergic neuronal populations in MPTP-treated zebrafish larvae. Firstly,

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treatment with PSB alone for 6 days at concentrations of 0.2-250 µM did not cause any

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death of zebrafish. Firstly, PSB (125 µM) did not affect the TH+ cell density in ventral

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diencephalic clusters in zebrafish. In line with previous study, we found that exposure

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of zebrafish embryos with MPTP significantly induced dramatic reduction of TH+ cell

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density (Figure 2A and 2B, all p < 0.01, versus control group). However, treatment with

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1, 5, 25, and 125 μM of PSB could significantly reverse the decreasing TH+ cell density,

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in a dose-dependent fashion (all p < 0.01, versus MPTP group).

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The dopaminergic neurotoxicity of MPTP that causes loss of dopamine stimulation

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results in deficits of locomotion behavior in zebrafish larvae. As shown in Figure 3A

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and 3B, MPTP significantly resulted in decrease of the movement distance in zebrafish

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(p < 0.01, versus control group). PSB treatment (1, 5, 25, and 125 μM of PSB)

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significantly suppressed MPTP-induced locomotive behavior deficits, increasing the

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movement distance, in a dose-dependent manner (all p < 0.01, versus MPTP group). In

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addition, PSB alone (125 μM) did not affect locomotion behavior in normal zebrafish.

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PSB provides protective actions against MPP+-induced apoptosis and death in SH-

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SY5Y cells

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We first examined the cytotoxicity of PSB in SH-SY5Y cells. As shown in Figure 4A

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and 4B, PSB alone at concentrations of 1-25 µM did not cause any apparent

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cytotoxicity to SH-SY5Y cells for 24 hours, but high doses (higher than 100 µM)

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showed obviously toxic effects. Our previous results showed that MPP+ exposure

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resulted in a dose- and time- dependent decrease of cell viability in SH-SY5Y cells

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(Supplementary Figure S1). Thus, treatment with 1.5mM MPP+ for 24 hours was used 12 ACS Paragon Plus Environment

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to induce neurotoxicity in current study. As shown in Figure 4C, pretreatments with 1, 5

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and 25 µM PSB for 2 h significantly suppressed MPP+-induced decrease of cell

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viability, in a dose-dependent fashion (both p < 0.01, versus MPP+ group), respectively.

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The LDH release assay also showed that 1, 5 and 25 µM PSB treatment significantly

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attenuated MPP+-induced LDH release (Figure 4D, all p < 0.05, versus MPP+ group),

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respectively. Moreover, the morphological changes of cells were also observed under a

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bright-field microscope. As shown in Figure 4E, MPP+ induced obvious cell damages,

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evidenced by roundness, neurite retraction and membrane blebbing. However, these

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alterations were reduced by treatment with 25 μM PSB.

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Next, the Annexin V and PI double-stain was used to detect anti-apoptotic action of

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PSB. As shown in Figure 5A and 5B, incubation with MPP+ resulted in significant

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increase of apoptotic cells (p < 0.01, versus control group). But this phenomenon was

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reversed by 1, 5 and 25 µM PSB treatments (both p < 0.05, versus MPP+ group). In

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addition, DNA fragmentation assay indicated that PSB (1, 5 and 25 μM) could

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significantly inhibit MPP+-induced increase of DNA fragmentation (Figure 5C, all p

Pinostrobin Exerts Neuroprotective Actions in Neurotoxin-Induced

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